# An Alkaliphilic Chitinase Unveils Environment-Dependent Variation in the Canonical Catalytic Machinery of Family-18 Glycoside Hydrolases

Jogi Madhuprakash, Bjørn Dalhus, Bastien Bissaro, Lal Duhsaki, Gustav Vaaje-Kolstad, Morten Sørlie, Åsmund K Røhr, Vincent G H Eijsink

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To cite this version:

Jogi Madhuprakash, Bjørn Dalhus, Bastien Bissaro, Lal Duhsaki, Gustav Vaaje-Kolstad, et al. An Alkaliphilic Chitinase Unveils Environment-Dependent Variation in the Canonical Catalytic Machinery of Family-18 Glycoside Hydrolases. Biochemistry, 2025, 64 (10), pp.2291-2305. 10.1021/acs.biochem.5c00082 . hal-05202769

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# An Alkaliphilic Chitinase Unveils Environment-Dependent Variation in the Canonical Catalytic Machinery of Family-18 Glycoside Hydrolases

Jogi Madhuprakash,* Bjørn Dalhus, Bastien Bissaro, Lal Duhsaki, Gustav Vaaje-Kolstad, Morten Sørlie, Åsmund K. Røhr, and Vincent G. H. Eijsink*

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Cite This: *Biochemistry* 2025, 64, 2291–2305

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Supporting Information

**ABSTRACT:** Chitinases belonging to glycoside hydrolase family-18 (GH18) employ substrate-assisted catalysis and typically have neutral/acidic pH-optima. We describe the structural and functional analysis of *CaChiA*, a chitinase from the anaerobic alkaliphilic bacterium *Chitinivibrio alkaliphilus* with an alkaline pH optimum (8.8) and unique active site features, including a noncanonical catalytic HxxDxE motif, which is DxDxDxE in other chitinases. Propka calculations indicated a significantly higher pKₐ for the catalytic acid/base, Glu148, in *CaGH18*, compared to other GH18 enzymes, aligning with its alkaline pH optimum. Both Propka calculations and functional studies of enzyme variants with mutations in the catalytic center suggested that not the change in the catalytic motif, but rather a unique glutamine, Gln57, modulating the properties of this motif, enables activity at alkaline pH. Further characterization of *CaChiA* unveiled additional peculiar enzyme properties, such as a unique ability to convert chitin to chitotriose. Thus, *CaChiA* adds novel catalytic capabilities to the widespread family of GH18 chitinases, made possible by adaptation of an intricate catalytic center.

<figure><img src="image_8.png" /><figcaption>CaGH18</figcaption></figure>

## INTRODUCTION

Chitin, a homopolymer of N-acetyl-D-glucosamine (GlcNAc) sugars linked through β-1,4 glycosidic bonds, is an abundant biopolymer found in the exoskeletons of insects and crustaceans and in fungal cell walls. Chitin is a source of nutrition for a plethora of microbes possessing enzymatic machineries for chitin degradation that are composed of exo- and endo-acting chitinases and lytic polysaccharide mono-oxygenases (LPMOs).¹ These enzymes are of industrial interest since they may be used for bioprocessing of chitin-rich biomass to produce, for example, chito-oligosaccharides with various potential applications, especially in the food, medical, and agriculture fields.²⁻⁴ Chitinases occur primarily in two glycoside hydrolase (GH) families of the CAZy database,⁵ namely families GH18 and GH19. GH18 chitinases are abundant in bacteria and fungi and are quite unique among glycoside hydrolases because they employ a ‘substrate-assisted’ catalytic mechanism, with the acetamido (N-acetyl) group of the sugar bound in the -1 subsite acting as the catalytic nucleophile⁶⁻⁹ (Figure 1).

The catalytic domains of family GH18 chitinases have a canonical (β/α)₈ TIM-barrel fold¹⁰⁻¹² and harbor the conserved sequence motifs DxDxDxE and SxGG that are diagnostic for the family. In the DxDxDxE motif, the first D is less conserved and its role in catalysis is not clear. The DxDxE

part is essential for catalysis, harboring a glutamate that acts both as an acid and a base during the catalytic cycle. The two conserved aspartates are involved in sequential protonation and deprotonation events that enable the necessary “cycling” of the pKₐ of the catalytic acid/base (Figure 1).⁸,⁹,¹³ The middle Asp rotates during catalysis, contributing to distortion and increased nucleophilicity of the catalytically crucial acetamido group of GlcNAc bound in the -1 subsite (Figure 1). Other conserved residues also contribute to catalysis, including a buried tyrosine and a buried serine (part of the SxGGW motif) which stabilize the rather buried charge on the first Asp in the DxDxE motif. Additionally, a conserved tyrosine stabilizes the distorted acetamido group, and another residue, either Asn or Asp, interacts with the -1 sugar and modulates the pKₐ of the catalytic acid (Figure 1).⁸,⁹ Clearly, catalysis in GH18 chitinases involves multiple residues, many of which are titratable.

Received: February 10, 2025

Revised: April 26, 2025

Accepted: April 28, 2025

Published: May 2, 2025

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<figure><img src="image_10.png" /><figcaption>**Figure 1.** Essential features of the catalytic mechanism of GH18 chitinases, based on the model described by Synstad et al. (2004)⁹ for *SmChiB*. During the catalytic cycle, buried Asp140 has varying and catalytically relevant interactions with other residues in the enzyme core.⁹ The structural contexts of Asp140 in *SmChiB* and of corresponding Glu144 in *CaChiA* are shown in Figure 3F,E, respectively. (A) Asp 142 (Asp146 in *CaChiA*) rotates away from Asp140 and forms hydrogen bonds with the substrate and the catalytic acid Glu144 (Glu148 in *CaChiA*) upon (B) substrate binding. Asp is stabilized by Tyr10 (Trp25 in *CaChiA*) and Ser93 (Cys102 in *CaChiA*), while Asp215 (Glu214 in *CaChiA*) and Tyr214 (Tyr213 in *CaChiA*), stabilize the oxazolinium ion intermediate by hydrogen bonds. (C) The catalytic base activates a water molecule that completes the hydrolysis followed by a rotation of Asp142 back to Asp140. Gln57 in *CaGH18* interacts with Asp146 (Asp142 in *SmChiB*) and may also play a role in intermediate stabilization (see text and Figures 3–5, below, for details). Gln57 corresponds to Ser50 in *SmChiB* (see Figure 3, below), which is not thought to play a role in catalysis. This figure is adapted from ref 8; copyright (2001) National Academy of Sciences.</figcaption></figure>

The structure, substrate binding and mechanism of GH18 chitinases have been studied quite extensively, focusing on enzymes that either have a close to neutral (pH 6.0–7.0) or slightly acidic pH optimum for activity.<sup>14–17</sup> It has been shown that the pH optimum of GH18 chitinases is sensitive to mutations in its complex catalytic center; in particular, it has been shown that enzymes containing an Asp in position 215 (Figure 1; residue numbering according to ChiB from *Serratia marcescens* (*SmChiB*)) have a neutral pH optimum, whereas enzymes carrying an Asn in this position, such as ChiC from *S. marcescens* (*SmChiC*),<sup>18</sup> have an acidic pH optimum.⁹ Most reported chitinases originate from aerobic chitinolytic bacteria, although enzymes from anaerobic Clostridia have also been described.<sup>19,20</sup> Little is known about microbial chitin degradation under anaerobic alkaliphilic conditions, while it would be of considerable interest to see how the well-studied catalytic center of GH18 enzymes could be adapted to such conditions.

In 2014, Sorokin et al.,²⁰ reported the first culturable members of the candidate phylum Termite Group 3 (TG3), which is closely related to the phylum Fibrobacterota. Of these, the bacteria *Chitinivibrio alkaliphilus* and *Chitinispirillum alkaliphilum*, within the novel classes *Chitinivibrionia* and *Chitinispirillia*, respectively, showed notable chitinolytic activity.²⁰,²¹ C. alkaliphilus, an obligate anaerobe, mediates chitin degradation by attaching firmly to chitin particles and by converting the polymer to chitooligomers that can be assimilated by the bacterium.²⁰ Chitin degradation by C. alkaliphilus involves two secreted chitinases, one GH18 (locus ID: CALK_07749) and one GH19 (locus ID: CALK_0031). The GH18 contains all typical features of a protein that is secreted by the type IX secretion system, which means that it is located extracellularly and possibly associated with the outer membrane.²⁰ This enzyme likely is a major contributor to the

chitinolytic power of C. *alkaliphilus*, which is anaerobic and seems to be lacking (oxygen-dependent) LPMOs.

To gain further insight into how the complex catalytic machinery of GH18 chitinases (Figure 1) may have adapted to extreme pHs, and to gain insight into novel chitinases with atypical properties, we have studied the GH18 chitinase from C. alkaliphilus, *CaChiA*. We have solved the crystal structure of the catalytic domain, *CaGH18*, in the apo-form and in complex with chitohexaose, and we have used the structures to identify features that may promote activity at high pH. The importance of these features was studied by site-directed mutagenesis of selected residues in the catalytic center and Propka analysis was used to assess the pKₐ values of catalytic residues. These analyses allowed us to putatively assign the high pH-optimum to specific structural properties. Catalytic properties at alkaline pH were assessed in reactions with chito-oligomers, as well as α- and β-chitin, for both the catalytic domain only and the full-length enzyme, which contains additional domains (see below), but lacks an apparent chitin-binding domain.

