Structural and Functional Analyses of the Tridomain-Nonribosomal Peptide Synthetase FmoA3 for 4-Methyloxazoline Ring Formation

Nonribosomal peptide synthetases (NRPSs) are attractive targets for bioengineering to generate useful peptides. FmoA3 is a single modular NRPS composed of heterocyclization (Cy), adenylation (A), and peptidyl carrier protein (PCP) domains. It uses alpha-methyl-l-serine to synthesize a 4-methyloxazoline ring, probably with another Cy domain in the preceding module FmoA2. Here, we determined the head-to-tail homodimeric structures of FmoA3 by X-ray crystallography (apo-form, with adenylyl-imidodiphosphate and alpha-methyl-l-seryl-AMP) and cryogenic electron microscopy single particle analysis, and performed site-directed mutagenesis experiments. The data revealed that alpha-methyl-l-serine can be accommodated in the active site because of the extra space around Ala688. The Cy domains of FmoA2 and FmoA3 catalyze peptide bond formation and heterocyclization, respectively. FmoA3's Cy domain seems to lose its donor PCP binding activity. The collective data support a proposed catalytic cycle of FmoA3.

Automated summary

The study determined the structure of FmoA3 using X-ray crystallography and cryogenic electron microscopy, revealing the accommodation of alpha-methyl-l-serine in the active site. The Cy domains of FmoA2 and FmoA3 catalyze different reactions, with the Cy domain of FmoA3 potentially losing its donor PCP binding activity.