Engineering a cytochrome P450 enzyme as a peroxygenase for selective hydroxylation of steroids

Domain: neuroscience
Published: 2026-02-07
Editorial outcome: major_revision

Abstract
Selective activation of steroid skeletons is crucial for the generation of pharmaceutically valuable compounds. Despite the success of engineering cytochrome P450 enzymes as peroxygenases to utilize cost-effective H2O2, their applications are limited due to poor H2O2 tolerance. Here, we report a cytochrome P450 enzyme, namely P450stri, from Streptomyces triculaminicus and its engineering as a peroxygenase with strong H2O2 tolerance for regioselective hydroxylation of steroids. We find that a conserved Phe above heme cluster predominantly determines the performance of peroxygenase activity and regioselectivity. Additionally, we reconstruct P450stri by two-dimensional engineering approach based on a Round Flask model to simultaneously increase selectivity and activity, yielding in the most effective 15β steroid hydroxylase variant using H2O2 as co-substrate. Moreover, we further transform the beneficial variants to corresponding residues in the members of “P450stri branch”, converting several P450 monooxygenases to peroxygenases with improved activity and selectivity. The present study provides insights into rational switch of enzymatic function, thus shedding a light on P450 enzymes on their biocatalytic applications.

Introduction
Selective activation of C(sp3)-H bonds represents a significant challenge in chemical synthesis, especially for substrates with inert carbon skeletons1,2. For instance, modifying non-active carbons in the core rings of steroids is an extremely difficult chemical reaction, due mainly to the energetically similar C(sp3)-H bonds3,4.

Because steroids are the second most commonly prescribed pharmaceuticals, their synthesis has attracted considerable interests5,6. Meanwhile, structural modifications of the core rings are a major route to diversify steroid structures, leading to approximately 300 approved steroid-based drugs to date7. Taken the hydroxy-functionalization as an example, direct hydroxylation of steroids at different sites typically requires harsh reaction conditions with environmental concerns3. Alternatively, biocatalytic methods for the preparation of hydroxylated steroids offer higher selectivity with better atom economy8–10. In the field of biocatalysis, cytochrome P450 monooxygenases (P450s) are ideal biocatalysts for C-H activation, which can facilitate the biosynthesis in a green and selective manner11. For example, the well-known cytochrome P450 (CYP102A1) from Bacillus megaterium (P450BM3) has been extensively investigated as a model P450 enzyme in biocatalysis over nearly fifty years12, and has been engineered for catalyzing a wide range of reactions13. Among its unnatural substrates, wild-type P450BM3 does not accept bulky steroid substrates, which requires enzyme engineering to play a key role for the change of substrate specificity with desired selectivity14. Unfortunately, engineering of P450s, through approaches such as directed evolution or semi-rational design, is labor-intensive and time-consuming15,16. In addition, their high dependency on NAD(P)H cofactor for O2 activation hinders their applications. Recently, the breakthrough comes from the engineering of several bacterial P450s as peroxygenases utilizing the H2O2 shunt pathway to overcome the NAD(P)H-dependence, in which H2O2 serves as a cost-effective oxidant for P450-catalyzed reactions17,18. However, the rapid inactivation of heme-mediated enzymes with poor H2O2 resistance becomes another major obstacle for practical applications19–23. Therefore, the increase of H2O2 tolerance to prevent heme oxidation is a vital issue for P450-derived peroxygenases on their catalytic efficiency and practical value.

In this work, we discovered a cytochrome P450 enzyme with native steroid hydroxylation activity and engineered this newly discovered P450 as a peroxygenase with strong H2O2 resistance and excellent regioselectivity. To facilitate the engineering efforts, the present study developed a Round Flask model separately focusing on heme environment and substrate accessing tunnels, in a two-dimensional fashion for simultaneous increase of selectivity and activity. Our study not only provides a feasible strategy for enzyme engineering, but also generates a valuable method for the preparation of hydroxylated steroids.

Results
Identification of a steroid hydroxylase P450stri
Bacterial P450s are typically expressed in water-soluble form, making them suitable for further engineering and applications24. Among different P450 systems, reactions catalyzed by the self-sufficient P450s (SSPs) with a heme domain and a CPR domain containing FAD and FMN are particularly operable and active12,25, but their substrates are usually less bulky, such as the case of P450BM3. Therefore, it would be valuable to find new P450 enzymes naturally using bulky steroids as substrates.

To simplify the screening efforts on obtaining steroid hydroxylases with high efficiency on electron transfer, we discovered a SSP, namely P450stri, from Streptomyces triculaminicus by genome mining using heme-associated nucleotide sequence as a probe. As shown in Fig. 1A, P450stri located at a distinct evolutionary clade compared to P450BM3 (41.4% amino acid sequence identity) and is classified into CYP102G subfamily. Notably, CYP102G subfamily has been demonstrated to differ in substrate scope from P450BM326,27, suggesting that P450stri may possess unique catalytic properties due to divergent evolutionary paths.

Next, we heterologously expressed P450stri in E. coli BL21(DE3) as a soluble protein with good yield (Fig. S1). The purified enzyme was characterized by CO binding assay, which displayed the typical cytochrome P450 spectrum with a characteristic absorbance peak at ~450 nm (Fig. 1B).

According to previous notion12, CYP102 family members possess remarkable activity toward fatty acids such as myristic acid (MA). Indeed, when MA was used as a substrate, P450stri could catalyze the ω-hydroxylation of MA at positions from ω−1 to ω−4 (Fig. 1C and Supplementary GC-MS data), and ω−2 position in MA was more favorable to be hydroxylated. Subsequently, testosterone (TST), a typical steroid, was served as the substrate to explore the possibility of hydroxylating steroids by P450stri. Under optimized conditions (Figs. S3–S5), we observed that P450stri preferentially utilizes less expansive NADH for catalysis of steroid substrates (Fig. S6), which is more advantageous for synthetic application compared to the use of NADPH by P450BM312. P450stri catalyzed the conversion of TST to three hydroxylated products with a NADH regeneration system. After purification and structural elucidation, the products were identified as C-15β, C-16β and C-2β in a ratio of 35:54:11 (Fig. 1D and Supplementary NMR data), suggesting the nature of multisite hydroxylation by this enzyme. Given the native steroid hydroxylation activity of P450stri, it could be served as a starting enzyme for regio- and stereo-selective hydroxylation of steroids.

Construction of a H2O2-driven biocatalytic system using P450stri
The applications of P450 enzymes typically suffer from poor selectivity, low catalytic efficiency, and reliance on expensive NAD(P)H cofactor. Alternatively, recent success on the conversion of P450s to peroxygenases paves a new way for C-H activation in a cost-effective manner18. However, the heme group in P450s is easy to be oxidized by high concentrations of H2O2, leading to a rapid enzyme inactivation. For instance, single mutation of P450BM3_F87A could convert a P450 enzyme to peroxygenase, but the H2O2 concentration typically had to be kept below 40 mM regardless of different reactions28–31, suggesting that high H2O2 tolerance for these enzymes is a key factor to decide their activity. Meanwhile, to avoid the direct use of H2O2, alternative methods, including employing an equimolar concentration of urea hydrogen peroxide or using H2O2 regeneration system, have been developed to slow down the heme oxidation rate for better peroxygenase activity20,32,33. Nevertheless, the inherited H2O2 sensitivity has been a major issue for practical utility of the peroxygenases. Given the low expression level of the oxygenase domain of P450stri, we chose full-length enzyme for the investigation. As previously implicated to be a crucial residue28,34,35, we changed Phe88 to a smaller Ala. The resulting mutant P450stri_F88A (M1) showed steroid hydroxylation in the presence of H2O2, indicating that same situation in P450BM3 is also applicable to P450stri. In our case, the highest product formation by M1 was achieved in the range of 120–200 mM H2O2 (Fig. 2A), which could be a collective outcome from the decrease of steric hindrance and hydrophobicity within the heme environment. In addition, compared to natural peroxygenases as well as other engineered peroxygenases36,37, such as P450BM3_F87A mutant, particularly on the hydroxylation of steroids, the tolerance of P450stri_F88A against H2O2 was much stronger, reflected by significantly increased product formation. Thus, 150 mM H2O2 was chosen for subsequent investigation. Compared to NADH-mediated reactions, M1 exhibited favorable properties to pH and temperature in the H2O2-diven system (Figs. S7–S9), which is beneficial to application.

Next, the performance of M1 mutant on TST hydroxylation was evaluated by H2O2-mediated peroxygenase. Compared to the use of NADH regeneration system, 15β-selectivity was improved from 35% to 94% by the single mutation at position 88 (Fig. 2C, D). Similarly, the product distribution of 15β-OH was changed from 81% to 94% by H2O2-diven hydroxylation, partially due to the reduction of further oxidation under NADH-dependent system (Figs. 2D and S11). Taken together, P450stri could be converted to a peroxygenase to show high H2O2 tolerance and increased regioselectivity.

Given that M1 mutant exhibited desired peroxygenase activity, high H2O2-tolerance, and good regioselectivity, UV–Vis spectral analysis was performed to characterize its substrate specificity in comparison with that of P450BM3. Compared to MA, changes in binding spectrum induced by TST could not be observed in the case of wild-type P450stri and P450BM3 (Figs. S17 and S22). P450stri seemed to favor fatty acids with multiple hydroxylation sites (Fig. 2E). After removing unfavorable steric hindrance above heme, M1 exhibited a larger absorbance shift upon TST binding than that of P450BM3_F87A, whereas the binding affinity toward MA by both variants was decreased. Meanwhile, M1 preferentially hydroxylated the ω−4 position in MA, differing from the hydroxylation at ω−5 position favored by P450BM3_F87A38, suggesting certain structural differences to influence the regioselectivity. In addition, M1 displayed a comparable peroxygenase activity toward MA as P450BM3_F87A, with each enzyme maintaining different site preferences as described above (Fig. S15).

As shown in Fig. 2F, G, M1 exhibited a higher binding affinity (Kd = 22.8 ± 1.6 μM) towards methandienone (MTS) than that for TST (Kd = 36.2 ± 4.6 μM), indicating that larger substitutions at C17 position likely enhance the binding to the active site (Figs. S19 and S20), and lead to higher peroxygenase activity (Fig. 2H). Thus, the steric volume of substitutions at C17 seemed to be crucial for substrate recognition.

Typically, titration of H2O2 to P450s results in heme bleaching, leading to oxidation of the heme thiolate ligand to sulfenic acid, the disruption of the conjugated tetrapyrrole rings as well as other radical-mediated reactions to damage the heme structure39–41. Thus, heme bleaching experiments were conducted to evaluate the stability of wild-type P450stri and M1 towards H2O2. Time-course of absorbance changes for heme decay of M1 were significantly lower than that of wild-type P450stri, especially in the presence of substrate TST (Fig. S25). These results were consistent to prior studies, suggesting that an increased free space in heme vicinity makes P450BM3_F87A more stable in H2O2 solutions28.

To gain more insights into the mechanism of P450stri-mediated hydroxylation, numerous efforts were made on solving its 3-D structure, but our efforts were not paid off. Alternatively, a 3-D structure of full-length P450stri was created by using AlphaFold342 (Fig. S10), and then molecular dynamic (MD) simulations for the heme domain of this enzyme were undertaken to explore potential structural features on these selectivity (Figs. S27 and S28). In the simulations, we recorded the distances of TST-C15—O=Fe-Heme and TST-C16—O=Fe-Heme in wild-type P450stri and M1 (Fig. 3A, B). Compared to wild-type P450stri (54% 16β-selectivity), the distance of TST-C15---O=Fe-Heme was decreased from 4.9 Å of wild-type M to 3.5 Å of M1 (94% 15β-selectivity). The shorter distance from TST to heme center in M1 was likely due to the removal of unfavorable steric hindrance above heme, implying a significant contribution of heme environment to regioselectivity.