## ■ RESULTS AND DISCUSSION

**Domain Architecture.** The full-length enzyme (*CaChiA*) consists of 651 amino acids and sequence analysis using Pfam and InterPro confirmed the presence of a signal peptide (residues 1–21), followed by an N-terminal GH18 domain (residues 22–295), two Flg_new_2 domains in tandem (D1: 301–373; D2: 380–452) and a C-terminal sorting domain (D3: 581–650) (Figure 2A). Flg_new_2 domains are commonly found in bacterial cell surface proteins and their function remains unknown. Inspection of these domains in an AlphaFold model of *CaChiA* (https://www.uniprot.org/uniprotkb/U7D706/entry) (Figure 2B) did not reveal features, such as exposed conserved aromatic amino acids on a relatively flat surface, that are typically seen in chitin-binding domains.
<figure><img src="image_11.png" /><figcaption>**Figure 2.** Domain architecture and phylogenetic analysis of CaChiA: (A) schematic representation of the domain organization of CaChiA from C. *alkaliphilus*. Abbreviations: SP, Signal Peptide; GH18, Glycoside Hydrolase family-18; D1 & D2, Flg_new 2 domains; D3, C-terminal sorting domain. The starting and ending amino acid positions are shown for each individual domain. (B) AlphaFold model of CaChiA showing the individual domains (in blue) and the disordered region (in red) between the domains D2 and D3. (C) A total of 214 sequences of GH18 catalytic domains found in bacterial, fungal, insect and plant genomes (branches and leaves colored according to origin; color provided in the figure) were used to prepare a MSA and phylogenetic tree (500 bootstraps) using the NGPhylogeny online platform. Clades have been defined on the basis of phylogenetic apparent divergence and conservation of the “DxxDxDxE” motif (displayed as a WebLogo) within the clade. The black and red arrows above the sequence motifs indicate significant occurrences of alternatives to the canonical “DxxDxDxE” motif; black arrows point at the first D, which generally is less conserved and whose role in catalysis is not very clear; red arrows point at Ds and/or Es in the DxDxE part that have well-described roles in catalysis.</figcaption></figure>

The C-terminal domain of the enzyme is identified as “secretion system C-terminal sorting domain” or “T9SS type A sorting domain” by the InterPro protein classification tool, which was also previously noted by Sorokin et al., (2014).²⁰ Such domains are known to mediate secretion by the T9SS, which can result in either release of the protein to the external environment or attachment to the outer membrane.²²

**The Canonical “DxxDxDxE” Catalytic Motif is Not Conserved in CaChiA.** Instead of the canonical catalytic “DxxDxDxE” motif, CaChiA carries HxxExxDxE. To investigate the occurrence of alternative catalytic centers within the GH18 family more broadly, we carried out a phylogenetic analysis of the catalytic domains of 214 GH18s from bacterial, fungal, plant and insect origin and made a detailed analysis of the conservation of the “DxxDxDxE” motif throughout the entire tree (Figure 2C). We defined 8 clades of phylogenetically close sequences within which the “DxxDxDxE”-like motif is relatively conserved (see WebLogo analysis, Figure 2). Strikingly, this analysis shows that several clades (1, 2, 4-ex, 6 and 7) contain or are composed exclusively of enzymes that do not harbor the canonical “DxxDxDxE” motif. The sequences in these clades come from plants, bacteria, and insects, but not

from fungi. Zooming in on the catalytically indispensable DxE part with the catalytic acid, it would seem that clade 1 and, to a lesser extent, clade 6 contain GH18 members that are not catalytically active. Clade 4 is a taxonomically diverse clade (from fungi, plants, and bacteria) containing GH18 enzymes carrying the canonical DxxDxDxE motif. Clade 4_ex contains members that would belong to clade 4 on the sole basis of phylogenetic clustering but that are exceptions because they do not possess the canonical “DxxDxDxE” motif. Clade 4_ex contains sequences from haloalkaliphilic anaerobic chitinolytic bacteria, namely *Chitinivibrio alkaliphilus* CaChiA (GeneBank ID: ERP38755.1) and *Chitinispirillum alkaliphilum* CalGH18_B (GeneBank ID KMQ52498.1), with “HxxExxDxE” and “DxxExxDxE” motifs, respectively.

A closer look at the occurrence of the DxxDxDxE-like motif in each clade showed that the D → H substitution is only found in CaChiA and in some GH18s of plant origin (clade 6). Furthermore, the “HxxExxDxE” motif found in CaGH18 is really unique, since it is not found in any of the other GH18s analyzed here. Interestingly, other alternatives to the otherwise conserved “DxxDxDxE” motif occur in clade 7 (“X1xxX2xDxE”, X1 = A, D, K, Q, R, S, T and X2 = C, D,
<table><caption>Table 1. Crystal Data, Data-Collection, and Refinement Statistics</caption><thead><tr><th>data collection</th><th>CaGH18</th><th>CaGH18-E148Q + (GlcNAc)<sub>6</sub></th></tr></thead><tbody><tr><td>beamline</td><td>ID30A-1 (ESRF, Grenoble)</td><td>ID30A-1 (ESRF, GRenoble)</td></tr><tr><td>wavelength (Å)</td><td>0.966</td><td>0.966</td></tr><tr><td>temperature (K)</td><td>100</td><td>100</td></tr><tr><td>space group</td><td>P2<sub>1</sub>2<sub>1</sub>2<sub>1</sub></td><td>P2<sub>1</sub></td></tr><tr><td>unit-cell parameters (Å, °)</td><td>a = 55.06, b = 62.34, c = 75.17<br/>α = β = γ = 90</td><td>a = 65.97, b = 62.42, c = 102.65<br/>α = γ = 90; β = 102.75</td></tr><tr><td>resolution (Å)</td><td>48.0–1.10 (1.11–1.10)</td><td>100.1–1.31 (1.33–1.31)</td></tr><tr><td>unique reflections</td><td>102,783 (1013)<sup>a</sup></td><td>192,384 (9230)</td></tr><tr><td>completeness (%)</td><td>99.6 (94.4)</td><td>98.6 (93.9)</td></tr><tr><td>multiplicity</td><td>5.0 (4.9)</td><td>3.1 (3.0)</td></tr><tr><td>mean I/σI</td><td>12.0 (2.8)</td><td>10.8 (2.1)</td></tr><tr><td>R<sub>meas</sub> (all I<sup>+</sup> and I<sup>-</sup>)<sup>b</sup></td><td>0.093 (0.607)</td><td>0.069 (0.653)</td></tr><tr><td>refinement statistics</td><td></td><td></td></tr><tr><td>resolution of data used in refinement</td><td>48.0–1.10</td><td>100.1–1.31</td></tr><tr><td>completeness for range (%)</td><td>99.6</td><td>98.6</td></tr><tr><td>R<sub>cryst</sub>/R<sub>free</sub> (%)<sup>c</sup></td><td>17.7/19.2</td><td>14.8/16.6</td></tr><tr><td>R.m.s.d. bonds (Å)</td><td>0.007</td><td>0.008</td></tr><tr><td>R.m.s.d. angles (°)</td><td>0.94</td><td>1.09</td></tr><tr><td>average B-factor (protein/ligand/solvent) (Å<sup>2</sup>)</td><td>10.4/16.0/23.9</td><td>15.4/18.2/33.9</td></tr><tr><td>number of non-H atoms</td><td></td><td></td></tr><tr><td>macromolecules</td><td>2293</td><td>4410</td></tr><tr><td>solvent water</td><td>341</td><td>848</td></tr><tr><td>ethylene glycol</td><td>8</td><td>24</td></tr><tr><td>NAG ligand</td><td></td><td>170</td></tr><tr><td>Na<sup>+</sup> ions</td><td></td><td>2</td></tr><tr><td>Ramachandran plot (%)<sup>d</sup></td><td></td><td></td></tr><tr><td>favorable region</td><td>97.9</td><td>98.0</td></tr><tr><td>additionally allowed</td><td>2.1</td><td>2.0</td></tr><tr><td>outliers</td><td>0.0</td><td>0.0</td></tr><tr><td>PDB code</td><td>7Z65</td><td>7Z64</td></tr></tbody></table>

<sup>a</sup>Values in parentheses are for the highest resolution shells. <sup>b</sup>R<sub>meas</sub> defined by Diederichs & Karplus. <sup>c</sup>R<sub>cryst</sub> = Σhkl ||F<sub>0</sub>|| - |F<sub>c</sub>||/Σhkl |F<sub>0</sub>|| where F<sub>0</sub> and F<sub>c</sub> are the observed and calculated structure factor amplitudes, respectively. R<sub>free</sub> is calculated from randomly chosen 4.88% and 5.02% sets of unique reflections not used in refinement for the native and E148Q mutant data sets, respectively. <sup>d</sup>Defined using MolProbity.<sup>28</sup>

H, N). These peculiar proteins would warrant further investigations.

