Enzymatic characterization of non-ribosomal peptide synthetases
condensation domains using an engineered adenylation domain
with an enlarged substrate-binding pocket
Fumihiro ISHIKAWA
Faculty of Pharmacy, Kindai University
Research objective
Non-ribosomal peptide synthetases (NRPSs) are large multifunctional proteins thatsynthesize peptide natural products, or their derivatives thereof, with considerable structuraland functional diversity. NRPS products include the antitumor agent, bleomycin; theantibiotic, vancomycin; and the immunosuppressant, cyclosporine A. Each NRPS module is
responsible for the activation, loading, and condensation of a single amino acid. A standard
NRPS module comprises three enzyme domains: the adenylation (A), thiolation (T; alsoknown as the peptidyl carrier protein), and condensation (C) domains. The A-domain
catalyzes the formation of an aminoacyl-adenylate (aminoacyl-AMP) intermediate using
adenosine triphosphate (ATP) and Mg2+. Next, the aminoacyl-AMP intermediate undergoes anucleophilic attack by the thiol group of the 4´-phosphopantetheine moiety of the holo-T
domain, forming the aminoacyl-S-T species. Finally, the C domain catalyzes peptide bondformation between the two aminoacyl-S-T species in the upstream and downstream NRPS
modules. The A-domain serves as an entry point for an amino acid in the NRPS machineryand is therefore an attractive target for the reprogramming the NRPS machinery for thebiosynthesis of non-native peptides. Furthermore, although the C domain plays an importantrole within the NRPS machinery, it remains poorly understood relative to other NRPS
domains. In this study, we conducted enzymatic characterization of NRPS condensation
domains using an engineered aryl acid adenylation domain with an enlarged substrate-binding pocket.
Methods1) Biochemical characterization of an engineered aryl acid adenylation domain with anenlarged substrate-binding pocket.
EntE is part of a three-module NRPS assembly line involving EntB, EntE, and EntF and is
responsible for the production of the siderophore enterobactin in Escherichia coli. EntE is astandalone aryl acid A-domain that catalyzes the activation of 2,3-dihydroxybenzoic acid(DHB) and transfers it to the aryl carrier protein (ArCP) domain of EntB. Recently, we havedescribed an engineered aryl acid A-domain with an enlarged substrate-binding site capable

of accepting a wide range of non-native aryl acids functionalized with nitro, cyano, chloro,bromo, and iodo groups (Fig. 1).1), 2) The structures of EntE N235G in complex with
3-cyanobenzoic acid-AMS and 2-nitrobenzoic acid-AMS demonstrated that the N235G
substitution enlarged the substrate-binding pocket by approximately 25 Å3 compared withthose of wild-type EntE (Figs. 1A and 1B).1),3) On the basis of these results, we designed andsynthesized a large number of disubstituted benzoic acid derivatives. Subsequently, weevaluated the substrate profiles of the wild-type EntE and the N235G variant toward 31
benzoic acid (BA) derivatives with different modifications at the 2- and 3-positions using a
non-radioactive high-throughput malachite green colorimetric assay. Furthermore, wedetermined the kinetic parameters of the adenylation reaction catalyzed by wild-type EntE
and the N235G variant.

Figure 1. Structural analysis of the N235G variant. (A) The substrate-binding pocket of theEntE–EntB (ArCP) complex (PDB ID: 3RG2) bound with a Sal-based vinyl sulfonamideinhibitor. The Asn235 side chain and Sal-based vinyl sulfonamide inhibitor are shown asstick models. (B) Structures of non-hydrolysable aryl-AMP analogues, 3-cyanoBA-AMS and
2-nitroBA- AMS. The 3-cyanoBA-AMS and 2-nitroBA-AMS molecules are shown as stickmodels. C) Aryl acid substrates accepted by the N235G variant.
2) Biochemical characterization of NRPS condensation domains using an engineeredaryl acid adenylation domain with an enlarged substrate-binding pocket.
To reconstitute the E. coli enterobactin biosynthetic pathway in vitro, we expressed
His-tagged wild-type EntE, the N235G variant, EntB, and EntF and purified these by affinity
chromatography. Next, we performed in vitro reconstruction of the enterobactin biosynthetic
pathway by including the components EntE, EntB, EntF, DHB, L-Ser, and ATP. Finally,using the N235G variant and non-native aryl acid substrates, we analyzed the production ofenterobactin derivatives and their shunt products using high-performance liquidchromatography (HPLC) and liquid chromatography/mass spectrometory (LC/MS).
Results1) Biochemical characterization of an engineered aryl acid adenylation domain with anenlarged substrate-binding pocket.
The substrate profile of the N235G variant demonstrated that it was capable of accepting 20of the 31 BA derivatives. This is noteworthy because of the wild-type EntE showed noenzymatic activity toward these aryl acids.
2) Biochemical characterization of the NRPS condensation domains using anengineered aryl acid adenylation domain with an enlarged substrate-binding pocket.
We successfully detected enterobactin and shunt products by HPLC and LC/MS. The
optimized conditions were as follows: EntE (1 μM), EntB (10 μM), EntF (2 μM), DHB (1mM), L-Ser (2 mM), and ATP (10 mM). Furthermore, we confirmed the biosynthesis ofenterobactin using the N235G variant. We investigated the production of enterobactin
derivatives using the N235G variant and non-native aryl acids to elucidate the substratespecificity of the C domain of EntF.
Conclusion
In this study, we described the limitations and scope of the substrate specificity of theN235G variant. We demonstrated that the N235G variant could accept a wide range ofbenzoic acid derivatives with different modifications at the 2- and 3-positions. Furthermore,
we reconstructed the enterobactin biosynthetic pathway using the N235G variant. Studies on
the biosynthesis of enterobactin derivatives are currently underway.
References
1）Ishikawa, F., Miyanaga, A., Kitayama, H., Nakamura, S., Nakanishi, I., Kudo, F., Eguchi, T.,and Tanabe, G. (2019) An engineered aryl acid adenylation domain with an enlarged substratebinding pocket. Angew. Chem. Int. Ed. 58: 6906-6910.
2）Ishikawa, F., Nohara, M., Nakamura, S., Nakanishi, I., and Tanabe, G. (2020) Precise probingof residue roles by NRPS code swapping: mutation, enzymatic characterization, modeling,and substrate promiscuity of aryl acid adenylation domains. Biochemistry 5: 351-363.
3）Ishikawa, F., Kitayama, H., Nakamura, S., Takashima, K., Nakanishi, I., and Tanabe, G.
(2021) Activity, binding, and modeling studies of a reprogrammed aryl acid adenylation
domain with an enlarged substrate binding pocket. Chem. Pharm. Bull. 69: 222-225.