Next, multiple sequence alignments based on approximately 250 homologous CYP102 sequences were performed to identify a common feature. It was found that the first about 50 residues in N-termini are generally not conserved (except for positions 25 and 26 based on P450stri numbering) to exhibit low evolutionarily conservation (Figs. 3D and S30). Given the fact that M1 outperformed variant P450BM3_F87A in both peroxygenase activity and H2O2 tolerance, we speculated that the variable N-termini could be a key factor to decide their different abilities against H2O2.

To identify the key elements in M1 responsible for its better peroxygenase activity under high H2O2 concentrations, we constructed three chimeric P450BM3_F87A variants using the N-terminal sequences of P450stri in different lengths (Fig. 3C and Supplementary Data 1). Since the N-termini exhibit non-conserved characteristics across CYP102 enzymes, they seemed to be feasible for engineering and the mutagenesis of N-terminal regions did not change the levels of protein expression. These variants were then tested in the hydroxylation of TST at high H2O2 concentrations. The initial screening results suggested that, besides P450BM3_F87A_C3, both P450BM3_F87A_C1 and P450BM3_F87A_C2 exhibited higher peroxygenase activity, particularly at 100 mM H2O2 (Table S1 and Figs. 3E and S26). Furthermore, five residues with low conservation scores and differing from P450stri were selected (Fig. S31) to replace with the corresponding residues from P450stri, resulting in double variants P450BM3_F87A/V26L and P450BM3_F87A/R47T with higher peroxygenase activity. These findings suggested that the peroxygenase activity of P450stri homologous enzymes is influenced by evolutionary variation in their N-terminal regions.

Next, the first 37 residues were exchanged with those of P450stri to generate P450BM3_F87A_C1 mutant, resulting in improved peroxygenase activity under H2O2 concentration up to 300 mM (Fig. 3G). The optimal H2O2 concentration was increased from 40 mM to 150 mM, suggesting a beneficial evolution in this region, especially regarding H2O2 tolerance. In the complex structure of TST-P450BM3 (Fig. 3F)43, two TST molecules were respectively bound to substrate tunnel (TST1) and substrate binding pocket (TST2), indicating that certain residues in the substrate entrance tunnel are important for its function. Based on this observation, non-conserved V26 (L27 in P450stri) pointing to TST1 was a possible residue to act as a “gatekeeper” for substrate recognition and directing H2O2 flow. Subsequently, corresponding P450BM3_F87A/V26L variant was constructed. The H2O2 tolerance of this variant indeed increased to above 100 mM, strongly supporting that V26 in P450BM3/L27 in P450stri is an essential residue for better peroxygenase activity under high H2O2 concentrations.

Engineering to improve peroxygenase activity based on a Round Flask model
In conventional enzyme engineering, the generation of a large number of mutants is a prerequisite to obtain desired enzyme mutant for various purposes, which is laborious and screening-dependent44. Recently, semi-rational engineering of residues around the substrate binding pocket or substrate tunnels18,45,46, namely Focused Rational Iterative Site-specific Mutagenesis (FRISM)47, has become an advanced approach to produce a smart library of mutants. However, FRISM usually focuses on a particular region for the increase of the efficiency on the engineering efforts. In our case, after generating the M1 variant with strong H2O2 tolerance and high regioselectivity, we had to deal with another important issue of increasing its peroxygenase activity. Based on the substrate entrance tunnels of M1 and the rigid cyclic structures of steroids, a model of Round Flask was introduced as a way for the semi-rational engineering of this enzyme, in which substrate binding pocket and substrate entrance tunnels were separately yet cooperatively engineered to achieve simultaneous increase of selectivity and activity in a “Two-dimensional” fashion. Meanwhile, MD simulations suggested that, compared to the binding pose in wild-type P450stri (Fig. 3A), TST is located closer to the substrate binding pocket in M1 (Fig. 3B). By eliminating unfavorable steric hindrance at position 88, the D ring was positioned above heme cluster, which pulls C15 forward to the CpdI center. This altered substrate binding pattern in M1 was likely to account for the improved 15β-selectivity. Although substrate binding site associated with heme environment is critical for determining regioselectivity, the substrate tunnels are also associated with the activity18,48. To simultaneously increase regioselectivity and activity, our focus was then centered around the substrate accessing tunnels in M1 variant. As shown in Fig. 4A, preliminary inspection of the substrate accessing tunnels of M1 by CAVER 3.0 plugin49 in Pymol50 based on heme domain of P450stri resulted in two substrate tunnels associated with a set of residues at the entrance for substrate recognition and binding. Eight residues were identified and most of which were located at the front-end of the N-terminus, clustering as substrate tunnels with the features of narrowly long, hydrophobic and non-aromatic. All these residues, except for P26, are variable in homologous sequences and thus more feasible for further engineering (Fig. 4B). Meanwhile, enabled by MD snapshots of M1-TST complex, we were able to observe that the positions or conformation of these residues are significantly changed during the simulations (Fig. 4C, D). These residues could decide the outcomes on how the hydrophobic TST reaches the heme center for hydroxylation. We therefore speculated that thoroughly engineering these residues could improve peroxygenase activity while maintaining or increasing regioselectivity.

Finally, a Round Flask model was constructed to guide the engineering of P450stri for both peroxygenase activity and 15β-selectivity. As shown in Fig. 4E, the Round Flask model could be divided into two parts as the “round bottom” and the “neck to cap”. The “round bottom” refers to enzyme active site and engineering residues in this region would reshape the enzyme’s response to substrate binding, and the “neck to cap” represents the substrate tunnel containing a cluster of ordered residues. The steric side chains of these residues in the “neck to cap” region appear to influence substrate accessibility and conformation, and thus this region could be engineered to enhance peroxygenase activity while keeping the desired regioselectivity by active site modification.

According to the model of “round bottom” and the 15β-selectivity of 94% possessed by mutant M1, we then used this variant as a template for the engineering of the “neck to cap” region to increase peroxygenase activity. Given the steric influence on the substrate entrance, we therefore utilized amino acid volume as the measure to scan eight residues in the substrate tunnels of M1, in which Ala, Leu, and Phe are respectively represented the volumes of “small”, “medium”, and “large”. Collectively, we came up with a Round Flask model based on modeled structure of P450stri and simulated interactions between the enzyme and substrate TST as illustrated in Fig. 4E, which could serve as a guideline for selecting appropriate amino acids for mutagenesis.

To accomplish two-dimensional engineering for simultaneous increase of both regioselectivity and activity, a series of double mutants were constructed and their properties were evaluated using TST as the substrate as summarized in Table S2. According to Fig. 5A, almost all the mutants maintained high 15β-selectivity derived from M1, confirming M1 as the selectivity controller for our engineering model. Meanwhile, engineering on residues P26, L27, and T48 at the suspected positions positively affected peroxygenase activity. By introducing Ala or Leu at position 26, the bioconversion was enhanced from 34.7% to 45.9% and 52.2%, respectively, suggesting that replacing the rigid Pro residues with hydrophobic side chains might alter the secondary structure composition at this site, resulting in increased peroxygenase activity. However, the F88A/P26F mutant significantly decreased activity, demonstrating that appropriate steric hindrance in the substrate tunnel is essential for substrate binding or product release. When the polar T48 was mutated to nonpolar Ala or Leu, peroxygenase activity improved while preserving high 15β-selectivity over 93%, indicating that the reconstructed hydrophobic environment in the substrate tunnel by mutants F88A/T48A (M5) or F88A/T48L (M6) might facilitate the binding of the strong hydrophobic TST. Surprisingly, residue L27 was identified to be crucial for peroxygenase activity, as that in the mutant P450BM3_F87A/V26L. The initial screening results showed a positive correlation between the peroxygenase activity and the steric hindrance at position 27 (F88A/L27F > M1 > F88A/L27A), confirming the rationality of our engineering strategy. Importantly, variant F88A/L27F (M4) achieved the highest peroxygenase activity and exhibited desired 15β-selectivity of 93%. Besides, the hydroxylation activity at M4 for C2 and C16 positions was 2.7 times and 2.0 times those of M1, indicating that L27F mutant also increased the peroxygenase activity to hydroxylate other sites. These results confirmed the critical role of hydrophobic side chain in the substrate tunnel, especially in facilitating the acceptance of nonpolar steroids for further regioselective hydroxylation in the substrate binding pocket. Additionally, NADH-mediated reactions were performed to investigate the product distribution of M4 (Fig. S34). Again, the portion of 15β-OH TST was varied from 70% to 93%, mainly due to the reduction of further oxidation.

To gain a deeper understanding of the enhanced utilization of H2O2 by M4, we performed additional kinetic experiments toward H2O2 (Fig. S35). M4 displayed a higher H2O2 binding affinity (Km = 80.1 mM) compared to M1 (Km = 113.3 mM), which positively correlates with peroxygenase activity. Subsequently, hydroxylation of TST and MTS by M4 at different concentrations of H2O2 was carried out to evaluate the H2O2 tolerance of M4 as well as the best TONs for the selected steroids. As shown in Fig. 5C, at all H2O2 concentrations tested, the TONs of MTS significantly outperformed that of TST, and their best TONs were obtained under high H2O2 concentrations (100–200 mM). In the case of substrate MTS, M4 maintained comparable TONs within a H2O2 concentration ranging from 200 to 400 mM, strongly suggesting its potential as a useful peroxygenase.

To further elucidate the molecular basis for improved catalytic performance of M4, MD simulations were performed to analyze conformational changes (Fig. S29). For variant M1 with desired 15β-selectivity, MD simulations showed a hydrogen bonding of the hydroxyl group of TST with I265. Meanwhile, the hydrogen bonding between A ring and surrounding residues was a crucial factor to lock a specific conformation for the substrate. TST molecule interacted with A337 and T359 through water-mediated hydrogen bond networks, thereby positioning C15 of TST optimally above catalytically active oxyferryl species (CpdI) in M1 (Fig. 5D). Conversely, upon substitution of Leu with sterically bulky Phe at position 27, TST molecule appeared to move forward and adapted a direct hydrogen bond with A337 in M4 (Fig. 5E), potentially contributing to a more stable binding pose. It is worth noting that the substrate binding pattern in M4 is similar to that of M1, due possibly to their favor on 15β-selectivity. Additionally, the hydrophobic phenyl ring at position 27 might facilitate the accommodation of hydrophobic TST molecule51, thus likely promoting the proper positioning of A ring. Compared to M1-TST complex, a comparable yet slightly more stable distance trajectory (TST-C15—O=Fe-Heme) was observed in M4-TST complex (Fig. 5B), suggesting the possibility of oxidizing C15. To verify these observations and postulations, Ala variants were then constructed and evaluated for the hydroxylation of TST (Fig. S33). As a result, both peroxygenase activity and 15β-selectivity were indeed decreased or even abolished by these mutants, thereby confirming the importance of these residues to the catalytic performance of M4, particularly I265 and T359.

Notably, emerging evidence has also indicated that water-accessing tunnels might be involved in the regulation of P450-derived peroxygenase activity52,53. Based on the similar physical and chemical properties of H2O2 and H2O, H2O could be served as a replacement for H2O2 to gain mechanistic insights53. Specifically, from MD simulations of M1, we found that water molecules flow through the tunnel between L27 and R50 to the active site within a few nanoseconds (Fig. S32), implying that there is a tunnel between L27 and R50 for H2O2 inflow. In the simulations of M4, F27 appeared to act as a hydrophobic “lid” that prevents H2O2 escape from the active site. This was also supported by the observation that two water molecules were retained under F27 for more than 60 ns, which is in agreement with the higher efficiency of H2O2 utilization by M4 (Fig. S35).

Based on the results from engineering, we subsequently performed multiple mutagenesis starting with 15β-selective mutants (Table S2). Despite a decreased in peroxygenase activity, the increase of 15β-selectivity by these triple mutants strongly supported the rationale on the regulation of regioselectivity by M1 variant. Therefore, using Round Flask engineering model, a variant M4 with promising peroxygenase activity and desired regioselectivity were obtained, confirming the effectiveness of this two-dimensional engineering strategy.