**CaChiA is Optimally Active at Alkaline pH.** The optimal pH and temperature for activity of the full-length CaChiA and its catalytic domain, CaGH18, were determined using (GlcNAc)<sub>6</sub> as the substrate in short reactions with maximally 10% substrate depletion. CaChiA showed optimal activity in a broad pH range, from pH 6.5 to 10.0, while the pH-activity for CaGH18 was similar albeit with a slightly more pronounced true optimum around pH = 9.0 (Figure S1A; see below for another pH-activity profile for CaGH18). These pH profiles, with high activity at pH 9–10, differ profoundly from the pH profiles typically observed for GH18 chitinases.<sup>9,18</sup> Temperature-activity profiles, derived from reactions run for 5 min, showed maximal activity at 50 °C for both CaChiA and CaGH18 (Figure S1B).

**Overall Structure of CaGH18.** Despite multiple crystallization trials, we could not obtain well diffracting crystals of full length CaChiA. This could be due to the multimodularity of CaChiA and the presence of a disordered long interconnecting region between the second Flg_new_2 domain and the C-terminal sorting domain as predicted by AlphaFold (Figure 2B). Therefore, further crystallization trials were done with a truncated enzyme containing the catalytic domain only, CaGH18. The structure of CaGH18 was solved by molecular replacement and refined to 1.1 Å resolution

(Table 1). The structure of CaGH18 comprises 283 residues, including the complete predicted 274 residue GH18 domain, an added N-terminal methionine, and 8 residues following the GH18 domain (the sequence is shown in Figure S2). The structure of CaGH18 shows the (β/α)<sub>8</sub> TIM barrel fold that is characteristic for GH18 proteins (Figure 3A). CaCH18 lacks the so-called (α + β) insertion domain that occurs in many GH18 chitinases, such as in SmChiA, SmChiB and SpChiD<sup>8,12,23,24</sup> where it contributes to shaping a deep substrate-binding cleft, or even tunnel, which is associated with exo-activity. Consequently, CaGH18 has a shallow substrate-binding cleft, similar to endo-acting chitinases such as SmChiC<sup>25</sup> but different from exo-acting, processive chitinases such as SmChiB (Figure 3B–D). Comparison of surface charges (Figure S3) showed that the surface of CaGH18 is more negatively charged compared to other GH18 chitinases. Variation in pH optima is commonly found to be associated with variation in surface charges, but the direction of the changes may vary between protein families.<sup>26</sup>

A structure-based sequence alignment of CaGH18 with the catalytic domains of other well-characterized GH18 chitinases confirmed the presence and canonical positioning of important catalytic residues but also revealed remarkable features that are unique to CaGH18 (Figure S2). Next to the deviating HxxExDxE motif (residues 141–148), a conserved tyrosine, Tyr10 in SmChiB (Figure 3F), is replaced by Trp25 (Figure
<figure><img src="image_12.png" /><figcaption>**Figure 3.** Overall structure and substrate-binding cleft of CaGH18 and comparison with the catalytic domains of other GH18 chitinases. (A) The catalytic domain of CaChiA displays the characteristic $(\beta/\alpha)_8$ TIM barrel fold; labels indicate the respective $\alpha$-helices and $\beta$-strands. The structure also shows the presence of three short-turn helices (in pink) and a $\beta$-hairpin (in brown). (B–D) show the varying surface topologies, in particular differences in the depth of the substrate binding clefts (indicated by black arrows), of CaGH18 (PDB ID: 7Z65) SmChiB (PDB ID: 1E15) and SmChiC (PDB ID: 4AXN), respectively. Figure S3 provides another view of these three enzymes and shows surface potentials. (E,F) show catalytically important residues/motifs in CaGH18 and SmChiB, respectively, revealing an atypical orientation of Glu148 and a loop connecting residues Cys102 and Trp110 (Ser93 and Trp97 in SmChiB), in what is a “disrupted” SxGGW motif in CaGH18, where this motif is CxxxxxxGW. Panel G shows that this loop (Ile103-His108) interacts with other parts of the protein, including through multiple hydrogen bonds indicated in the figure by dashed lines.</figcaption></figure>

3E). Furthermore, we observed a significant difference in the typically conserved SxGGW motif (for example in SmChiB, Figure 3F), of CaGH18. Of note, Ser93 in this motif is substituted by Cys102, which is the only cysteine in CaGH18 (Figure 3E,F). Subsequently, the structures diverge up to Val115 in CaGH18 and Tyr110 in SmChiB (see Figure S2). Of the two glycines in the SxGGW motif, only the last one, Gly109 is conserved in CaGH18. The loop that connects Cys102 to Gly109 encompasses six residues (Ile103-His108) and is stabilized by an intricate H-bonding network, as shown in Figure 3G. Nevertheless, it is worth noting that the side chains of Trp110 in CaGH18 and Trp97 in SmChiB, are structurally equivalent (Figure 3E,F). Consequently, the two enzymes employ distinct mechanisms to position Trp in this location.

Inspection of the catalytic center showed an atypical orientation of the catalytic acid/base, Glu148, which seems due to the hydrophobic steric clash with the bulky side chain of Trp110 that follows Gly109 in the disrupted SxGGW motif (Figure 3E). A similar unusual positioning of the catalytic glutamate has been described previously for SmChiC (PDB ID: 4AXN) and a chitinase from Lactococcus lactis (LlChi18A, PDB ID: 3IAN).²⁵ The unusual features of the catalytic center of CaGH18 are discussed in more detail below.

**Substrate Binding.** To obtain insight into the mode of action of this unique chitinase, we prepared a catalytically inactive mutant, CaGH18-E148Q, which was used for

cocrystallization experiments. Despite multiple attempts to cocrystallize CaGH18-E148Q with chito-oligosaccharides of varying lengths, we only obtained well diffracting crystals for CaGH18-E148Q in complex with the longest oligomer, (GlcNAc)₆. This may indicate a preference of the enzyme for longer chito-oligosaccharides, as is confirmed by oligomer degradation studies described below. The complex was refined to 1.31 Å resolution, which revealed the presence of (GlcNAc)₆ bound to subsites -3 to +3 (Figure 4A; Table 1). Of note, this is a unique substrate-binding mode that has not been observed previously with GH18 chitinases. The overall structure of CaGH18-E148Q in complex with (GlcNAc)₆ is very similar to the structure of CaGH18, with an RMSD of 0.17 Å for the Cª atoms (analyzed using the PyMOL “Align” function).

Figure 4A shows that binding of (GlcNAc)₆ to CaGH18 involves a multitude of hydrophobic and polar contacts. Hydrophobic π-CH stacking interactions are most prominent in subsites -3, -1 and +3, involving Tyr29, Trp273 and Trp189, respectively. Other potential hydrophobic interactions, involve Trp110 in subsite -1, and Tyr213 and Phe184 in subsite +1. The side chains of Asn151 and Glu215 are hydrogen bonded to the O3 (3.1 Å) and O6 (2.8 Å) of GlcNAc bound in the +3 subsite, respectively (Figure 4A), which suggests that sugar binding in subsite +3 is strong. Glu215 also forms a hydrogen bond with the N atom (2.9 Å) of the acetamido group of GlcNAc in the +2 subsite. Polar
<figure><img src="image_13.png" /><figcaption>**Figure 4.** (A) Crystal structure of CaGH18-E148Q in complex with (GlcNAc)₆ (PDB ID: 7Z64). The picture shows the side chains of residues interacting with the substrate, which occupies subsites -3 to +3. Residues with orange carbons show the catalytically crucial Asp146 and the catalytic acid/base Glu148 (mutated to Gln). Aromatic residues have yellow carbons and other residues have cyan carbons. The substrate (GlcNAc)₆ is shown with light-gray carbons. (B) A closer view on a unique interaction involving the side chains of Gln57, Trp110, Asp146, and Trp273 that helps positioning GlcNAc bound in the -1 subsite. (C) Superposition of the CaGH18 apoenzyme (carbons in wheat color) and the CaGH18-E148Q + (GlcNAc)₆ complex (carbons in cyan color), showing residues involved in substrate-binding and catalysis that vary in conformation.</figcaption></figure>

interactions promoting binding of GlcNAc in the -2 subsite involve Arg278 (2.9 Å) and the hydroxyl group of Tyr29 (3.7 Å). Binding of GlcNAc in the -3 subsite is promoted by interactions with the residues Arg72 (3.8 Å) and Tyr75 (2.7 Å) (Figure 4A).

As expected, based on studies with other GH18 chitinases,⁸ the GlcNAc unit bound in the -1 subsite is distorted toward a ¹,⁴B conformation. The binding and the distortion of the -1 sugar involve residues and interactions that are well-known from studies of other GH18 enzymes (Trp110, Asp146, Tyr213, Trp273, all conserved) (Figure 4B). For example, the carbonyl oxygen from the acetamido group of the -1 sugar makes a strong H-bond (2.6 Å) with the hydroxyl group of

Tyr213, as observed in other GH18-substrate complexes. Importantly, sugar binding in the -1 subsite involves one residue that is unique for CaChiA, namely Gln57 (Figure 4B).

