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

Abstract

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 α‐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 α‐methyl‐l‐seryl‐AMP) and cryogenic electron microscopy single particle analysis, and performed site‐directed mutagenesis experiments. The data revealed that α‐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.

Structures and function of a tailoring oxidase in complex with a nonribosomal peptide synthetase module

“…Similar to the situation with BmdB, this dimerization is not necessary for function and does not significantly increase product formation 72 . Very recently, an elegant structure of a Cy-A-PCP construct of FmoA3 showed it has a head-to-tail homodimer architecture with a massive dimerization interface along the back side of the Cy and A domains 73 . The Cy-A domain interface observed in BmdB is similar to that seen in FmoA3 73 and in other C-A-containing structures 67 , 74 – 76 .…”

“…Very recently, an elegant structure of a Cy-A-PCP construct of FmoA3 showed it has a head-to-tail homodimer architecture with a massive dimerization interface along the back side of the Cy and A domains 73 . The Cy-A domain interface observed in BmdB is similar to that seen in FmoA3 73 and in other C-A-containing structures 67 , 74 – 76 . The BmdB M2 –BmdC complex structures (Fig.…”

Understanding the Diversity and Molecular Basis of Biosynthesis of Heterocyles in Natural Products Produced by Actinobacteria

“…38 To elucidate these functions, we attempted to analyze the functions of the FmoA2 and FmoA3 Cy domains by site-directed mutagenesis. 49 As a result, we found that the Cy domain of FmoA2 only catalyzes the peptide bond formation while FmoA3 only catalyzes the heterocyclization (Figure 13). In addition to FmoA2 and A3, there are several NRPSs that have two Cy domains but only have one corresponding heterocycle in their products.…”

“…The FmoA3 variant in which two of these glutamate residues were replaced with glycine resulted in a gain in peptide synthesis ability. 49 These results suggest that FmoA3 potentially can synthesize peptide bonds and has evolved into a Cy domain specialized for heterocyclization catalysis by losing its ability to interact with the upstream CP domain.…”

“…We next attempted to use structural biology to analyze why the Cy domain of FmoA3 does not catalyze peptide bond formation reactions. 49 We succeeded in analyzing the structure of FmoA3 by two methods:…”