Subsequently, the kinetic parameters were measured using the mutants with increased substrate conversions (Table S3 and Fig. S36). All tested mutants exhibited higher binding affinities (lower Km) compared to that of wild-type P450stri and M1, among which M7 showed the lowest Km value of 0.26 mM. The improvement of catalytic efficiency (kcat/Km) for TST by M2 and M3 was due possibly to the increased binding affinity. Because of the changes of residue L27, the catalytic efficiency of peroxygenase by M4 reached to 3.5 times that of M1, and was 4.9-fold higher than that of wild-type P450stri in NADH-mediated system. Such a catalytic efficiency possessed by variant M4 was the highest thus far, which outperforms previously reported 15β-hydroxy steroid hydroxylase (B4-M5, kcat/Km = 3.5 × 105 M−1 min−1, 85% 15β-selectivity)54. Meanwhile, M4 also exhibited a higher kcat value (253 min−1) than a recent report of CYP154C5 variant AAA/T248D (90 min−1) on TST hydroxylation51.

Semi-preparative scale enzymatic synthesis of 15β-hydroxy-steroids
To explore the synthetic potential of P450stri_M4 at a semi-preparative scale, regio- and stereo-selective hydroxylations of steroids (TST and MST) were carried out, using the simple and cost-effective H2O2-driven biocatalytic system catalyzed by M4 variant at room temperature. As showed in Figs. 6 and S37, decent conversions of 90% for TST and 100% for MTS were achieved. As illustrated in Fig. 6A, excellent 15β-selectivity of 92% was retained for TST in a 100 mL scale with an isolated yield of 75% (15.2 mg), confirming the scalability of engineered M4 variant for the biosynthesis. In the case of MTS, the product with an additional 15β-hydroxy group was produced (Supplementary NMR data) and purified with an isolated yield of 82% (18.8 mg), which showed 15β-selectivity (91%) with 150 mM H2O2 in addition to 100% conversion. Collectively, both substrates of TST and MTS demonstrated the synthetic potential of variant M4 on the preparation of 15β-hydroxy-steroids using inexpensive H2O2 as the driving force, further highlighting the biocatalytic utility of this engineered peroxygenase.

Expansion of two-dimensional engineering approach to “P450stri branch”
Since P450 enzymes ubiquitously occur in nature, it was important to address the distribution and evolutionary relationship of P450stri-associated enzymes. Therefore, based on approximately 250 homologous sequences, we performed EFI-SSN analysis55 for P450stri-associated enzymes. According to Fig. 7A, most of the enzymes are located in the phylum of Actinomycetota, followed by Bacillota and Pseudomonadota, suggesting that the homologous proteins of P450stri generally occur in bacterial kingdom. According to evolutionary distinctions, the “P450stri branch” is composed of twelve potential P450 enzymes. Given their sequence homolog, we speculated that the enzymes in this branch might generally possess the capability of catalyzing steroid hydroxylation. To test this hypothesis and to verify the feasibility of our engineering approach, we randomly selected three P450s among this branch, namely P450sabi, P450sdsm, and P450sgri, for further investigation (Figs. S1, S2, S38–S42). These enzymes share about 41% sequence identity with P450BM3 and all belong to the less-studied CYP102G subfamily26. To test whether the Round Flask model could be applied to the engineering of other enzymes in the same branch with a two-dimensional fashion, the beneficial information on M1 and M4 variants of P450stri was then transformed to P450sabi, P450sdsm, and P450sgri at their corresponding positions, generating six mutants for peroxygenase comparisons under optimal H2O2 concentrations (Figs. S43 and S45). As a result, all variants using M1 as the template, including P450sabi_F87A (P450sabi_M1), P450sdsm_F86A (P450sdsm_M1), and P450sgri_F88A (P450sgri_M1), indeed acquired peroxygenase activity with higher 15β-selectivity. This strongly suggested that on the change of conserved Phe in the region of “round bottom” is sufficient to convert a typical P450 monooxygenase as a peroxygenase with higher regioselectivity (Fig. 7B and Table S4). For example, the construction of mutant P450sgri_M1 successfully turned P450sgri to a 15β-hydroxylase, along with the enhancement of regioselectivity from 28% to 87%. Moreover, when this residue was combined a beneficial residue located in the substrate tunnel for mutagenesis, the conversions by resulting variants with double mutations were profoundly increased (Fig. 7C), further indicating the broad utility of the present approach on the improvement of peroxygenase activity originated from different P450s.

After determining the kinetic parameters of the variants generated from the Round Flask model (Fig. S46), the catalytic efficiencies of these variants with double mutations, exemplified by P450sabi_F87A/L26F (P450sabi_M4), P450sdsm_F86A/L25F (P450sdsm_M4), and P450sgri_F88A/L27F (P450sgri_M4), were enhanced two to sixfold, while their regioselectivity was also remarkably increased compared to wild-type enzymes (Fig. 7D). This simple substitutions at two residues could achieve a two-dimensional improvement of the enzymes in the “P450stri branch”, demonstrating the rationality of the Round Flask model for steroid peroxygenase engineering and the versatility of this engineering approach.

In conclusion, we have discovered a cytochrome P450 enzyme, namely P450stri, from Streptomyces triculaminicus with natural hydroxylation of testosterone to produce 2β-, 15β- and 16β-hydroxylated products. To establish a viable biocatalytic system using inexpensive H2O2, we have successfully engineered P450stri as a strong peroxygenase (M1) with H2O2 tolerance up to 300 mM, showing potential for practical applications. Additionally, sequence-structure-function analyses have revealed that the N-terminus of P450stri is crucial for its high H2O2 tolerance and peroxygenase activity, which is demonstrated by the peroxygenase activity at high H2O2 concentrations from the chimeric P450BM3_F87A_C1, an enzyme of P450BM3 with N-terminal sequence of P450stri. Furthermore, to accomplish simultaneous increase of selectivity and activity in enzyme engineering, we have further engineered variant M1 based on a Round Flask model with a limited set of mutants to achieve enhanced hydroxylation activity and excellent regioselectivity. Moreover, M4-catalyzed reactions have been scaled up for semi-preparation of different 15β-hydroxylated steroids. Finally, the Round Flask engineering model was implemented to convert several P450s in the “P450stri branch” as peroxygenases with improved selectivity and activity in a “two-dimensional” manner. Collectively, this study enables practical regioselective hydroxylation of steroids and offers a new approach for the engineering of P450 biocatalysts for selective C-H activation.

Methods
Site-directed mutagenesis
Mutagenesis was performed using the Gibson Assembly method by ClonExpress Ultra One Step Cloning Kit V3-C117 (Vazyme Biotech Co., Ltd). Site-directed mutations were introduced by PCR in 25 μl scale and the PCR product was purified for recombination. The mutated genes in the pET28a vector were then transformed into E. coli BL21(DE3) for protein expression. The primers used for mutagenesis are listed in Supplementary Data 1.

Cloning, expression and purification of P450s and their variants
The pET28a plasmids harboring codon-optimized P450 genes and their variants, with NdeI and XhoI restriction enzyme sites, were transformed into BL21(DE3) E. coli cells. The cells were incubated in 200 mL TB medium containing 50 μg/mL kanamycin at 37°C and 220 rpm until OD600 reached 0.6~0.8. Protein expression was induced by adding IPTG, FeSO4 and α-aminolevulinic acid (ALA) to the final concentrations of 0.5 mM, 0.1 mM, and 0.5 mM, respectively, and the cells were then incubated overnight at 18°C and 200 rpm for 20 h. Afterward, the cells were harvested by centrifugation (5000 × g, 10 min, 4°C), washed with PBS buffer (pH 7.4), and stored at −80°C for further use.

The harvested cell pellets containing P450s were resuspended in 50 mM Tris-HCl buffer (10 mM imidazole, 300 mM NaCl, 5% glycerol, pH 8.0) and lysed by ultrasonication. Cell debris was removed by centrifugation twice at 12,000 × g for 40 min at 4°C and the enzymes were purified through Ni-NTA affinity chromatography. The fractions obtained from elution buffer (50 mM Tris-HCl, 300 mM imidazole, 300 mM NaCl, 5% glycerol, pH 8.0) were desalted by PD-10 columns (Cytiva, USA) using the storage buffer (50 mM Tris-HCl, 10% glycerol, pH 8.0). The enzymes were subsequently concentrated using a 100 kDa Amicon Ultra-15 Centrifugal Filter Unit (Millipore, USA). The purified proteins were examined by SDS-PAGE with Coomassie blue staining. The active P450 concentration was determined by CO difference spectra as described previously56.

Computational study
The predicted 3D structure of heme-bound P450stri complex was generated by AlphaFold342. To set up substrate-bound systems, docking simulations by Molecular Operating Environment (MOE)57 were performed using standard default parameter settings in MOE. The partial atomic charges were received using restrained electrostatic potential (RESP) calculations at the HF/6-31G* level of theory with Gaussian0958,59. The force field for the CpdI species was taken from published parameters60. MD simulations with water molecules were performed using AMBER22 package61. The P450stri enzymes were parameterized using AMBER ff19SB force field62. The general Amber force field (GAFF)63 was utilized for substrate TST. The topology files and initial coordinates were prepared by the tleap module of AMBER22. The enzyme-TST complexs were solvated in an equilibrated box of TIP3P water and neutralized by adding Na+ ions to the system. After the system setup was complete, the complex was subjected to energy minimization using 10,000 steps of steepest descent and 10,000 steps of conjugate gradient algorithm, respectively. Subsequently, the system was gently heated from 0 to 300 K under the NVT ensemble for 300 ps. To gain a uniform density, 1.0 ns of density equilibration was performed under the NPT ensemble (300 K and 1.0 atm). Finally, 200 ns productive MD simulations under the NPT ensemble were performed, with the simulation time length typical to previous computational studies of P450s. All the covalent bonds involving hydrogen atoms during MD simulations were constrained using SHAKE algorithm64, with an integral step of 2 fs, and the nonbonded cutoff length was set to 10 Å. The conformational PCA of M1-TST complex was analyzed using AmberTools2261 and all productive trajectories were combined for further analysis using VMD65, ChimeraX66 and PyMOL50.

GC-MS analyses using myristic acid as the substrate
For NAD(P)H-supported reactions, the hydroxylation of myristic acid was performed at 35°C on 400 μL scale in a glass tube with 0.2 μM P450 enzyme, 0.5 mM myristic acid and 4 mM NAD(P)H in phosphoric acid buffer (100 mM, 5% glycerol, pH 8.0). After 30 min, reactions were quenched and acidified by adding 20 μL HCl (1 M) for further gas chromatography-mass spectrometry (GC-MS) analysis as follows: the samples were extracted with ethyl acetate (3 × 400 μL) and the combined organic extracts were further dried over anhydrous MgSO4 and then evaporated under N2 gas. The residue was dissolved in 350 μL anhydrous ethyl acetate and 50 μL of BSTFA/TMCS (99:1) for TMS-derivatization at 75°C for 30 min. The products were confirmed by GC-MS.

For H2O2-driven reactions, the hydroxylation of myristic acid was performed at 35°C on 400 μL scale in a glass tube with 2 μM P450 enzyme, 0.2 mM myristic acid and H2O2 concentration of 10 mM for P450BM3_F87A and 40 mM for M1, respectively, in phosphoric acid buffer (100 mM, 5% glycerol, pH 8.0) for 2 h. The reaction samples were processed as described previously and analyzed immediately by GC-MS. The products were confirmed with authentic dodecanoic acid as the internal standard via peak area integration. A control reaction containing all the components except the P450 enzyme was conducted.