Binding of (GlcNAc)₆ is accompanied by multiple changes in side chain orientations, including residues Gln57, Arg72, Trp110, Glu214, Trp189 and the catalytically crucial residues Asp146 and Gln148 (Figure 4C). In particular, Gln148 adopts a position that is compatible with catalysis, while Asp146 rotates from a position facing toward Glu144 to a position facing toward Gln148. The oxygen atom of the scissile glycosidic bond is positioned at 2.8 Å away from the headgroup of Gln148.

### Peculiar Features of the Catalytic Center of CaGH18.

It is well-known that catalysis by family GH18 chitinases involves rotation of the side chain of the aspartate that is closest to the catalytic glutamate (i.e., the second D in the “DxDxE” motif, Asp146 in CaGH18, Asp142 in SmChiB; Figure 1). Detailed studies of SmChiB have shown that, upon substrate binding, the side chain of this Asp rotates from a “downwards” position where it interacts with Asp140 (Glu 142 in CaGH18) toward an upward position, where it interacts with the substrate and the catalytic acid/base glutamate (Glu148 in CaGH18; Glu144 in SmChiB).⁸,⁹ In SmChiB Asp142 and Asp140 share a proton when Asp142 is in the downward position, whereas the negative charge of Asp140 is compensated by interactions with the hydroxyl groups of Tyr10 and (rotating) Ser93 when Asp142 is in the upward position.⁹ Remarkably, while Tyr10 and Ser93 are conserved in GH18 chitinases, CaGH18 instead features a tryptophan (Trp25) and a cysteine (Cys102), respectively (Figure 5). In addition, the buried aspartate, Asp140 in SmChiB, is a glutamate (Glu144) in CaGH18.

<figure><img src="image_14.png" /><figcaption>**Figure 5.** Comparison of the catalytic centers in CaChiA and SmChiB bound to substrate. The picture shows a superposition of the structures of the CaGH18-E148Q + (GlcNAc)₆ complex (PDB ID: 7Z64, carbons in green color) and the SmChiB-E144Q + (GlcNAc)₅ complex (PDB ID: 1E6N, carbons in cyan color). This superposition shows strong similarity for the carboxylic residues in the D/ExxDxE motifs but reveals differences in electrostatics near the scissile glycosidic bond and in binding of GlcNAc in the -1 subsite (see text for details). The substrate, (GlcNAc)₆ in the structure of CaGH18-E148Q + (GlcNAc)₆, is shown with gray carbons and the subsites are indicated by numbers.</figcaption></figure>
Another difference concerns Glu214, which in CaGH18 replaces the aspartate (Asp215 in SmChiB) that is commonly found in GH18 chitinases with a neutral pH optimum, and which affects the pKₐ of the catalytic acid during the catalytic cycle as shown by mutagenesis studies and comparing naturally occurring enzymes with a neutral (Asp at this position) or an acidic (Asn at this position) pH optimum.⁹,¹⁸ The closest Asp215-Glu144 distance in SmChiB is 6.8 Å, whereas the closest distance between Glu148 and Glu214 in CaGH18 is as much as 12 Å (measured in the structures with bound substrate). This difference would lead to considerable changes in active site electrostatics between the two enzymes. Of note, a neighboring Glu, Glu215, is somewhat closer to the catalytic Glu148, but the distance is still considerable (10.7 Å). In contrast to Asp215 in SmChiB, it seems unlikely that these two glutamates affect electrostatics in the catalytic center.

All in all, the catalytic center of CaGH18 shows multiple unusual features that may affect catalysis and its pH dependency.

**Insights into the Alkaline pH Optimum of CaChiA: pH-Activity Profiles of Mutants and pKₐ Calculations.** In an attempt to identify the residues responsible for the alkaline pH optimum and to study the impact of the variations in and around the DₓₓDₓDₓE motif, several mutants of CaGH18 were generated and mutational effects on the pH-activity profile and activity at pH 8.8 were assessed. Mutation of residues His141 or Glu144 in the HₓₓExxDₓE motif aspartate had only minor effects on activity and on the pH-activity profile, with, if anything, a slight shift toward a more alkaline pH optimum (Figure 6). It would thus seem that the variations in the canonical DₓₓDₓDₓE motif seen in CaGH18 do not explain on their own the high pH optimum of the enzyme. It is worth noting the limited effect of the relatively dramatic His → Asp mutation at the first position in the motif; this position shows relatively large natural variation (see Figure 2), and our data suggest that this residue, which is far away from the catalytic center has little effect on catalysis. Of note, pKₐ calculations discussed in detail below, show that a His and an Asp in this position have similar pKₐ values, between 4 and 5.

On the other hand, a considerable reduction in activity and a shift in the optimum pH from ‘basic to acidic’ were observed for the Q57A mutant, which showed a pH optimum around pH 6.0 (Figure 6). More in-dept kinetic analyses are needed to fully unravel the structural features that determine the special properties of CaGH18, for example analyses aimed at separating between substrate-binding effects and rate effects. The present results do show, however, that the unique glutamine at position 57 is critical for the activity of CaGH18 and likely contributes to the alkaline pH optimum of the enzyme.

A possible change in the pKₐ value of the catalytic diad, DₓE, relative to other GH18 chitinases, was assessed experimentally using ITC. Deprotonation of this diad will promote a favorable electrostatic interaction with positively charged allosamidin, a known inhibitor of GH18 chitinases²⁹,³⁰ and was investigated by testing the contribution from buffer ionization to the change in binding enthalpy (ΔHᵣ).³⁰,³¹ ITC experiments were carried out at identical buffer concentrations at pH 8.0 with four reaction buffers having different ionization enthalpies at 30 °C (Figure S4). The ΔHᵣ obtained was fitted as a function of the ionization enthalpy of the buffer (eq 1).

<figure><img src="image_15.png" /><figcaption>**Figure 6.** Mutational effects on the optimal pH of CaGH18. The impact of point mutations on the optimal pH of CaGH18 was analyzed using 0.5 mM (GlcNAc)₆ as the substrate in a 50 mM solution of various buffers at 37 °C. The enzyme exclusively converts (GlcNAc)₆ to two molecules of (GlcNAc)₃ and, therefore, the catalytic activity of each mutant at different pH was quantified by monitoring the production of (GlcNAc)₃. Product formation was assessed after 5 min of incubation. Note that maximally approximately 10% of the substrate was converted in these reactions, which means that the reported product levels reflect the initial reaction rates, except at the most extreme pH values, where enzyme inactivation may have occurred. All reactions were performed in triplicates and the standard deviations are shown as error bars.</figcaption></figure>

$$ \Delta H_r = \Delta H_{\text{ind}} + nH^+ \times \Delta H_{\text{ion}} \quad (1) $$

In this equation, ΔHᵣ is the buffer independent enthalpy change and nH⁺ is the number of protons taken up or released by the enzyme upon ligand binding. The slope of the linear regression demonstrated that 0.55 protons are transferred from the enzyme–ligand complex to the buffer (nH⁺) = -0.55 (0.05), which indicates a pKₐ of 8.09 of the DₓE diad. As a control, the same approach was undertaken for SmChiB indicating a pKₐ value of 6.95, in accordance with previously published data for this enzyme.³⁰ This upward shift in the apparent pKₐ of the catalytic diad is compatible with the higher pH optimum for activity of CaGH18. Of note, the Kₐ for binding of allosamidin by CaGH18 (2 µM) was similar to the Kₐ determined for endo acting SmChiC.³²

Calculations of pKₐ values in the apo enzyme and the enzyme–substrate complex for both CaChiA and SmChiB, which has a neutral pH optimum, showed clear differences (Table 2). Of note, these calculations should be interpreted with care, especially in the present case where crucial ionizable side chains are coupled (Table 2). While deriving true pKₐ values from these Propka calculations is associated with uncertainty, as illustrated by some calculated values being unlikely high, the calculations can indicate trends and differences. Looking at the catalytic acid/base in CaGH18 (E148) and SmChiB (E144), the calculations indicate a
<table><caption>**Table 2.** pK<sub>a</sub> Calculations for Wild-Type and Mutant CaGH18 (Alkaline pH Optimum) and Wild-Type SmChiB (Neutral pH Optimum)<sup>a</sup></caption><thead><tr><th></th><th colspan="4">CaGH18 with (GlcNAc)<sub>6</sub></th></tr><tr><th></th><th>WT</th><th>Q57A</th><th>H141D</th><th>E144D</th></tr></thead><tbody><tr><td>His141</td><td>4.53</td><td>4.53</td><td>4.25 (Asp)</td><td>4.53</td></tr><tr><td>Glu144</td><td>8.85*</td><td>8.83</td><td>8.85*</td><td>6.45 (Asp)</td></tr><tr><td>Asp146</td><td>6.56*</td><td>11.54*</td><td>6.56*</td><td>7.30*</td></tr><tr><td>Glu148 cat</td><td>12.28*</td><td>7.66*</td><td>12.28*</td><td>12.14*</td></tr><tr><th></th><th colspan="4">CaGH18 without substrate</th></tr><tr><td>His141</td><td>4.45</td><td>4.45</td><td>4.95 (Asp)</td><td>4.45</td></tr><tr><td>Glu144</td><td>11.88*</td><td>11.86*</td><td>11.87*</td><td>11.03* (Asp)</td></tr><tr><td>Asp146</td><td>4.61*</td><td>4.98*</td><td>4.61*</td><td>4.75*</td></tr><tr><td>Glu148</td><td>11.34</td><td>11.32</td><td>11.34</td><td>11.53</td></tr><tr><th></th><th colspan="4">WTSmChiB with and without substrate</th></tr><tr><th></th><th>with</th><th>with</th><th>without</th><th>without</th></tr><tr><td>Asp137</td><td>3.42</td><td></td><td>3.21</td><td></td></tr><tr><td>Asp140</td><td>5.52</td><td></td><td>4.73*</td><td></td></tr><tr><td>Asp142</td><td>7.26*</td><td></td><td>11.68*</td><td></td></tr><tr><td>Glu144</td><td>11.87*</td><td></td><td>7.94</td><td></td></tr></tbody></table>

