Identification of Key Amino Acid Residues in the Adenylation Domains of Polymyxin Synthetase Responsible for l‑Threonine Adenylation in Paenibacillus polymyxa NBRC3020.

In nonribosomal peptide synthetase (NRPS) research, expanding our understanding of the relationship between active-site amino acid residues in adenylation (A) domains and their adenylation activity and substrate specificity remains essential, as detailed biochemical characterization is still limited. In this study, we investigated two l-threonine (l-Thr)-specific A domains, A2 and A10, from the NRPS responsible for polymyxin D biosynthesis in Paenibacillus polymyxa NBRC 3020. To preserve enzymatic functionality, these domains were heterologously expressed in Escherichia coli BAP-1 as larger modules containing adjacent functional domains. Adenylation activity was evaluated indirectly by quantifying pyrophosphate generated during the reaction. Both modules not only exhibited the highest activity toward l-Thr but also showed measurable activity toward structurally related amino acids, including l-serine (l-Ser), l-allo-threonine (l-allo-Thr), and d-alanine (d-Ala), indicating relatively broad substrate specificity. Docking simulations based on AlphaFold3-predicted structures identified three residues within the 10-amino-acid Stachelhaus codePhe, Met, and Hisas key determinants of l-Thr side-chain recognition. Alanine substitution of these residues caused marked reductions in adenylation activity, confirming their essential roles experimentally. Comparative analysis of A domains with strict versus broader l-Thr specificity revealed that, although first-shell residues were highly conserved, distinct differences were present in second-shell residues influencing active-site geometry. Guided by these observations, introduction of the corresponding double mutation into A10 enhanced its selectivity for l-Thr over l-Ser. These findings provide new insights into the molecular basis of substrate recognition in NRPS A domains.