Substrate binding assays
Substrate binding assays of M1 were performed based on substrate-induced change in heme environment, measured by UV–vis spectrophotometer (SHIMADZU, UV-1780) at 25°C. The medium scan rate was applied from 300 nm to 600 nm with the interval of 1.0 nm. The baseline spectrum was initially recorded using 1 mL of phosphoric acid buffer (100 mM, 10% glycerol, pH 8.0). DMSO stock of substrates were added to 1 mL of phosphoric acid buffer containing 4 μM P450 enzyme until no further spectral shift occurring. The absorbance difference was plotted against substrate concentration, and the data were fitted to Michaelis–Menten equation to determine the dissociation constant Kd.

Kinetic analysis for heme decay
The heme decay kinetics of P450stri enzymes were determined using UV–vis spectrophotometer (SHIMADZU, UV-1780) at 25°C. H2O2 was added to freshly prepared P450 enzyme solution (4 μM in 100 mM phosphoric acid buffer containing 10% glycerol, pH 8.0, with or without 200 μM testosterone) to a final concentration of 80 mM in 1 mL. The absorbance changes at 418 nm were immediately recorded after thoroughly and gently pipetting the mixture, and the decay rate (1/t) was calculated by fitting a curve of absorbance decrease against time using the single exponential function in Origin 2022: y=y0+Aexp(−kt)”.

Kinetic parameters determination of P450s for testosterone
The kinetic determination of P450s with testosterone under NADH-mediated reactions was conducted as follows: the reaction mixture containing 0.8 μM P450 enzyme, testosterone at concentrations from 0 to 1.5 mM, and 4 mM NADH in 200 μL of phosphoric acid buffer (100 mM, 5% glycerol, pH 8.0), was incubated at 35°C and 850 rpm for 2 min. After termination of the reactions by adding equal volume of methanol, the yields of hydroxylated products were quantified by HPLC, and the initial reaction rates at various testosterone concentrations were calculated using Michaelis–Menten equation.

The kinetic determination of peroxygenases with testosterone under H2O2-driven reactions was conducted as follows: the reaction mixture containing 1 μM P450 enzyme, testosterone at concentrations from 0 to 1.5 mM, and 150 mM H2O2 in 200 μL of phosphoric acid buffer (100 mM, 5% glycerol, pH 8.0), was incubated at 35°C and 850 rpm for 1 min. After termination of the reactions by adding equal volume of methanol, the yields of hydroxylated products were quantified by HPLC, and the initial reaction rates at various testosterone concentrations were calculated using Michaelis–Menten equation.

Biocatalytic reactions at a semi-preparative scale
For structural elucidation of three hydroxylation products from TST, reactions catalyzed by P450stri were performed individually in eight parallel 50 mL batches (400 mL in total), using an NADH regeneration system (4 μM P450stri, 0.5 mM TST, 4 mM glucose, 1 mM NAD+, and 0.5 mg/mL GDH) at 35°C and 120 rpm for 12 h. The reactions were terminated by addition of ethyl acetate (3 × 400 mL) to extract the products (72% conversion). The organic phase was evaporated and the residue was further purified by reverse-phase C18 chromatography using acetonitrile and H2O (1:3, v/v) as a mobile phase. The eluates containing the products were combined, evaporated, and freeze-dried, with isolated yields of 65% (10.1 mg, P1-TST), 78% (18.4 mg, P2-TST), and 46% (2.2 mg, P3-TST), respectively. The purified hydroxylation products were dissolved in deuterated chloroform (CDCl3) and subjected to NMR analysis.