<sup>a</sup>The pK<sub>a</sub> values were calculated using propka3 version 3.4.0, as described in the Materials & Methods section. An * (assigned by propka3) indicates that ionizable side chains are coupled. Each protein variant (apo-enzyme or complex) represents a different PDB entry. Prior to the calculations, the glutamine residue in the enzyme-substrate complexes (Gln148 in CaGH18, Gln144 in SmChiB) was reverted to the wild-type catalytic glutamate. These point mutations were introduced using the mutagenesis-wizard in PyMOL. The calculations for SmChiB were done for comparative purposes. An in-depth analysis of the mutational effects on pK<sub>a</sub> values and the pH-dependency of catalytic activity for SmChiB is described in Synstad et al., (2004).<sup>9</sup> Prominent changes in pK<sub>a</sub> values upon mutations in CaGH18 are marked with bold font.

drastically higher pK<sub>a</sub> value for CaGH18 in the absence of substrate (11.34 versus 7.94 for SmChiB). The pK<sub>a</sub> value for

CaGH18 is also higher in the presence of substrate, but in this case the difference is small (12.28 vs 11.87). The higher pK<sub>a</sub> of the catalytic acid/base is consistent with the higher pH-optimum observed for CaGH18 compared to SmChiB. According to Propka, the general explanation for the elevated pK<sub>a</sub> value of Glu148 in CaGH18 can be traced back to the atypical position of this residue (Figure 3E<sub>7</sub>F), giving it a more buried nature and different solvent exposure compared to other GH18 chitinases such as SmChiB.

Decomposition of the effects resulting in the elevation of the pK<sub>a</sub> value of the catalytic acid/base Glu148 (pK<sub>a</sub> 11.34) in CaGH18 (without substrate) showed contributions of desolvation (+6 pH units) and Coulombic interaction with Asp146 (+0.8 pH units). For the corresponding Glu144 (pK<sub>a</sub> 7.94) in SmChiB, for which pK<sub>a</sub> cycling during catalysis has been suggested,<sup>9</sup> contributions from desolvation (+3 pH units) and a small Coulombic interaction with Asp215 (+0.2 pH units) result in an elevated pK<sub>a</sub> value, which, however, is considerably lower compared to Glu148 in CaGH18.

When substrate is bound, the catalytic glutamate is closed off from the solvent, and the rotamers of the Glu148 and Asp146 in CaGH18 (see above) and of Asp142 in SmChiB<sup>9</sup> change. These alterations in the active site environment impact the pK<sub>a</sub> value of the catalytic glutamate further. The contributions to the pK<sub>a</sub> value for Glu148 in CaGH18 (pK<sub>a</sub> 12.28) are desolvation (+5.5 pH units), hydrogen bonding to substrate (−1 pH unit) and hydrogen bonding and Coulombic interaction with Asp146 (+1.3 and +2 pH units, respectively). For the corresponding Glu144 in SmChiB (pK<sub>a</sub> 11.87), the values are desolvation (+5.7 pH units), hydrogen bonding to substrate (−0.75 pH unit), hydrogen bonding with Asp142 (+1 pH unit) and Coulombic interactions with Asp142 and Asp215 (+2 and +0.35 pH units, respectively). Clearly, desolvation by the substrate plays an important role in modulating the pK<sub>a</sub> of the catalytic acid/base during the catalytic cycle, as pointed out previously for SmChiB.<sup>9</sup> CaChiA

<figure><img src="image_16.png" /></figure>

**Figure 7.** Product profiles for CaGH18 acting on different oligomeric substrates. The mode of action of CaGH18 was analyzed using a time-course reaction with 0.5 mM of different substrates [(GlcNAc)<sub>2−6</sub>] in 50 mM Tris-HCl, pH 8.8, 0.1 mg/mL BSA at 50 °C. CaGH18 did not show any activity on (GlcNAc)<sub>2</sub> and (GlcNAc)<sub>3</sub>. Panels A–C show reactions with (GlcNAc)<sub>4</sub>, (GlcNAc)<sub>5</sub> and (GlcNAc)<sub>6</sub>, respectively; reliable quantification of monomeric products was not possible. Note that the enzyme concentrations used were 500 nM, 40 nM and 4 nM, respectively, and that the catalytic rates for the various substrates thus differ substantially. Also note that transglycosylation reactions are commonly observed for GH18 chitinases<sup>34,35</sup> and that such reactions may explain why product levels not always add up. For example, formation of (GlcNAc)<sub>4</sub> in the reaction with (GlcNAc)<sub>5</sub> may result from direct conversion of the pentamer to a tetramer and a monomer, or from the formation of an octameric transglycosylation product [(GlcNAc)<sub>5</sub> + its product (GlcNAc)<sub>3</sub>], which is then converted to two molecules of (GlcNAc)<sub>4</sub>. The product profiles for CaChiA were similar to those for CaGH18 (data not shown). The error bars represent the standard deviations for three independent experiments.
is special in that the elevated $pK_a$ of Glu148 does not seem to depend on the presence of substrate.

As shown in Figure 6, the mutants H141D and E144D did not alter the pH optimum profile of the CaGH18 enzyme, while the Q57A mutation resulted in an acidic shift. Interestingly, the $pK_a$ calculations confirmed the special role of Q57. For the apoenzyme all mutants gave predicted $pK_a$ values similar to those predicted for the WT. The H141D and E144D mutations did not affect predicted $pK_a$ values for the enzyme-substrate complex neither. However, the predicted values for residues 146 and 148 in the Q57A mutant ($pK_a$ Glu148 = 7.66, $pK_a$ Asp146 = 11.54) with substrate bound, when these two residues are electrostatically coupled, were more or less interchanged with the WT values ($pK_a$ Glu148 = 12.28, $pK_a$ Asp146 = 6.56). Thus, the calculations show a major effect of the Q57A mutation on active site electrostatics that aligns with the experimentally observed shift in the pH optimum. In the Q57A mutant with substrate bound, the contribution from desolvation (+5 pH units) to the $pK_a$ of Glu148 remains large, whereas hydrogen bonding with Asp146 (-1.3 pH units) and substrate (-1 pH unit) and the absence of Coulombic effects result in the low $pK_a$ value of 7.66. When replacing Gln57 with alanine, the 3.0 Å hydrogen bond between Gln57 and Asp146 is eliminated (Figure 4A,B), and this has large effects on the $pK_a$ predictions. Thus, the interplay between Gln57 and Asp146 likely is an important determinant of the catalytic properties of CaChiA.

**Substrate Preferences and Product Profiles.** Reactions of CaChiA or CaGH18 with oligomeric substrates of different lengths showed essentially similar product profiles for both enzyme variants. The enzyme does not cleave (GlcNAc)₂ and (GlcNAc)₃ (data not shown) and shows low activity on (GlcNAc)₄, which is primarily converted to (GlcNAc)₃, with some formation of (GlcNAc)₂ (Figure 7A). The enzyme is more active on (GlcNAc)₅, producing primarily (GlcNAc)₃ and (GlcNAc)₂ (Figure 7B). Reactions with (GlcNAc)₆ are at least 1 order of magnitude faster compared to (GlcNAc)₅ (see below) and (GlcNAc)₃ is the by far major product (Figure 7C), which is compatible with the binding mode observed in the crystal structure. Preliminary ITC experiments to assess binding of (GlcNAc)₆ to CaGH18-E148Q suggested a $K_d$ for this substrate is in the 0.5 mM range, meaning that the affinity for this substrate is low compared to other well-studied endo- and exo-acting chitinases.³³ Taken together, these observations suggest that CaChiA is optimized to act on larger chitin-structures, i.e., larger than (GlcNAc)₆.

Determination of specific activities at pH 8.8 (Table 3) confirmed that both CaChiA and CaGH18 strongly prefer (GlcNAc)₆ over (GlcNAc)₅. The enzymes display an as much as ~40-fold higher specific activity with (GlcNAc)₆, which is remarkable, and strongly suggests that the enzyme has evolved to act on longer (polymeric) substrates, as is also suggested by its shallow substrate binding surface/cleft (see above). In accordance with the pH profiles of Figure 6, the H141D and E144D mutants showed similar specific activities, whereas the specific activity of the Q57A mutant, with its shifted pH-optimum, was reduced (from 59.6 ± 2.7 s⁻¹ to 2.23 ± 0.05 s⁻¹).

