Exploring the Substrate Selectivity of the Condensation Domain VibH through Engineered Adenylation Domains, Non-Native Amide Synthesis, and Substrate Recognition Analysis.

Authors

Fumihiro Ishikawa, Hiroyuki Izuno, Shinya Nakamura, Ryotaro Wakano, Shinta Ijichi, Shumpei Asamizu, Nao Miyazaki, Saaya Kusuhara, Hiroyasu Onaka, Isao Nakanishi, Genzoh Tanabe

Published in

ACS chemical biology. Sep 07, 2026. Epub Sep 07, 2026.

Abstract

Nonribosomal peptide synthetases (NRPSs) are modular assembly-line enzymes that generate structurally diverse and biologically active natural products. Although adenylation (A) domain selectivity has been extensively characterized and engineered, the specificity of condensation (C) domains, which catalyze peptide bond formation and can constrain NRPS reprogramming, remains less well understood. Here, we report a systematic functional analysis of VibH, a stand-alone C domain VibH from the vibriobactin biosynthetic pathway. By integrating VibH with wild-type and engineered variants of the upstream aryl acid A domain EntE, we bypassed intrinsic A-domain constraints and independently interrogated donor- and acceptor-site substrate tolerance. The donor site of VibH exhibited stringent specificity, accepting only mono- and disubstituted benzoic acid derivatives closely resembling 2,3-dihydroxybenzoic acid (DHB), whereas bulkier aryl substrates were not processed. In contrast, the acceptor site displayed broad tolerance toward structurally diverse amines. Monoamines showed clear chain-length dependence, with productive turnover restricted to medium-chain substrates, whereas diamines were more broadly accepted. Systematic analysis further revealed distance-dependent steric tolerance, in which bulky substituents were accommodated when positioned distal to the reactive amine but were restricted beyond an upper steric threshold. This engineered reconstitution strategy enabled the synthesis of numerous non-native amide conjugates and uncovered an asymmetric substrate-recognition architecture characterized by a stringent donor site and a permissive yet spatially constrained acceptor site. Molecular docking analysis provided a structural rationale for these trends by suggesting distinct binding modes for native and non-native donor substrates and distance-dependent accommodation of bulky amine acceptors. These findings provide mechanistic insights into C-domain specificity and establish a framework for rational NRPS reprogramming.

PMID:
42704106
Bibliographic data and abstract were imported from PubMed on 07 Sep 2026.

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