To explore the synthetic potential of P450stri_M4, scale-up reactions were conducted at 100 mL using 4 μM P450stri_M4, 0.8 mM substrate, and 150 mM H2O2. The reactions were conducted at 80 rpm at room temperature. Time-course experiments were carried out by taking 200 μL of the reaction mixtures at different time points to determine substrate conversion and regioselectivity. The reactions were terminated by addition of ethyl acetate (3 × 100 mL) to extract the products, subsequent purification and freeze-drying were carried out as described above, and final dry products were weighted. The resulting hydroxylated MTS derivatives were dissolved in CDCl3 and subjected to NMR analysis for structural characterization.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "This study presents a semi-rational engineering approach for enhancing the hydrogen peroxide-dependent peroxygenase activity and regioselectivity of a steroid-hydroxylating P450 enzyme (P450stri). The authors employed a multi-stage strategy: (1) Initial site-directed mutagenesis targeting a conserved phenylalanine residue yielded a single mutant with baseline peroxygenase activity and improved regioselectivity; (2) Sequence alignment identified putative H₂O₂-tolerance determinants, which were functionally validated through chimeric P450BM3 construction; (3) Substrate channel engineering via key residue mutagenesis generated double mutants with significantly enhanced activity, constituting the novel \"Round Flask\" strategy. The approach's potential generalizability was demonstrated through successful application to three additional steroid-hydroxylating P450s among twelve homologs mined with P450stri as a template.\nWhile this work makes valuable contributions to biocatalysis and protein engineering, several aspects require clarification prior to publication:\n1. Many NAD(P)H-dependent P450 enzymes feature a conserved phenylalanine residue near the heme active center. Replacing this residue with smaller side-chain amino acids (e.g., alanine) has been shown to markedly enhance peroxygenase activity—a phenomenon first demonstrated in P450 BM3 (see Ref. 26 in this paper). The prevailing hypothesis attributes this effect to reduced steric hindrance and hydrophobicity in the active site, thereby accommodating H₂O₂. However, emerging evidence suggests an alternative or complementary mechanism: the mutation may open water-accessible channels that facilitate H₂O₂ inflow to the heme center (Zhao P. et al. J. Am. Chem. Soc. 2023, 145(9), 5506-5511; Zhao P et al. Chem. Sci. 2024, 15, 8062-8070; Ref.48 in this study). Please comments these viewpoints and provides a multidimensional understanding for the role of Phe mutation in the improvement of P450 peroxygenase activity.\n2. In Figure 2A, the comparison of peroxide-dependent peroxygenase activity between P450stri_F88A and P450BM3_F87A is inequitable. This is because P450stri is a native steroid hydroxylase, which P450BM3 is a fatty acid hydroxylase. I will suggest to re-examine the peroxygenase activity of P450BM3_F87A by using one of fatty acid, such as lauric acid as a model substrate.\n3. From Line187-192, the purpose and significance of this experiment, as well as the conclusion drawn from it, are not very clear. Please clarify.\n4. Table S2 lists the heme oxidation rate constant of P450stri_F88A as “0” at 80 mM H2O2. How about the wild-type P450stri (WT)? The detailed experimental condition should be clarified. Is it in the presence of substrate (testosterone)? In addition, the time-dependent spectroscopic change of heme decay in P450stri_F88A and WT should be provided in the presence/absence substrate.\n5. It is unclear for the product distribution in testosterone hydroxylation catalyzed by P450BM3_F87A and other P450BM3 variants including those chimeras. However, the peroxygenase activities of these enzymes always being compared. Please provide the corresponding experimental evidences.\n6. Please clarify how to calculate the relative peroxygenase activity of P450 variants in Figure 3I.\n7. Semi-rationally engineering the residue sites around substrate pocket/tunnel/channel by replacing limited residue to construct “smart library” has been well utilized in recent investigations on enzyme engineering by many groups (Ref: Chen J et al ACS Catal 2019, 7350-7355; Xu J et al. J. Am. Chem. Soc. 2019, 141, 7934-7945), which has been named as “FRISM (Focused Rational Iterative Site-specific Mutagenesis)” by Reetz M (Method Enzymol. 2020, 643, 225-242). The approach of “Round Flask” named in the current study is similar to that of “FRISM”. Please comment this point in the manuscript and provide a balance view for the readership.\n8. The role of L27F mutation in M4 is not still clear. From the results, it almost did not alter regioselectivity for 15β-hydroxylation (even slightly decreased 1%) but total conversion rate increased compared with M1, suggesting that the catalytic activity enhancement for other sites (2β and 16β) by the introduction of F27 is even higher than that for 15β. While only 15β-hydroxylation was discussed in the main-text and Figure 5. This should be clarified.", "reviewer_2": "The article reports the hydroxylation of testosterone using a new self-sufficient P450 enzyme from a streptomyces ‘P450stri’. The enzyme is a member of the well characterised CYP102 family. The enzyme was engineered to improve the selectivity of testosterone hydroxylation to favor hydroxylation at C15. The mutation of a phenylalanine to an alanine enabled this reaction using just the heme domain and hydrogen peroxide. This is in agreement with what has been reported previously for similar CYP102 enzymes.\nOverall, it is a thorough piece of work using known strategies and methods. Most of the work is well done and presented. The work is well referenced, and these are up to date. It is on the whole well written, but some aspects need clarification before publication. My major comment on the outcomes would be that the authors focus on the high peroxide stability of this enzyme compared to others. This is interesting but doesn’t translate into improved levels of steroid product per enzyme molecule (total turnover numbers appro 175-250 compared to >1000 already reported for other P450 steroid peroxygenase systems). Noteworthy results are the high stability of the enzyme to peroxide and the good selectivity for 15-hydroxysteroid formation. There is a lot of useful data but impact may be limited by the availability of whole cell systems to do effective hydroxylation and cofactor regeneration and the low turnover numbers of the peroxygenase versus other reported systems and low number of steroids used in the study.\nQuestions/Comments\n2. Figure 1 page 6. It is not at all clear what P450s have been included in part a of the figure and what the breakdown is between the different branches. Can different branches be labelled using the standard P450 classification methods (families and subfamilies, see https://p450atlas.org/ and other references on P450 nomenclature). The authors should name P450stri, and the other enzymes studied here. It seems similar to CYP102G family members which have been studied and demonstrated to have a different substrate range to P450Bm3 https://doi.org/10.1016/j.bbapap.2017.08.002 )\nOther comments\nAbstract and page 15 . – The round flask model is not well described until later and it is not clear what it actually is. It should be clearly defined earlier.\nPage 5 line 93. Why is the SSP novel. It is new but it seems to be similar in behavior to P450Bm3 and other CYP102 enzymes. See comments on CYP102G enzymes above.\nPage 7 line 129 “Modify P450s is easy to be malfunctioned by” needs to be changed.\nPage 8 line 153. Structure of P450stri has been ‘predicted’ using AlphaFold3 rather than generated (or a predicted structure of P450Stri has been generated). Is it the structure of the heme domain? Was the docking undertaken with the WT enzyme or the F88A mutant?\nPage 8 line 198 The oxidation of the heme thiolate is not the only mechanism of heme degradation by peroxide. Information on these other routes should be added.\nPage 13 line 246-247 What are the first 50 residues that are not conserved being compared with? Is it P450Bm3 or CYP102 enzymes in general? Do the changes the authors make at the N-terminus alter the levels of protein production (when replacing large segments of the enzyme).\nFigure 5A page 21 does n.d. mean not detected or not determined. If the latter, it should be determined for comparison.\nTable S4 Check the number of significant figures that can be justified (reduce from 4 and 5).\nFigure S23 – what concentrations of peroxide and enzyme were used. Reduce the number of significant figures reported from 4 and 5.\nFigure S29-S31.The conversion levels for these other p450s seem much lower than that of P450stri. Is there a reason why? How similar are these proteins. – A sequence alignment of these enzymes and CYP102A1 would be useful in the SI (heme domain at least).", "reviewer_3": "This study focuses on the engineering of a newly discovered cytochrome P450 enzyme, P450stri from Streptomyces triculaminicus, into a peroxygenase for the selective hydroxylation of steroids. The researchers aimed to overcome limitations of current P450 peroxygenases, such as poor H2O2 tolerance, to create a cost-effective biocatalytic system for generating pharmaceutically valuable steroid compounds.\nThe study identified P450stri as a novel self-sufficient P450 with native steroid hydroxylation activity, capable of converting testosterone into three hydroxylated products (C-15β, C-16β, and C-2β) using NADH. However, similar to other P450s, its application was hindered by reliance on the expensive NAD(P)H cofactor. To address this, P450stri was engineered into a peroxygenase (M1) by mutations(F88A) and M4 by F88A/L27F, enabling it to utilize cost-effective H2O2 as an oxidant.\nThe manuscript showed an enzyme engineering for practical applications in steroid modification. However, plenty of such bioengineering works have already been published (for different P450 members), and therefore, I am not sure whether it is novel enough to be published in Nature Communications. My other concern is that mechanistic insights are based on the Alphafold models, which provide a poor confidence score for the Metalloenzymes. In fact, in Alpha fold reconstructed models, the position of heme-porphyrin is never predicted. Therefore, structural information based on such poor facts is only speculative. Authors must provide experimental structures (Crystal structure) for a mechanistic insight. The MD simulations are not up to mark and seem to be using incorrect models. My other concerns are as follows:\n1) On Page 8, line 157, the authors say that the Alphafold was used to generate the 3D structure of the mutant, and then molecular docking with the substrate was performed to study the selectivity. Furthermore, the authors claim that after the mutation, M1, the distance of the C15 from the heme center decreased from 12 to 7 angs. Such a claim is preliminary and may be inaccurate for two reasons: 1) Alphafold is not recommended for the metalloenzyme, and therefore, the confidence score of an Alphafold-generated structure may be incomplete. 2) Molecular docking is not a tool for conformational study; conformational study must be discussed after the MD simulations. For the second case, I see the authors have performed the MD simulations, but on page 8, claims are made based on docking, and therefore, it is inaccurate.\n2) Figure 3B, the distances are misleading. Figure caption says that distances are from the TST and heme center. Note that heme center ( center of mass of heme) may not be the heme iron and substrate's center of mass may not be the C15. Therefore, such a distance plot is completely misleading.\n3) Figure 3 C and D. How does the enhanced water population play a role in peroxidase activity? It is well known that during peroxidase activity, catalytic cycles don't need 2e and 2 protons as in the mono-oxygenase. In such a case, the role of water becomes less important. Therefore, I am curious to know the mechanism if water populations increase.\n4) It seems that the authors have performed all MD simulations in the resting state. This is an inaccurate approach. In the presence of the H2O2 ( peroxide shunt pathways), H2O2 coordinates with the distal heme iron, forming Fe-H2O2, which ultimately converted to Cpd I. Therefore, MD simulation must be performed in Heme-Fe-H2O2. The parameterization of heme-H2O2 should be done properly, which is not done here.\nSince most of the following results are based on incorrect computational model (that is the resting state without H2O2 coordinated to Heme-iron), I am not sure about their validity."}, "author_response": "Response: We greatly appreciate your positive feedbacks and thank you for your time on evaluating our manuscript.\n1. Many NAD(P)H-dependent P450 enzymes feature a conserved phenylalanine residue near the heme active center. Replacing this residue with smaller side-chain amino acids (e.g., alanine) has been shown to markedly enhance peroxygenase activity—a phenomenon first demonstrated in P450BM3 (see Ref. 26 in this paper). The prevailing hypothesis attributes this effect to reduced steric hindrance and hydrophobicity in the active site, thereby accommodating H O . However, emerging evidence suggests an alternative or complementary mechanism: the mutation may open water-accessible channels that facilitate H O inflow to the heme center (Zhao P. et al. J. Am. Chem. Soc. 2023, 145(9), 5506-5511; Zhao P. et al. Chem. Sci. 2024, 15, 8062-8070; Ref. 48 in this study). Please comments these viewpoints and provides a multidimensional understanding for the role of Phe mutation in the improvement of P450 peroxygenase activity.\nResponse: Thanks for your insightful comment on the mechanistic interpretation of Phe mutation-mediated enhancement of peroxygenase activity. The substitution of phenylalanine (Phe) with smaller side-chain amino acids (e.g., alanine), either in the water-accessing tunnels (Zhao P. et al. J. Am. Chem. Soc. 2023, 145(9), 5506-5511) or in the heme environment (P450BM3_F87A and M1 in this study), could significantly increase the peroxygenase activity. In our opinions, these two routes may act synergistically to boost peroxygenase activity by facilitating H O inflow to the heme center. Based on your valuable suggestions, we have revised the manuscript to provide a potential explanation with the citation of these references (Refs.32 and 33).\nPages 7-8, lines 142-150:\n“Notably, emerging evidence has also indicated that water-accessing tunnels might be involved in the regulation of P450-derived peroxygenase activity.32-33 Specifically, substituting Phe at position 88 with smaller Ala was hypothesized to reduce the steric hindrance and hydrophobicity within heme environment, thereby promoting efficient H O inflow. In our case, the highest product formation by M1 was achieved in the range of 120 to 200 mM H O (Figure 2A), which could be a collective outcome from the decrease of steric hindrance and hydrophobicity combined with larger accessibility of H O .”