As an extra control, we verified by circular dichroism that mutation of Gln57 to alanine does not affect the overall structure of CaGH18 (Figure S5). Taken together, the observations made for the Q57A mutant confirm the important role of Gln57 in modulating the catalytic properties

<table><caption>Table 3. Specific Initial Activity of CaChiA and Its Variants Towards (GlcNAc)₅ and (GlcNAc)₆ at pH 8.8 and 50 °Cᵃ</caption><thead><tr><th>enzyme</th><th>specific activity (s<sup>-1</sup>)<br>(GlcNAc)<sub>5</sub> as substrate</th><th>% of wild type</th></tr></thead><tbody><tr><td>CaChiA</td><td>1.65 ± 0.09</td><td>100</td></tr><tr><td>CaGH18</td><td>1.60 ± 0.13<br>(GlcNAc)<sub>6</sub> as substrate</td><td>97</td></tr><tr><td>CaChiA</td><td>66.1 ± 4.3</td><td>100</td></tr><tr><td>CaGH18</td><td>59.6 ± 2.7<br>(GlcNAc)<sub>6</sub> as substrate</td><td>90</td></tr><tr><td>CaGH18</td><td>59.6 ± 2.7</td><td>100</td></tr><tr><td>Q57A</td><td>2.23 ± 0.05</td><td>3.7</td></tr><tr><td>H141D</td><td>64.5 ± 5.0</td><td>108</td></tr><tr><td>E144D</td><td>63.0 ± 0.3</td><td>106</td></tr></tbody></table>

ᵃThe substrate concentration was 0.5 mM. No activity was detected for the catalytic mutants E148Q and E148A. The catalytic rates were derived from linear progress curves and are expressed as mol product per mol enzyme per second.

of CaGH18. An arrangement similar to that of Gln57 in CaGH18 (Figure 4B) has not been described for any other Chitinase.

**Activity on Polymeric Chitin.** Degradation reactions with α- and β-chitin (Figure 8) showed that the enzyme primarily produces (GlcNAc)₃. Production of (GlcNAc)₃ as the major end product is a unique feature of this alkaliphilic chitinase. GH18 chitinases, regardless of whether they are endo- or exo-acting, tend to produce (GlcNAc)₂ as the dominant product.³⁶ Interestingly, while CaChiA was equally active on α- and β-chitin, CaGH18 was more active toward β-chitin than α-chitin (Figure 8). The two enzyme forms showed similar activity on α-chitin, whereas the truncated enzyme was more active than the full-length enzyme on β-chitin, a substrate that is more accessible and easier to degrade. This intriguing difference between the two enzyme variants warrants further investigations. None of the additional domains in CaChiA have an obvious function in chitin-binding. It is conceivable that, with certain substrates, such as β-chitin, these domains can engage in nonproductive binding, which will hamper catalysis.

## ■ CONCLUSION

The present study shows that CaChiA is an active chitinase with a specific activity on (GlcNAc)₆ that is similar to the specific activities of other chitinases. This chitinase has remarkable properties, such as a noncanonical catalytic sequence motif, an exceptionally high pH optimum and the ability to primarily produce trimeric products. Our results suggest that the noncanonical H¹⁴¹ₓₓE¹⁴⁴ₓD¹⁴⁶ₓE¹⁴⁸ sequence motif is not the main determinant of the elevated pH optimum of CaChiA. In fact, the results suggest that the rather drastic Asp → His(141) change at the first position and the quite conservative Asp → Glu(144) change at the second position hardly affect catalysis. Notably, the catalytic center of CaChiA shows other unusual structural features that are less easy to grasp and address experimentally. One of these is the rather drastically changed environment of the most buried carboxylic acid (Glu144 in CaChiA; Asp in most other GH18s). Another deviation concerns the nature and position of an Asp or Asn that is known to modulate the $pK_a$ of the catalytic acid and the pH optimum in GH18s and that lacks in CaChiA, where it is “replaced” by Glu214 and Glu215 that are located more than 10 Å away from the catalytic acid.
<figure><img src="image_17.png" /><figcaption>**Figure 8.** Degradation of α-chitin (A) and β-chitin (B) by CaChiA and CaGH18 at alkaline pH. All the reactions were performed with 1 µM enzyme and 10 mg/mL substrate, in 50 mM Tris–HCl, pH 8.8, 0.1 mg/mL BSA at 50 °C in triplicates, and the products obtained were quantified using liquid chromatography. Black solid lines represent products generated by CaChiA and blue dashed lines represent products generated by CaGH18. Squares: (GlcNAc)₂, circles: (GlcNAc)₃, triangles: (GlcNAc)₄. The insets highlight product levels in the initial phase of the reaction.</figcaption></figure>

Further work is needed to assess whether these latter variations, outside the catalytic H<sup>141</sup>xxE<sup>144</sup>xD<sup>146</sup>xE<sup>148</sup> motif, contribute to the alkaline pH optimum of this unique chitinase. In this study we pinpoint another structural feature that is unique for CaChiA, namely Gln57, that is clearly important for enzyme performance and likely promotes activity at alkaline pH. Gln57 is in close contact with two conserved tryptophan residues that interact with the substrate and, in both the apoenzyme and the enzyme–substrate complex, the glutamine headgroup is at only 3.0 Å from catalytically crucial Asp146. In the enzyme–substrate complex, Gln57 is at only 4.1 Å from the nitrogen atom of the acetamido group of the –1 sugar that evolves to a positively charged oxazolinium ion during catalysis (Figure 1). The experimental and Propka results consistently suggest that Gln57 contributes to the elevated pH optimum of CaChiA.

CaChiA is also special in terms of its domain structure, which suggests the absence of a chitin-binding domain and secretion via the Type IX system, which could potentially imply that the protein is anchored to the outer membrane.³⁷ The catalytic domain of the enzyme is special in that the substrate-binding “groove” or “cleft” is exceptionally shallow. The enzyme nevertheless interacts well with substrates as shown by the crystal structure of the complex with (GlcNAc)₆ and the catalytic rates obtained with this substrate. The clear preference for producing trimeric products is, while suggested previously,³⁸ highly unusual and may reflect biologically relevant functionalities, related to chitin degradation or perhaps other processes,³⁹ which remain to be unraveled.

## MATERIALS AND METHODS

**Bacterial Strains, Biochemicals, and Enzymes.** Chemically competent OneShot *Escherichia coli* TOP10 cells and OneShot BL21 Star (DE3) *E. coli* cells were purchased from Invitrogen (Carlsbad, CA). Q5 DNA polymerase and the master mix for polymerase chain reactions were procured from New England Biolabs (Ipswich, MA). All chemicals used in the present study were of analytical grade and procured from Sigma-Aldrich (MO, USA), GE Health care (Uppsala, Sweden), HiMedia laboratories (Mumbai, India) or Thermo Fisher Scientific (USA). Polymeric substrates like α-chitin

(extracted from shrimp shells) and β-chitin (extracted from squid pen), were procured from Mahatani Chitosan Pvt. Ltd. (Veraval, India). Chitooligosaccharides with different degrees of polymerization were purchased from Megazyme (Co. Wicklow, Ireland).

**Phylogenetic Analysis.** To construct the pool of GH18 sequences, the sequence of the catalytic domain of CaChiA, CaGH18 (residues 22–303), was used as query for protein–protein-BLAST (blastp) searches against the nonredundant NCBI database, with a threshold sequence identity values of 35%. In addition to the resulting pool of sequences, GH18–encoding sequences found in plants,⁴⁰ insects,⁴¹ and fungal genomes were included resulting in a total of 214 sequences.

The GH18 catalytic domains (as defined by PFAM) of these sequences were extracted by removing the signal peptide identified using SignalP 6.0⁴² and by removing N-terminal and C-terminal extensions on the basis of a multiple sequence alignment (ClustalOmega) and, for less clear cases, structural predictions using Rosetta (https://www.rosettacommons.org/software). The phylogenetic tree was built (500 bootstraps) using the online platform NGPhylogeny,⁴³ where sequences were aligned with MAFFT,⁴⁴ the MSA further curated with BMGE,⁴⁵ and the tree inferred with FastME 2.0.⁴⁶ The Phylogenetic tree was visualized using the iTOL platform.⁴⁷ Sequences defining each phylogenetic subgroup were realigned using ClustalOmega and manually verified before generating a conservation logo for the region containing the “DxxDxDxE” motif, using the WebLogo 3.0 online tool.⁴⁸

**Molecular Cloning of CaChiA and Its Variants.** The gene encoding the full-length enzyme, CaChiA (residues 1 to 651; UniProt identifier U7D706) was codon optimized for *E. coli* and procured from GenScript (Piscataway, NJ, USA). Further, gene specific primers (see Table S1) with sequence overhangs pertaining to the prelinearized pNIC–CH expression vector (Addgene, Cambridge, MA, USA) were used to amplify genes encoding CaChiA and the catalytic domain only (CaGH18; residues 22–303 preceded by a methionine) and expression vectors were obtained by ligation-independent cloning as described by Aslanidis & de Jong, (1990).⁴⁹ In the resulting expression plasmid, the CaChiA and CaGH18 genes exclude the region corresponding to the signal peptide (residues 1–21) and include a 6x-His-tag at the C-terminus.
The reaction mixture was transformed into chemically competent OneShot *E. coli* TOP10 cells (Invitrogen, Carlsbad, CA) and transformants were selected by using LB agar plates supplemented with 5% sucrose and 50 µg/mL kanamycin (final concentrations). Transformants were picked and inoculated in LB broth containing 50 µg/mL kanamycin and grown overnight at 37 °C with shaking at 200 rpm. These fully grown overnight cultures were used for isolation of plasmid DNA using the NucleoSpin plasmid kit from Macherey-Nagel. The integrity of the cloned sequences was confirmed through Sanger sequencing services from GATC Biotech AG (Constance, Germany).

