Identification of Key Amino Acids in the A Domains of Polymyxin Synthetase Responsible for 2,4-Diaminobutyric Acid Adenylation in Paenibacillus polymyxa NBRC3020 Strain

Mai Nemoto; Wataru Ando; Taichi Mano; Minjae Lee; Satoshi Yuzawa; Toshihisa Mizuno

doi:10.1021/acschembio.4c00553

Identification of Key Amino Acids in the A Domains of Polymyxin Synthetase Responsible for 2,4-Diaminobutyric Acid Adenylation in Paenibacillus polymyxa NBRC3020 Strain

ACS Chem Biol. 2025 Jan 17. doi: 10.1021/acschembio.4c00553. Online ahead of print.

Authors

Mai Nemoto¹, Wataru Ando¹, Taichi Mano¹, Minjae Lee^{2

3}, Satoshi Yuzawa^{4

5}, Toshihisa Mizuno^{1

6}

Affiliations

¹ Department of Life Science and Applied Chemistry, Graduate School of Engineering, Nagoya Institute of Technology, Gokiso-cho, Showa-ku, Nagoya, Aichi 466-8555, Japan.
² Faculty of Environment and Information Studies, Keio University, Fujisawa, Kanagawa 252-0882, Japan.
³ Department of Chemistry, University of California, Berkeley, Berkeley, California 94720, United States.
⁴ Systems Biology Program, Graduate School of Media and Governance, Keio University, Fujisawa, Kanagawa 252-0882, Japan.
⁵ Institute for Advanced Biosciences, Keio University, Tsuruoka, Yamagata 997-0017, Japan.
⁶ Department of Nanopharmaceutical Sciences, Graduate School of Engineering, Nagoya Institute of Technology, Gokiso-cho Showa-ku, Nagoya, Aichi 466-8555, Japan.

PMID: 39818748
DOI: 10.1021/acschembio.4c00553

Abstract

Developing novel nonribosomal peptides (NRPs) requires a comprehensive understanding of the enzymes involved in their biosynthesis, particularly the substrate amino acid recognition mechanisms in the adenylation (A) domain. This study focused on the A domain responsible for adenylating l-2,4-diaminobutyric acid (l-Dab) within the synthetase of polymyxin, an NRP produced by Paenibacillus polymyxa NBRC3020. To date, investigations into recombinant proteins that selectively adenylate l-Dab─exploring substrate specificity and enzymatic activity parameters─have been limited to reports on A domains found in enzymes synthesizing l-Dab homopolymers (pldA from S. celluloflavus USE31 and pddA from S. hindustanus NBRC15115), which remain exceedingly rare. The polymyxin synthetase in NBRC3020 contains five A domains specific to l-Dab, distributed across five distinct modules (modules 1, 3, 4, 5, 8, and 9). In this study, we successfully obtained soluble A domain proteins from modules 1, 5, 8, and 9 by preparing module-specific recombinant proteins. These proteins were expressed in E. coli BAP-1, purified via Ni-affinity chromatography, and demonstrated high specificity for l-Dab. Through sequence homology analysis, three-dimensional structural modeling, docking simulations to estimate substrate-binding sites, and functional validation using alanine mutants, we identified Glu281 and Asp344 as critical residues for recognizing the side chain amino group of l-Dab, and Asp238 as essential for recognizing its main chain amino group in the A domain. Notably, these key residues were conserved not only across the A domains in modules 1, 5, 8, and 9 of P. polymyxa NBRC3020 but also in those of the P. polymyxa PKB1 strain, as confirmed by sequence homology analysis. Interestingly, in pldA and pddA, the key residues involved in recognizing the side-chain amino group of l-Dab, which are conserved among polymyxin synthetases of NBRC3020 and PKB1 strain, were not observed. This suggests a potentially different mechanism for l-Dab recognition.