\n2. In Figure 2A, the comparison of peroxide-dependent peroxygenase activity between P450stri_F88A and P450BM3_F87A is inequitable. This is because P450stri is a native steroid hydroxylase, which P450BM3 is a fatty acid hydroxylase. I will suggest to re-examine the peroxygenase activity of P450BM3_F87A by using one of fatty acid, such as lauric acid as a model substrate.\nResponse: Thanks for your valuable comments. However, we did not perform the experiment to examine the peroxygenase activity of P450 _F87A using a fatty acid BM3 as substrate with following reasons: 1) this study was designed to investigate steroid hydroxylation by engineered peroxygenases from CYP102 enzymes regardless of their original substrate specificity; 2) in Figure 2A, the comparison with P450BM3_F87A was to assess the impact of a homologous Phe mutation on steroid hydroxylation; 3) P450BM3_F87A mutant was previously reported to possess the activity of steroid hydroxylation (Ref.14). Therefore, we feel that the comparison on fatty acid hydroxylation could not provide valuable information to guide the engineering of peroxygenase in this study, which is also not relevant to the present purpose. However, based on your comments, we have revised the manuscript for clarification.\nPage 8, lines 150-153:\n“In addition, compared to natural peroxygenases as well as other engineered peroxygenases,34-35 such as P450 _F87A mutant, particularly on the hydroxylation of steroids, the tolerance of P450stri_F88A against H O was much stronger, reflected by significantly increased product formation.”\n3. From Line187-192, the purpose and significance of this experiment, as well as the conclusion drawn from it, are not very clear. Please clarify.\nResponse: We thank the reviewer for this important reminder and apologize for the confusion. According to your comments, we have revised the manuscript to describe the experimental results more clearly.\nPage 10, lines 185-191:\n“As shown in Figure 2E, M1 produced typical CO-difference spectra in the absence of H O , confirming the activity as a monooxygenase. Then, we examined whether M1 still possess CO-binding activity in the presence of H O . No residual CO-binding activity was observed after H O titration, even with prolonged CO bubbling for several times. These results strongly suggested that the monooxygenase and peroxygenase modes of M1 are incompatible with each other.”\n4. Table S2 lists the heme oxidation rate constant of P450stri_F88A as “0” at 80 mM H O . How about the wild-type P450stri (WT)? The detailed experimental condition should be clarified. Is it in the presence of substrate (testosterone)? In addition, the time-dependent spectroscopic change of heme decay in P450stri_F88A and WT should be provided in the presence/absence substrate.\nResponse: We apologize for this confusion and acknowledge that this section should be clearly presented. We have carefully redesigned the heme-bleaching experiments by lowering the enzyme concentrations for wild-type P450stri and M1, in the presence or absence of TST as described in detailed experimental conditions. The results demonstrated that, either in the presence or absence of substrate, F88A substitution significantly increased its stability towards H O . Detailed experiment procedure and heme bleaching results have been added in the revised manuscript.\nPage 31, lines 608-615:\n“Heme bleaching assay\nThe H O tolerance capacity of P450stri enzymes was determined using UV-vis spectrophotometer (SHIMADZU, UV-1780) at 25°C. The medium scan rate was applied from 300 nm to 600 nm with the interval of 1.0 nm. The baseline spectrum was initially recorded using 1 mL of phosphoric acid buffer (100 mM, 10% glycerol, pH 8.0). Spectra of the mixture containing 4 μM enzyme and 80 mM H O in a total volume of 1 mL phosphoric acid buffer (100 mM, 10% glycerol, pH 8.0) was recorded to monitor the rate of heme degradation over 15 min.”\nPages 10-11, lines 199-206:\n“Thus, heme bleaching experiments were conducted to evaluate the stability of wild type P450stri and M1 towards H O . UV-vis spectra indicated that the soret heme peak of M1 remained stable without noticeable degradation in the presence of TST under H O concentration up to 80 mM, and time-dependent spectroscopic changes for heme decay of M1 were significantly lower than that of wild type P450stri (Figure 3A). These results suggested that, F88A substitution can increase the stability toward H O , possibly by impeding H O accessibility to heme environment.”\nFigure 3A:\nFigure 3. The molecular basis for the high H O tolerance of M1. (A) Effects of H O on heme bleaching of wild type P450stri and M1. Spectral analysis was recorded during the incubation with H O at different time intervals (0-15 min);\nSupplementary Figure S14:\nFigure S14. Comparison of the rates of heme bleaching signal of wild type P450stri and M1 under 80 mM H O . A) The heme bleaching rates of wild type P450stri in the absence of TST; B) The heme bleaching rates of wild type P450stri in the presence of TST; C) The heme bleaching rates of M1 in the absence of TST; D) The heme bleaching rates of wild type P450stri in the presence of TST.\n5. It is unclear for the product distribution in testosterone hydroxylation catalyzed by P450BM3_F87A and other P450BM3 variants including those chimeras. However, the peroxygenase activities of these enzymes always being compared. Please provide the corresponding experimental evidences.\nResponse: Thanks for your valuable comments. According to your comments, we have performed additional experiments to analyze the product distribution in TST hydroxylation catalyzed by P450BM3 variants including the chimeras, revealing that P450stri_F88A mutant (94% 15β-selectivity) also outperformed P450BM3_F87A mutant (80% 15β-selectivity). The corresponding experimental results are presented as Supplementary Table S2 and Supplementary Figure S15 in the revision.\nSupplementary Table S2:\nTable S2. The peroxygenase activity and regioselectivity of P450 variants on the BM3 hydroxylation of testosterone (TST)[a]\nMutants Conversion (%) P1 (%) P2 (%) P3 (%)\nP450 _F87A 6.5 ± 0.5 80 - 20\nBM3\nP450 _F87A_C1 11.3 ± 0.2 80 10 10\nBM3\nP450 _F87A_C2 7.9 ± 0.2 81 8 11\nBM3\nP450 _F87A_C3 2.7 ± 0.1 80 0 20\nBM3\nP450 _F87A/V26L 14.4 ± 1.3 83 5 12\nBM3\nP450 _F87A/F42R n.d. - - -\nBM3\nP450 _F87A/A44L 4.6 ± 0.2 85 - 15\nBM3\nP450 _F87A/R47T 10.6 ± 0.7 80 5 15\nBM3\nP450 _F87A/T49R 3.3 ± 0.5 82 - 18\nBM3\n[a]Reaction conditions: enzyme (4 μM), testosterone (0.5 mM) and 100 mM H O in 100 mM phosphate buffer (pH 8.0, 5% glycerol) with the volume of 200 μl at 35℃ for 8 h; n.d.: not detectable.\nSupplementary Figure S15:\nFigure S15. Representative variants of P450 to mediate the bioconversion of testosterone BM3 (TST) in H O -driven biocatalytic system. P1 represents 15β-OH TST, P2 represents 16β-OH TST, and P3 represents 2β-OH TST. Reaction conditions: enzymes (4 μM), TST (0.5 mM) and 100 mM H O in 100 mM phosphate buffer (pH 8.0, 5% glycerol) with the volume of 200 μl at 35℃ for 8 h.\n6. Please clarify how to calculate the relative peroxygenase activity of P450 variants in Figure 3I.\nResponse: We appreciate your comment. We have revised manuscript to indicate the way of calculation in the figure legend.\nPage 12, lines 223-225:\n“The relative peroxygenase activity was calculated based on the peroxygenase activity of P450 _F87A variant under 40 mM H O .”\nBM3 2 2\n7. Semi-rationally engineering the residue sites around substrate pocket/tunnel/channel by replacing limited residue to construct “smart library” has been well utilized in recent investigations on enzyme engineering by many groups (Ref: Chen J. et al. ACS Catal. 2019, 7350-7355; Xu J. et al. J. Am. Chem. Soc. 2019, 141, 7934-7945), which has been named as “FRISM (Focused Rational Iterative Site-specific Mutagenesis)” by Reetz M (Method Enzymol. 2020, 643, 225-242). The approach of “Round Flask” named in the current study is similar to that of “FRISM”. Please comment this point in the manuscript and provide a balance view for the readership.\nResponse: Thanks for the valuable comments. While we acknowledge the similarity between our “Round Flask” model and FRISM approach, our engineering model features a “Two-step” semi-rational compatibility process for the engineering of regioselective and activity in a “two-dimensional” fashion, achieving a dual-benefit in a concise way. In the revised manuscript, we have elaborated on the key feature and major difference to provide a balanced view for the readership.\nPages 14-15, lines 280-294:\n“In conventional enzyme engineering, the generation of a large number of mutants is a prerequisite to obtain desired enzyme mutants for various purposes, which is laborious and screening-dependent.43-45 Recently, semi-rational engineering of residues around the substrate binding pocket or substrate tunnels,18, 46-47 namely Focused Rational Iterative Site-specific Mutagenesis (FRISM),48 has become an advanced approach to produce a smart library of mutants. However, FRISM usually focuses on a particular region for the increase of the efficiency on engineering efforts. In our case, after generating the M1 variant with strong H O tolerance and high regioselectivity, we had to deal with another important issue of increasing its peroxygenase activity. Based on the substrate entrance tunnels of P450stri_F88A and the rigid cyclic structures of steroids, a model of “Round Flask” was introduced as a way for the semi-rational engineering of this enzyme, in which substrate binding pocket and substrate entrance tunnels were separately yet cooperatively engineered to achieve simultaneous increase of selectivity and activity in a “Two-dimensional” fashion.”\n8. The role of L27F mutation in M4 is not still clear. From the results, it almost did not alter regioselectivity for 15β-hydroxylation (even slightly decreased 1%) but total conversion rate increased compared with M1, suggesting that the catalytic activity enhancement for other sites (2β and 16β) by the introduction of F27 is even higher than that for 15β. While only 15β-hydroxylation was discussed in the main-text and Figure 5. This should be clarified.\nResponse: Thank you for your comment and pointing out the confusion regarding the role of L27F mutation in M4 as well as its enhancement for other hydroxylated products. We apologize for the confusion. To clarify the actual role of this mutant, we have conducted additional MD simulations for F88A/L27F mutant to explore its dynamic changes in structure. Compared to M1, M4 exhibited a different substrate binding mode, which may contribute to its improved catalytic performance. Regarding the improved peroxygenase activity towards different sites (2β and 16β), we reanalyzed the differences on product distribution between M1 and M4. Indeed, the hydroxylation at M4 at C2 and C16 positions was 2.7 times and 2.0 times higher than those by M1, which explains the negative impact on the selectivity by L27F mutant. We have revised the text and Figure 5 in the manuscript accordingly.\nPage 19, lines 368-371:\n“Besides, the hydroxylation activity at M4 for C2 and C16 positions was 2.7 times and 2.0 times those of M1, indicating that L27F mutant also increased the peroxygenase activity to hydroxylate other sites.”\nPages 20-21, lines 388-405:\n“To further elucidate the molecular basis for improved catalytic performance of M4, MD simulations were performed to analyze conformational changes. Compared to the M1-TST complex, a comparable yet slightly more stable distance trajectory (TST-C15---HO -O (H)-Fe-Heme) was observed in M4-TST complex (Figure 5D), which may account for their similar 15β-selectivity. On the other hand, upon substitution of Leu with bulky Phe at position 27, TST molecule adapted different binding modes in M1 and M4 (Figure 5E and 5F), contributing to the variation on their catalytic performance. Additionally, MD simulations also showed that the hydrogen bonding between A ring and key residues is a crucial factor to lock a specific substrate conformation. Moreover, the hydrophobic phenyl ring at position 27 in M4 could facilitate the interaction between A ring and R50 for easier accommodation of hydrophobic TST molecule, thus promoting the formation of proper hydrogen bonds with R50 and A337. Consequently, D ring was directly faced Heme-Fe-H O center in a more vertical manner, potentially benefiting the oxidation at C15 position. To confirm this observation and speculation, M4_R50A mutant was constructed for the hydroxylation of TST. As a result, both peroxygenase activity and 15β-selectivity (88% 15β-selectivity for M4_R50A mutant) were decreased (Figure 5G), thus demonstrating the involvement of R50 in the catalytic performance of M4.”\nPages 21-22, lines 414-429:\nFigure 5. Bioconversion of TST by P450stri mutants and molecular basis of the selected mutants. (A) Product formations of P450stri mutants for TST hydroxylation. n.d. represents “not detected”; Reaction conditions: enzyme (2 μM), testosterone (0.5 mM) and 150 mM H O in 100 mM phosphate buffer (pH 8.0, 5% glycerol) with a volume of 200 μl at 35℃ for 8 h. The 15β-selectivity of each variant is indicated above the corresponding column; (B) Kinetic parameters comparision of M1 and M4 using varied H O concentrations. Reaction conditions: 1 μM enzyme, 0.5 mM testosterone, H O (0-600 mM), reactions were allowed to proceed in the initial 1 min with shaking at 850 rpm at 35℃.(C) Hydroxylation of TST by M4 at different H O concentrations. Reaction conditions: 1 μM enzyme and 1 mM substrate under varied H O concentrations in 100 mM phosphate buffer (pH 8.0, 5% glycerol) with the volume of 200 μl at 35℃ for 8 h; (D) The distance (Å) of TST-C15---HO -O (H)-Fe-Heme for M1 and M4 during MD simulations; (E) Representative snapshot of M1-TST binding mode during MD simulations; (F) Representative snapshot of M4-TST binding mode during MD simulations; (G) Relative peroxygenase activity of P450stri mutants for TST hydroxylation. n.d. represents “not detected”."}, {"round": 2, "reviewer_comments": {"reviewer_1": "After carefully checking the revised manuscript and the author's response, I feel that there are still some questions. After suitable revisions, I believe the manuscript deserve to publish in nature communications.\n1. For the response to Comment 3 of Reviewer 1, I still cannot understand the significance of the experiment to examine the CO binding ability of M1 in the presence of hydrogen peroxide. From the revised manusript, it seems that the author intends to demonstrate that “the monooxygenase and peroxygenase modes of M1 are incompatible with each other.” Firstly, I do not think this conclusion is correct as there are many reports indicating that some P450 enzymes can function as both monooxygenases and peroxidases (please carefully review the literature). Secondly, the experimental itself also has its shortcomings. In the absence of substrate, heme will be bleached by hydrogen peroxide. It is likely that this situation leads to the inability to detect the binding of CO.