The expression plasmid pNIC-CH-CaGH18 was used as a template for generation of CaGH18 point mutants. Mutagenesis was performed using the QuikChange II site-directed mutagenesis kit (Agilent Technologies), as described by the manufacturer. The primer combinations used for mutagenesis are listed in Table S1 and insertion of the desired mutations, as well as the overall correctness of the gene sequence, were verified using the Sanger sequencing services from GATC Biotech AG (Constance, Germany).

**Protein Expression and Purification.** CaChiA and its variants were overexpressed using OneShot BL21 Star (DE3) *E. coli* cells. Starter cultures for protein overexpression were prepared by inoculating the transformants into 10 mL LB broth supplemented with 50 µg/mL kanamycin and growing overnight at 37 °C, 200 rpm. 1% of the overnight culture was then transferred into 500 mL of LB broth with 50 µg/mL kanamycin followed by incubation at 37 °C, 200 rpm, until the OD₆₀₀ reached between 0.4–0.6, at which point protein expression was induced with 0.4 mM isopropyl-b-D-thiogalactopyranoside (IPTG), followed by incubation for 16 h at 18 °C with shaking at 200 rpm. Subsequently, cells were harvested by centrifugation at 5,000g for 15 min, at 4 °C, and the cell pellet was resuspended in 50 mL 50 mM Tris-HCl (pH 8.0) supplemented with 500 mM NaCl and 5 mM imidazole. Prior to sonication phenylmethylsulfonyl fluoride to a final concentration of 0.1 mM was added and cell disruption was carried out on ice using a Vibracell sonicator, model CV33, equipped with a 3 mm probe (Sonics, Newtown, CT). The conditions used for sonication were as follows: 25% amplitude with 30 pulses of 5 s each and with a 20 s delay between pulses. The cell debris was pelleted by centrifugation at 12,500g for 30 min, at 4 °C, and the supernatant was sterilized using 0.2 µm filters. Protein purification with a 5 mL HisTrapHP column was carried out as previously described by.⁵⁰ Following SDS-PAGE analysis, protein fractions with highest purity were pooled, and then concentrated and buffer exchanged to 50 mM Tris-Cl, pH 8.0, using Amicon Ultra Centrifugal filters (10 kDa cutoff, Millipore, Billerica, MA). Purified proteins were stored at 4 °C.

**Crystallization of CaGH18 and CaGH18-E148Q + (GlcNAc)₆.** Wild-type CaGH18 was crystallized using the hanging drop vapor diffusion method in a 96-well plate. Each hanging drop was prepared by mixing 1 µL of CaGH18 (at 20–25 mg/mL in Tris-HCl pH 8.0) with an equal volume of a reservoir solution from different commercially available crystal screens. Crystals appeared in several conditions, but well diffracting crystals appeared when using 30% (w/v) PEG 8000, 100 mM Imidazole/Hydrochloric acid pH 8.0, 200 mM NaCl as precipitant (from Rigaku Wizard Classic 2, MiTeGen, LLC, Ithaca, NA).

To obtain crystals of CaGH18-E148Q in complex with (GlcNAc)₆, the mutant enzyme, at a concentration of 25 mg/ mL in 20 mM Tris-HCl, pH 8.0, was supplemented with 3 mM (GlcNAc)₆ and the samples were stored at 4 °C for 24 h to ensure complete binding. These preformed complexes were used for vapor-diffusion crystallization screening in 96-well sitting drop trays, using a Mosquito crystallization robot (TTP Labtech, UK) and commercially available screens. Crystals appeared in several conditions, but well diffracting crystals appeared when using 0.15 M potassium bromide, 30% w/v Polyethylene glycol monomethyl ether 2000 (from Index screen, HR2–144, Hampton Research, USA).

**Diffraction Data Collection, Structure Determination and Model Refinement.** Crystals were flash frozen in liquid nitrogen after a short soak in reservoir solution supplemented with 20% ethylene glycol. X-ray diffraction data for CaGH18 and CaGH18-E148Q cocrystallized with (GlcNAc)₆ were collected at the ID30A-1 "Massif" beamline at the European Synchrotron Radiation Facility (ESRF), in Grenoble, France. The data was integrated, scaled and analyzed using XDS,⁵¹ Aimless⁵² and CCP4i.⁵³ The structures were solved by molecular replacement with the Phaser module in the PHENIX software,⁵⁴ using the AlphaFold structure as the search model. The refinement was done using PHENIX⁵⁴ and each refinement cycle was interspersed with rebuilding and manual adjustments using Coot.⁵⁵

The asymmetric unit of the final model of CaGH18 contains one protein chain of 283 residues (sequence provided in Figure S2), two ethylene glycol molecules and 341 water molecules (Table 1). The asymmetric unit of the final model of CaGH18-E148Q + (GlcNAc)₆ contains two protein chains with 278 residues each, as well as two (GlcNAc)₆ molecules, 6 ethylene glycol molecules, 2 sodium ions and 848 water molecules (Table 1). The final atomic coordinates and structure factors have been deposited in the Protein Data Bank with accession codes 7Z65 (native CaGH18) and 7Z64 (CaGH18-E148Q + (GlcNAc)₆).

Electrostatic surfaces were generated using the Adaptive Poisson-Boltzmann Solver (APBS)⁵⁶ plugin in PyMOL, which employs the pdb2pqr program⁵⁷ to assign partial charges at pH 7.0. The resulting Poisson-Boltzmann surfaces were then color-mapped to visualize the electrostatic potential for each protein.

**Product Analysis by UPLC.** Chromatographic separation and quantification of chitooligomers ((GlcNAc)₂⁻₆) were performed using a long hydrophilic interaction chromatography (HILIC) column (Acquity UPLC BEH Amide, 300 Å, 1.7 µm, 2.1 mm × 150 mm), as described previously by Madhuprakash et al., (2018),³⁵ with minor modifications. The injection volume was 7.0 µL and the flow rate was 0.4 mL/min. The analysis was executed using a gradient with 20% eluent A (15 mM Tris-HCl pH 8.0) and 80% eluent B (100% acetonitrile) as the starting condition. The gradient was as follows: 0–5 min, starting conditions; 5–12 min, linear gradient to 70% B; 12–16 min, linear gradient to 55% B; 16–18 min, linear gradient back to the starting conditions; 18–22 min, starting conditions. The absorbance at either 195 or 205 nm was monitored for detection of products. Detection at 205 nm was used in most cases, since this gave better signal-to-noise ratios, peak integration and product quantification. A concentration range in which peak area and solute concentration were linearly correlated was established for each oligomer, i.e., (GlcNAc)₂⁻₆, and standard curves were
generated for the respective oligomers. The resulting standard curves had R² values above 0.998, allowing the molar concentrations of (GlcNAc)₂⁻₆ to be determined with confidence.³⁵

**Determination of Optimal Parameters for Chitinase Activity.** The pH optimum for chitinase activity was determined using 0.5 mM (GlcNAc)₆ as the substrate, which was incubated with 4 nM of either CaChiA or CaGH18 and 0.1 mg/mL BSA for 5 min in a 50 mM solution of various buffers. The buffers were: ammonium acetate (pH 3.0, 4.0, 5.0), BisTris (pH 5.8, 6.5, 7.0), Tris–HCl (pH 8.0, 8.8) and CAPS (pH 9.7, 10, 10.5, 11). Essentially the same conditions were used to determine the pH optima of CaGH18 mutants, except for the Q57A mutant, for which the enzyme concentration was 80 nM. The temperature optima of CaChiA or CaGH18 were determined using 50 mM Tris–Cl, pH 8.8. The reaction mixture was preheated for 5 min at the respective temperatures before adding the enzyme, and the reaction conditions were as described above. All reactions were performed in triplicates and catalytic activity was quantified by monitoring the production of (GlcNAc)₃. Under these conditions, maximally approximately 10% of the substrate was converted in the reactions.

**Specific Activity Measurements with (GlcNAc)₅ or (GlcNAc)₆ as the Substrate.** Specific activity measurements were performed using 0.5 mM (GlcNAc)₅ or 0.5 mM (GlcNAc)₆ as substrate, in 50 mM Tris–HCl, pH 8.8, 0.1 mg/mL BSA. All reactions were performed in triplicates at 50 °C. Care was taken to have true initial conditions, meaning that the rate of hydrolysis of the substrate (GlcNAc)₅ or (GlcNAc)₆ was constant over time and that the substrate concentration remained above 80% of the starting concentration. For reactions with (GlcNAc)₅ the enzyme concentration was 40 nM. For reactions with (GlcNAc)₆ the enzyme concentration was 4 nM, except for the Q57A mutant for which the concentration was 80 nM. Samples were collected at regular time intervals (5–20 min) and the reaction was stopped by adding an equal volume of 100% acetonitrile. Samples were stored at –20 °C until UPLC analysis of reaction products.