\n2. Figure S12 and S13 are more likely the different spectra upon substrate titration rather than the original substrate-bound spectra of M1.\n3. The response to comment 4 is still somewhat vague. In fact, the related discussions and figures in the revised manuscript are based on the experimental results of Figure S14. Figure S14 shows a qualitative result, but it is difficult to draw quantitative conclusions from this result. By monitoring the kinetic curve of the maximum absorption of heme at a specific wavelength (usually 418 nm) as it decays over time, the degradation rate constant of heme can be quantitatively calculated. Additionally, a fifteen-minute reaction time is a bit short. Finally, there are some irregularities in the spectral changes of Figure S14D, with almost no change in the first ten minutes and a sudden decrease in the fifteenth minute. This is completely different from the results of Figure S14A-C, indicating that the degradation rate of P450stri in the presence of substrate TST is different. The sudden decrease observed now is likely due to baseline changes in light diffraction caused by poor solubility of TST. Therefore, it is necessary to conduct more precise experiments again.\n4. I am pleased that the author agrees with Reviewer 2's statement in Comment 7. Namely, “the increased stability of P450stri to H2O2 seems to be derived from lack of access of H2O2 to the heme (and lower total number of turnovers) rather than an inherently higher stability of the heme itself.” And the authors revised their description as “These results suggested that, F88A substitution can increase the stability toward H2O2, possibly by impeding H2O2 accessibility to heme environment.” Therefore, I suggest to remove the phrase “with strong H2O2 tolerance” from the title. Because the qualitative description can easily lead readerships to misunderstand that the stability to H2O2 comes from the inherent properties of P450stri and its mutants themselves.", "reviewer_2": "In the revised manuscript the authors have addressed a good deal of the comments and concerns raised by the reviewers.\nMost parts of the manuscript have been improved.\nWhen such a large number of queries are raised and addressed others can arise. I have made comment on those below.\n1) Line 482/483 While the CYP102 family has been less studied it has been studied. The authors need to add references.\nhttps://doi.org/10.1016/j.bbapap.2017.08.002.\nThey should also include the sequence of CYP102G4 in there analysis Figure S25, S26 and S27 (DNA sequence GenBank: LY804624.1)\n2) Figure 3, Figure 5 and Page 30/31. The authors have added MD simulations based on an AlfaFold model. These were done using the Fe(III)HO-OH (peroxide complex). Why did they use this intermediate? In peroxygenases this is converted to Cpd I the active intermediate that will catalyse the first step of the hydroxylation reaction. These MD simulations of generated model structures with this intermediate don’t really add anything significant to this manuscript in terms of activity or binding. There really isn’t enough detail provided to assess fully the quality of this work and the use of this intermediate in the model is not appropriate for determining the position of hydroxylation. Therefore this part of manuscript doesn’t take the complexity of enzyme structure/function, the heme intermediates and motion into full account. There is no real evidence or justification for the large changes observed in the binding mode in these simulations (C16 seems to be closer than C15). This paper does not really need these simulations or benefit from them. I don’t think this addition improved the manuscript.\n3) Line 185-191 M1 has been demonstrated to work as a monooxygenase and a peroxygenase in this paper (Figure S11 and Figure 2). Therefore this is a confusing section. The experiment described here demonstrates is that the titration of peroxide in this experiment has potentially denatured the enzyme.\n4) Line 143-148 The F87/88 residue here is really an active site residue not access channel. There maybe an increase in peroxide flow but the role in F87 in P450Bm3 has been known for a while. As well as reference 26 work extensive work was done by others. E.g. https://doi.org/10.1002/cbic.200300660 and https://doi.org/10.1002/anie.200351434 and these are probably more relevant than those added. These reference should be added.\n5) One reviewer suggested doing turnovers with one fatty acid. Given the nature of CYP102 enzymes I thought this was an excellent suggestion and was disappointed that it wasn't included. A comparison with one fatty acid should rellay be included in the initial parts of manuscript to show the preference of P450 stri for steroids versus fatty acids in context with that of P450Bm3. A reaction and analysis of the products and analysis of the binding of the fatty acid to the WT and F87A mutant would be sufficient. Based on previous work (https://doi.org/10.1016/j.bbapap.2017.08.002) palmitic acid seems to be the best for CYP102G enzymes. I think this is important in determining the preference of P450stri for testosterone or fatty acids (the WT). I note here the CYP102G4 enzyme has been show to catalyse other non-fatty acid substrate and including this reference is also important.\nMinor comments\na) Line 375-376 remove the term cost-effective. This has not been proven and it is easy to prevent further oxidation by adding more substrate or less NAD(P)H.\nb) Figure S12/S13 what is the concentration of the enzyme in micromolar. The magnitude of the absorbance change is also greater. Showing the absolute spectra of the protein before and after addition of substrate (not the difference spectrum) would help here.", "reviewer_3": "I found most of the concerns were addressed by the authors, and I see the revised manuscript is significantly enhanced relative to its earlier version.\nHowever, I still have a concern regarding how water molecules affect the reactivity of the peroxidase. Authors have responded that they have deleted that part, but this is not a solution. Authors need to extend their computations/experiments to dig more for the mechanistic insight. The rest looks good to me. But I would like to see the water effect before I feel it could be accepted for the publication."}, "author_response": "Point to Point Responses to Reviewers:\nReviewer #1 (Remarks to the Author):\nThis study presents a semi-rational engineering approach for enhancing the hydrogen\nperoxide-dependent peroxygenase activity and regioselectivity of a steroid-\nhydroxylating P450 enzyme (P450stri). The authors employed a multi-stage strategy:\n(1) Initial site-directed mutagenesis targeting a conserved phenylalanine residue yielded\na single mutant with baseline peroxygenase activity and improved regioselectivity; (2)\nSequence alignment identified putative H O -tolerance determinants, which were\n2 2\nfunctionally validated through chimeric P450 construction; (3) Substrate channel\nBM3\nengineering via key residue mutagenesis generated double mutants with significantly\nenhanced activity, constituting the novel “Round Flask” strategy. The approach’s\npotential generalizability was demonstrated through successful application to three\nadditional steroid-hydroxylating P450s among twelve homologs mined with P450stri\nas a template.\nWhile this work makes valuable contributions to biocatalysis and protein engineering,\nseveral aspects require clarification prior to publication.\nResponse: We greatly appreciate your positive feedbacks and thank you for your time\non evaluating our manuscript.\n1. Many NAD(P)H-dependent P450 enzymes feature a conserved phenylalanine\nresidue near the heme active center. Replacing this residue with smaller side-chain\namino acids (e.g., alanine) has been shown to markedly enhance peroxygenase\nactivity—a phenomenon first demonstrated in P450BM3 (see Ref. 26 in this paper).\nThe prevailing hypothesis attributes this effect to reduced steric hindrance and\nhydrophobicity in the active site, thereby accommodating H O . However, emerging\n2 2\nevidence suggests an alternative or complementary mechanism: the mutation may open\nwater-accessible channels that facilitate H O inflow to the heme center (Zhao P. et al.\n2 2\nJ. Am. Chem. Soc. 2023, 145(9), 5506-5511; Zhao P. et al. Chem. Sci. 2024, 15, 8062-\n8070; Ref. 48 in this study). Please comments these viewpoints and provides a\nmultidimensional understanding for the role of Phe mutation in the improvement of\nP450 peroxygenase activity.\nResponse: Thanks for your insightful comment on the mechanistic interpretation of\nPhe mutation-mediated enhancement of peroxygenase activity. The substitution of\nphenylalanine (Phe) with smaller side-chain amino acids (e.g., alanine), either in the\nwater-accessing tunnels (Zhao P. et al. J. Am. Chem. Soc. 2023, 145(9), 5506-5511) or\nin the heme environment (P450BM3_F87A and M1 in this study), could significantly\nincrease the peroxygenase activity. In our opinions, these two routes may act\nsynergistically to boost peroxygenase activity by facilitating H O inflow to the heme\n2 2\ncenter. Based on your valuable suggestions, we have revised the manuscript to provide\na potential explanation with the citation of these references (Refs.32 and 33).\nPages 7-8, lines 142-150:\n“Notably, emerging evidence has also indicated that water-accessing tunnels might be\ninvolved in the regulation of P450-derived peroxygenase activity.32-33 Specifically,\nsubstituting Phe at position 88 with smaller Ala was hypothesized to reduce the steric\nhindrance and hydrophobicity within heme environment, thereby promoting efficient\nH O inflow. In our case, the highest product formation by M1 was achieved in the\n2 2\nrange of 120 to 200 mM H O (Figure 2A), which could be a collective outcome from\n2 2\nthe decrease of steric hindrance and hydrophobicity combined with larger accessibility\nof H O .”\n2 2\n2. In Figure 2A, the comparison of peroxide-dependent peroxygenase activity between\nP450stri_F88A and P450BM3_F87A is inequitable. This is because P450stri is a native\nsteroid hydroxylase, which P450BM3 is a fatty acid hydroxylase. I will suggest to re-\nexamine the peroxygenase activity of P450BM3_F87A by using one of fatty acid, such\nas lauric acid as a model substrate.\nResponse: Thanks for your valuable comments. However, we did not perform the\nexperiment to examine the peroxygenase activity of P450 _F87A using a fatty acid\nBM3\nas substrate with following reasons: 1) this study was designed to investigate steroid\nhydroxylation by engineered peroxygenases from CYP102 enzymes regardless of their\noriginal substrate specificity; 2) in Figure 2A, the comparison with P450BM3_F87A\nwas to assess the impact of a homologous Phe mutation on steroid hydroxylation; 3)\nP450BM3_F87A mutant was previously reported to possess the activity of steroid\nhydroxylation (Ref.14). Therefore, we feel that the comparison on fatty acid\nhydroxylation could not provide valuable information to guide the engineering of\nperoxygenase in this study, which is also not relevant to the present purpose. However,\nbased on your comments, we have revised the manuscript for clarification.\nPage 8, lines 150-153:\n“In addition, compared to natural peroxygenases as well as other engineered\nperoxygenases,34-35 such as P450 _F87A mutant, particularly on the hydroxylation\nBM3\nof steroids, the tolerance of P450stri_F88A against H O was much stronger, reflected\n2 2\nby significantly increased product formation.”\n3. From Line187-192, the purpose and significance of this experiment, as well as the\nconclusion drawn from it, are not very clear. Please clarify.\nResponse: We thank the reviewer for this important reminder and apologize for the\nconfusion. According to your comments, we have revised the manuscript to describe\nthe experimental results more clearly.\nPage 10, lines 185-191:\n“As shown in Figure 2E, M1 produced typical CO-difference spectra in the absence of\nH O , confirming the activity as a monooxygenase. Then, we examined whether M1\n2 2\nstill possess CO-binding activity in the presence of H O . No residual CO-binding\n2 2\nactivity was observed after H O titration, even with prolonged CO bubbling for several\n2 2\ntimes. These results strongly suggested that the monooxygenase and peroxygenase\nmodes of M1 are incompatible with each other.”\n4. Table S2 lists the heme oxidation rate constant of P450stri_F88A as “0” at 80 mM\nH O . How about the wild-type P450stri (WT)? The detailed experimental condition\n2 2\nshould be clarified. Is it in the presence of substrate (testosterone)? In addition, the time-\ndependent spectroscopic change of heme decay in P450stri_F88A and WT should be\nprovided in the presence/absence substrate.\nResponse: We apologize for this confusion and acknowledge that this section should\nbe clearly presented. We have carefully redesigned the heme-bleaching experiments by\nlowering the enzyme concentrations for wild-type P450stri and M1, in the presence or\nabsence of TST as described in detailed experimental conditions. The results\ndemonstrated that, either in the presence or absence of substrate, F88A substitution\nsignificantly increased its stability towards H O . Detailed experiment procedure and\n2 2\nheme bleaching results have been added in the revised manuscript.\nPage 31, lines 608-615:\n“Heme bleaching assay\nThe H O tolerance capacity of P450stri enzymes was determined using UV-vis\n2 2\nspectrophotometer (SHIMADZU, UV-1780) at 25°C. The medium scan rate was\napplied from 300 nm to 600 nm with the interval of 1.0 nm. The baseline spectrum was\ninitially recorded using 1 mL of phosphoric acid buffer (100 mM, 10% glycerol, pH\n8.0). Spectra of the mixture containing 4 μM enzyme and 80 mM H O in a total volume\n2 2\nof 1 mL phosphoric acid buffer (100 mM, 10% glycerol, pH 8.0) was recorded to\nmonitor the rate of heme degradation over 15 min.”\nPages 10-11, lines 199-206:\n“Thus, heme bleaching experiments were conducted to evaluate the stability of\nwild type P450stri and M1 towards H O . UV-vis spectra indicated that the soret heme\n2 2\npeak of M1 remained stable without noticeable degradation in the presence of TST\nunder H O concentration up to 80 mM, and time-dependent spectroscopic changes for\n2 2\nheme decay of M1 were significantly lower than that of wild type P450stri (Figure 3A).\nThese results suggested that, F88A substitution can increase the stability toward H O ,\n2 2\npossibly by impeding H O accessibility to heme environment.”\n2 2\nFigure 3A:\nFigure 3. The molecular basis for the high H O tolerance of M1. (A) Effects of H O on\n2 2 2 2\nheme bleaching of wild type P450stri and M1. Spectral analysis was recorded during the\nincubation with H O at different time intervals (0-15 min);\n2 2\nSupplementary Figure S14:\nFigure S14. Comparison of the rates of heme bleaching signal of wild type P450stri and M1\nunder 80 mM H O . A) The heme bleaching rates of wild type P450stri in the absence of TST;\n2 2\nB) The heme bleaching rates of wild type P450stri in the presence of TST; C) The heme\nbleaching rates of M1 in the absence of TST; D) The heme bleaching rates of wild type P450stri\nin the presence of TST.