**Activity of CaChiA and Its Variants toward Oligo-meric and Polymeric Chitin Substrates.** The mode of action of CaChiA or CaGH18 was analyzed by carrying out reactions with 0.5 mM of (GlcNAc)₄⁻₆ in 50 mM Tris–HCl, pH 8.8, 0.1 mg/mL BSA that were incubated at 50 °C. The enzyme concentrations were optimized for each oligomeric substrate before performing time-course degradation experiments. The final enzyme concentrations were as follows: 4 nM for (GlcNAc)₆, 40 nM for (GlcNAc)₅ and 0.5 µM for (GlcNAc)₄. Furthermore, to verify the impact of point mutations on the mode of action of CaGH18, the activity of all the mutants was analyzed with 0.5 mM (GlcNAc)₆ as the substrate.

Activity toward polymeric α- or β-chitin substrates was assessed using reaction mixtures containing 10 mg/mL chitin, 1 µM enzyme and 0.1 mg/mL BSA in 50 mM Tris–HCl, pH 8.8. All the reactions were performed in triplicates. Samples were collected at regular intervals and the reactions were stopped by adding an equal volume of 100% acetonitrile after which products were analyzed by UPLC as described above.

**ITC Measurements.** ITC experiments were performed with a VP-ITC system from Microcal, Inc. (Northampton, MA). Solutions were thoroughly degassed prior to experiments

to avoid air bubbles in the calorimeter. Experiments were done using 20 mM potassium phosphate buffer, HEPES, tricine, or Tris-Cl, all at pH of 8.0. The heats of ionization of these buffers are as follows: potassium phosphate, 1.22 kcal/mol;⁵⁸ HEPES, 5.02 kcal/mol;⁵⁸ Tricine, 7.64 kcal/mol,⁵⁸ and Tris-Cl, 11.2 kcal/mol.⁵⁹ For a typical titration of CaGH18 with allosamidin,²⁹ ¹⁵ µM CaGH18 was placed in the reaction cell with a volume of 1.4214 mL, and 500 µM of allosamidin was placed in the ITC syringe. Aliquots of 4 µL were injected into the reaction cell at 150 s intervals with a stirring speed of 260 rpm. The titrations were normally completed after 70 injections. As a control, the same experiments were performed with SmChiB instead of CaGH18.

For binding of (GlcNAc)₆, ¹⁵ µM CaGH18-E148Q was placed in the reaction cell with a volume of 1.4214 mL, and 500 µM of (GlcNAc)₆ was placed in the ITC syringe. Aliquots of 8 µL were injected into the reaction cell at 150 s intervals with a stirring speed of 260 rpm. The titrations were normally completed after 50 injections.

ITC data were collected automatically using the Microcal Origin v.7.0 software accompanying the VP-ITC system. All data were corrected for heat of dilution by subtracting the heat remaining after the saturation of binding sites on the enzyme prior to further data analysis. Data were fitted using a nonlinear least-squares algorithm using a single-site binding model employed by the Origin software that accompanies the VP-ITC system. All data from the binding reactions fitted well with the single-site binding model, yielding the stoichiometry (n), equilibrium binding association constant (Kₐ), and the enthalpy change (ΔHₜ) of the reaction.

**Calculation of pKₐ Values.** The pKₐ values of selected ionizable amino acids in or near the active sites of CaGH18 and SmChiB were calculated using propka3 version 3.4.0.⁶⁰,⁶¹ PDB entries 7Z65 and 1E15 were used when calculating pKₐ values of CaGH18 and SmChiB without substrate, and PDB entries 7Z64 and 1E6N were used to calculate the corresponding pKₐ values for the enzyme–substrate complexes. Prior to the calculations the glutamine residue in 7Z64 and 1E6N was reverted to the wild-type catalytic glutamate. The mutagenesis-wizard in PyMOL was used to introduce mutations in the structures, and the rotamer of the mutated amino acid side chain that provided the best match to the (experimentally observed) rotamer of the wild-type residue without creating steric clashes was always selected. Some of the ionizable side chains in or near the active site are close to other ionizable side chains in the structure and are thus coupled (marked with an asterisk by propka3).

**Circular Dichroism (CD) Spectroscopy.** The CD spectra of CaGH18 and its Q57A mutant were acquired at 25 °C using a Jasco J-1500 spectropolarimeter (Jasco International Co. Ltd., Tokyo, Japan) with a 2 mm path length quartz cuvette. Far-UV spectra (190–250 nm) were collected using 0.17 mg/ mL protein solutions in 5 mM Tris–HCl, pH 8.0, by accumulation of three consecutive scans at a rate of 20 nm/ min and a bandwidth of 2 nm. The unfolding of the chitinases was studied by heating the protein solutions in 5 mM Tris– HCl, pH 8.0 from 20 to 99 °C using a Peltier heating system, with a heating rate of 1 °C/min. All CD spectra were baseline- corrected using a spectrum recorded for the Tris–HCl buffer (5 mM, pH 8.0).
■ ASSOCIATED CONTENT

Supporting Information

The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.biochem.5c00082.

Includes detailed information about the primers used in
the study (Table S1); optimal conditions for CaChiA
and CaGH18 activity (Figure S1); structure-based
sequence alignment (Figure S2); APBS surface charges
(Figure S3); ITC profile displaying allosamidin binding
(Figure S4); and CD analysis (Figure S5) (PDF)

Accession Codes

Accession ID: UniProt Accession ID of the protein CaChiA:
U7D706.

■ AUTHOR INFORMATION

Corresponding Authors

**Jogi Madhuprakash** - Department of Plant Sciences, School of Life Sciences, University of Hyderabad, Hyderabad 500046 Telangana, India; ● orcid.org/0000-0001-6491-0423;
Phone: +91-40-23134566; Email: jmpsl@uohyd.ac.in

Vincent G. H. Eijsink - Faculty of Chemistry, Biotechnology and Food Science, Norwegian University of Life Sciences (NMBU), 1432 Ås, Norway; ● orcid.org/0000-0002-9220-8743; Phone: +47-67232463; Email: vincent.eijsink@nmbu.no

Authors

Bjørn Dalhus - Department of Medical Biochemistry, Institute for Clinical Medicine, University of Oslo, N-0424 Oslo, Norway; Department of Microbiology, Clinic for Laboratory Medicine, Oslo University Hospital, Rikshospitalet, N-0424 Oslo, Norway

Bastien Bissaro - INRAE, Aix Marseille Université, UMR1163 Biodiversité et Biotechnologie Fongiques, 13009 Marseille, France; ● orcid.org/0000-0001-8354-3892

Lal Duhsaki - Department of Plant Sciences, School of Life Sciences, University of Hyderabad, Hyderabad 500046 Telangana, India

Gustav Vaaje-Kolstad - Faculty of Chemistry, Biotechnology and Food Science, Norwegian University of Life Sciences (NMBU), 1432 Ås, Norway; ● orcid.org/0000-0002-3077-8003

Morten Sørlie - Faculty of Chemistry, Biotechnology and Food Science, Norwegian University of Life Sciences (NMBU), 1432 Ås, Norway; ● orcid.org/0000-0001-7259-6710

Åsmund K. Røhr - Faculty of Chemistry, Biotechnology and Food Science, Norwegian University of Life Sciences (NMBU), 1432 Ås, Norway; ● orcid.org/0000-0002-4956-4865

Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.biochem.5c00082

Author Contributions

JM, BD, BB, LD, MS, and AKR carried out the experiments.
BB performed the phylogenetic analysis. AKR conducted the
pKₐ analysis. LD performed the CD analysis. JM, BD, BB, LD,
GVK, MS, AKR, and VGHE. interpreted the data. JM and
VGHE conceptualized the study, designed the experiments,
and supervised the work. JM wrote the first draft, and BB, MS
and AKR, VGHE contributed to the draft. JM and VGHE

finalized the manuscript. All authors reviewed and approved
the final version of the manuscript.

Notes

The authors declare no competing financial interest.

■ ACKNOWLEDGMENTS

V.G.H.E. is grateful to the Research Council of Norway for
support to SFI-IB, a research-based innovation centre for
industrial biotechnology (309558). J.M. extends thanks to the
Science and Engineering Research Board, Government of India
(GoI) (CRG/2019/006426), the Council of Scientific &
Industrial Research, GoI (38(1503)/21/EMR-II), and the
University of Hyderabad-Institution of Eminence (UoH-IoE)
(RC1-20-020) for their support with research grants. J.M. also
wishes to acknowledge the Department of Science and
Technology, GoI, for the Funds for Infrastructure in Science
and Technology, Level C support to the Department of Plant
Sciences, UoH. BD is grateful for the support provided by the
South-Eastern Norway Regional Health Authority core facility
for Structural Biology (grant 2015095 to BD). The
computations were conducted using resources provided by
Sigma2, projects NN1003K and NS1003K—the National
Infrastructure for High-Performance Computing and Data
Storage in Norway.

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