\n5. It is unclear for the product distribution in testosterone hydroxylation catalyzed by\nP450BM3_F87A and other P450BM3 variants including those chimeras. However, the\nperoxygenase activities of these enzymes always being compared. Please provide the\ncorresponding experimental evidences.\nResponse: Thanks for your valuable comments. According to your comments, we have\nperformed additional experiments to analyze the product distribution in TST\nhydroxylation catalyzed by P450BM3 variants including the chimeras, revealing that\nP450stri_F88A mutant (94% 15β-selectivity) also outperformed P450BM3_F87A\nmutant (80% 15β-selectivity). The corresponding experimental results are presented as\nSupplementary Table S2 and Supplementary Figure S15 in the revision.\nSupplementary Table S2:\nTable S2. The peroxygenase activity and regioselectivity of P450 variants on the\nBM3\nhydroxylation of testosterone (TST)[a]\nMutants Conversion (%) P1 (%) P2 (%) P3 (%)\nP450 _F87A 6.5 ± 0.5 80 - 20\nBM3\nP450 _F87A_C1 11.3 ± 0.2 80 10 10\nBM3\nP450 _F87A_C2 7.9 ± 0.2 81 8 11\nBM3\nP450 _F87A_C3 2.7 ± 0.1 80 0 20\nBM3\nP450 _F87A/V26L 14.4 ± 1.3 83 5 12\nBM3\nP450 _F87A/F42R n.d. - - -\nBM3\nP450 _F87A/A44L 4.6 ± 0.2 85 - 15\nBM3\nP450 _F87A/R47T 10.6 ± 0.7 80 5 15\nBM3\nP450 _F87A/T49R 3.3 ± 0.5 82 - 18\nBM3\n[a]Reaction conditions: enzyme (4 μM), testosterone (0.5 mM) and 100 mM H O in 100 mM\n2 2\nphosphate buffer (pH 8.0, 5% glycerol) with the volume of 200 μl at 35℃ for 8 h; n.d.: not\ndetectable.\nSupplementary Figure S15:\nFigure S15. Representative variants of P450 to mediate the bioconversion of testosterone\nBM3\n(TST) in H O -driven biocatalytic system. P1 represents 15β-OH TST, P2 represents 16β-OH\n2 2\nTST, and P3 represents 2β-OH TST. Reaction conditions: enzymes (4 μM), TST (0.5 mM)\nand 100 mM H O in 100 mM phosphate buffer (pH 8.0, 5% glycerol) with the volume of 200\n2 2\nμl at 35℃ for 8 h.\n6. Please clarify how to calculate the relative peroxygenase activity of P450 variants in\nFigure 3I.\nResponse: We appreciate your comment. We have revised manuscript to indicate the\nway of calculation in the figure legend.\nPage 12, lines 223-225:\n“The relative peroxygenase activity was calculated based on the peroxygenase activity\nof P450 _F87A variant under 40 mM H O .”\nBM3 2 2\n7. Semi-rationally engineering the residue sites around substrate pocket/tunnel/channel\nby replacing limited residue to construct “smart library” has been well utilized in recent\ninvestigations on enzyme engineering by many groups (Ref: Chen J. et al. ACS Catal. 2019, 7350-7355; Xu J. et al. J. Am. Chem. Soc. 2019, 141, 7934-7945), which has been\nnamed as “FRISM (Focused Rational Iterative Site-specific Mutagenesis)” by Reetz M\n(Method Enzymol. 2020, 643, 225-242). The approach of “Round Flask” named in the\ncurrent study is similar to that of “FRISM”. Please comment this point in the manuscript\nand provide a balance view for the readership.\nResponse: Thanks for the valuable comments. While we acknowledge the similarity\nbetween our “Round Flask” model and FRISM approach, our engineering model\nfeatures a “Two-step” semi-rational compatibility process for the engineering of\nregioselective and activity in a “two-dimensional” fashion, achieving a dual-benefit in\na concise way. In the revised manuscript, we have elaborated on the key feature and\nmajor difference to provide a balanced view for the readership.\nPages 14-15, lines 280-294:\n“In conventional enzyme engineering, the generation of a large number of mutants is a\nprerequisite to obtain desired enzyme mutants for various purposes, which is laborious\nand screening-dependent.43-45 Recently, semi-rational engineering of residues around\nthe substrate binding pocket or substrate tunnels,18, 46-47 namely Focused Rational\nIterative Site-specific Mutagenesis (FRISM),48 has become an advanced approach to\nproduce a smart library of mutants. However, FRISM usually focuses on a particular\nregion for the increase of the efficiency on engineering efforts. In our case, after\ngenerating the M1 variant with strong H O tolerance and high regioselectivity, we had\n2 2\nto deal with another important issue of increasing its peroxygenase activity. Based on\nthe substrate entrance tunnels of P450stri_F88A and the rigid cyclic structures of\nsteroids, a model of “Round Flask” was introduced as a way for the semi-rational\nengineering of this enzyme, in which substrate binding pocket and substrate entrance\ntunnels were separately yet cooperatively engineered to achieve simultaneous increase\nof selectivity and activity in a “Two-dimensional” fashion.”\n8. The role of L27F mutation in M4 is not still clear. From the results, it almost did not\nalter regioselectivity for 15β-hydroxylation (even slightly decreased 1%) but total\nconversion rate increased compared with M1, suggesting that the catalytic activity\nenhancement for other sites (2β and 16β) by the introduction of F27 is even higher than\nthat for 15β. While only 15β-hydroxylation was discussed in the main-text and Figure\n5. This should be clarified.\nResponse: Thank you for your comment and pointing out the confusion regarding the\nrole of L27F mutation in M4 as well as its enhancement for other hydroxylated products.\nWe apologize for the confusion. To clarify the actual role of this mutant, we have\nconducted additional MD simulations for F88A/L27F mutant to explore its dynamic\nchanges in structure. Compared to M1, M4 exhibited a different substrate binding mode,\nwhich may contribute to its improved catalytic performance. Regarding the improved\nperoxygenase activity towards different sites (2β and 16β), we reanalyzed the\ndifferences on product distribution between M1 and M4. Indeed, the hydroxylation at\nM4 at C2 and C16 positions was 2.7 times and 2.0 times higher than those by M1,\nwhich explains the negative impact on the selectivity by L27F mutant. We have revised\nthe text and Figure 5 in the manuscript accordingly.\nPage 19, lines 368-371:\n“Besides, the hydroxylation activity at M4 for C2 and C16 positions was 2.7 times and\n2.0 times those of M1, indicating that L27F mutant also increased the peroxygenase\nactivity to hydroxylate other sites.”\nPages 20-21, lines 388-405:\n“To further elucidate the molecular basis for improved catalytic performance of M4,\nMD simulations were performed to analyze conformational changes. Compared to the\nM1-TST complex, a comparable yet slightly more stable distance trajectory (TST-C15-\n--HO -O (H)-Fe-Heme) was observed in M4-TST complex (Figure 5D), which may\n2 1\naccount for their similar 15β-selectivity. On the other hand, upon substitution of Leu\nwith bulky Phe at position 27, TST molecule adapted different binding modes in M1\nand M4 (Figure 5E and 5F), contributing to the variation on their catalytic\nperformance. Additionally, MD simulations also showed that the hydrogen bonding\nbetween A ring and key residues is a crucial factor to lock a specific substrate\nconformation. Moreover, the hydrophobic phenyl ring at position 27 in M4 could\nfacilitate the interaction between A ring and R50 for easier accommodation of\nhydrophobic TST molecule, thus promoting the formation of proper hydrogen bonds\nwith R50 and A337. Consequently, D ring was directly faced Heme-Fe-H O center in\n2 2\na more vertical manner, potentially benefiting the oxidation at C15 position. To\nconfirm this observation and speculation, M4_R50A mutant was constructed for the\nhydroxylation of TST. As a result, both peroxygenase activity and 15β-selectivity\n(88% 15β-selectivity for M4_R50A mutant) were decreased (Figure 5G), thus\ndemonstrating the involvement of R50 in the catalytic performance of M4.”\nPages 21-22, lines 414-429:\nFigure 5. Bioconversion of TST by P450stri mutants and molecular basis of the selected\nmutants. (A) Product formations of P450stri mutants for TST hydroxylation. n.d. represents\n“not detected”; Reaction conditions: enzyme (2 μM), testosterone (0.5 mM) and 150 mM\nH O in 100 mM phosphate buffer (pH 8.0, 5% glycerol) with a volume of 200 μl at 35℃ for\n2 2\n8 h. The 15β-selectivity of each variant is indicated above the corresponding column; (B)\nKinetic parameters comparision of M1 and M4 using varied H O concentrations. Reaction\n2 2\nconditions: 1 μM enzyme, 0.5 mM testosterone, H O (0-600 mM), reactions were allowed to\n2 2\nproceed in the initial 1 min with shaking at 850 rpm at 35℃.(C) Hydroxylation of TST by M4\nat different H O concentrations. Reaction conditions: 1 μM enzyme and 1 mM substrate\n2 2\nunder varied H O concentrations in 100 mM phosphate buffer (pH 8.0, 5% glycerol) with the\n2 2\nvolume of 200 μl at 35℃ for 8 h; (D) The distance (Å) of TST-C15---HO -O (H)-Fe-Heme\n2 1\nfor M1 and M4 during MD simulations; (E) Representative snapshot of M1-TST binding\nmode during MD simulations; (F) Representative snapshot of M4-TST binding mode during\nMD simulations; (G) Relative peroxygenase activity of P450stri mutants for TST\nhydroxylation. n.d. represents “not detected”."}, {"round": 3, "reviewer_comments": {"reviewer_1": "All of my concerns have been well resolved in the revised version. This reviewer suggests to accept the manuscript to publish as it is.", "reviewer_2": "The revised article addresses the majority of the points and is much improved.\nSome relatively minor things that need fixing before acceptance/publication.\n1) Page 25. Do the authors state anywhere in the manuscript what the isolated amount of metabolite was (in mg). They have an isolated yield, but I couldn’t find the actual amount in mg.\n2) Page 35-36 – How did the authors get the sample from reverse phase HPLC into the correct solvent (chloroform) for NMR. This sections describes the method for large scale isolation of the metabolites using the mutant and hydrogen peroxide. On page 6 the authors use the WT enzyme and an NADPH regenerating system to characterize the three metabolites. This method needs to be described as well.\n3) Supporting information page 3. The NMR data – the authors should add references to previous NMR characterization. They use a mix of o, m (multiplet) and then complete assignments. Can they define what o is here? If peaks are overlapping how where the assigned?\nOther Minor fixes\n1) Line 52 date not data.\n2) line 100 as a soluble protein with good yield (remove water)\n3) line 107-108 the oxidation at the omega-2 position is the most abundant not necessarily easier to be hydroxylated.\n4) Page 7 figure legend. It is worth highlighting these were derivatized and linking to the methods and analysis in the SI. The authors should also provide references for the MS fragmentation analysis and how they were assigned.\n5) Page 10. Does the data (product distribution) agree with the F87A variant of P450Bm3. Please comment and add references.\n6) Page 13 lines 236-238 should be rewritten to correct grammar.", "reviewer_3": "This is the 2nd revision, and in the first revision, I had only one concern about the role of water. This seems addressed as the authors have performed a new set of MD simulations. I don't have any further comments, and I believe the work is in good shape to be accepted for publication."}, "author_response": "Point to Point Responses to Reviewers:\nReviewer #1 (Remarks to the Author):\nAll of my concerns have been well resolved in the revised version. This reviewer\nsuggests to accept the manuscript to publish as it is.\nResponse: We appreciate your time and positive evaluation on our work.\nReviewer #2 (Remarks to the Author):\nThe revised article addresses the majority of the points and is much improved. Some\nrelatively minor things that need fixing before acceptance/publication.\nResponse: We appreciate your positive assessment and valuable suggestions.\n1. Page 25. Do the authors state anywhere in the manuscript what the isolated amount\nof metabolite was (in mg). They have an isolated yield, but I couldn’t find the actual\namount in mg.\nResponse: According to your comments, the information has been added on Pages 18-\n19, lines 399 and 402.\n2. Pages 35-36: How did the authors get the sample from reverse phase HPLC into the\ncorrect solvent (chloroform) for NMR. This section describes the method for large scale\nisolation of the metabolites using the mutant and hydrogen peroxide. On page 6 the\nauthors use the WT enzyme and an NADPH regenerating system to characterize the\nthree metabolites. This method needs to be described as well.\nResponse: Thanks for your comments. We have incorporated the experiment details on\nPage 27, lines 586-596 and Page 27, lines 601-604.\n3. Supporting information Page 3. The NMR data-the authors should add references to\nprevious NMR characterization. They use a mix of o, m (multiple) and then complete\nassignments. Can they define what o is here? If peaks are overlapping how where the\nassigned?\nResponse: We appreciate your comments. References on NMR characterization have\nbeen added in the general method of SI (Page 2). We apologize for the misuse of “o,\noverlapped” in NMR data interpretation. We have rechecked all proton peak\nassignments via 2D-HSQC and revised to use standard notation (“m” for multiplet)\nin SI (Pages 3-4).\nOther Minor fixes\n1. Line 52 date not data.\nResponse: We have changed in text.\n2. Line 100 as a soluble protein with good yield (remove water)\nResponse: We have removed in text.\n3. Line 107-108 the oxidation at the omega-2 position is the most abundant not\nnecessarily easier to be hydroxylated.\nResponse: We have changed in text.\n4. Page 7 figure legend. It is worth highlighting these were derivatized and linking to\nthe methods and analysis in the SI. The authors should also provide references for the\nMS fragmentation analysis and how they were assigned.\nResponse: Thank you for your comments. Detailed descriptions and references have\nbeen incorporated in Figure legends of the text (Page 37, line 857), and general\nmethods in SI (Page 2).\n5. Page 10. Does the data (product distribution) agree with the F87A variant of\nP450bm3. Please comment and add references.\nResponse: References have been added and discussed in the revision. Page 8, lines\n170-172.\n6. Page 13, lines 236-238 should be rewritten to correct grammar.\nResponse: We have revised in text.\nReviewer #3 (Remarks to the Author):\nThis is the 2nd revision, and in the first revision, I had only one concern about the role\nof water. This seems addressed as the authors have performed a new set of MD\nsimulations. I don’t have any further comments, and I believe the work is in good shape\nto be accepted for publication.\nResponse: We appreciate your time and positive evaluation on our work."}]

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