# Mapping the nonribosomal specificity code through promiscuity-guided A-domain engineering

## TL;DR

Researchers studied how to redesign the specificity of nonribosomal peptide synthetases by analyzing mutations in their active sites, enabling the design of new bioactive peptides.

## Contribution

The study reveals how multispecificity and promiscuity in A-domains can be harnessed to guide the engineering of nonribosomal peptide synthetases.

## Key findings

- Mutant libraries of the A-domain revealed three key sequence positions that strongly influence substrate specificity.
- Promiscuity in A-domains enables evolvability and neofunctionalization for new peptide design.
- Computational modeling explained the impact of specific mutations on functional flexibility.

## Abstract

Nonribosomal peptide synthetases (NRPSs) assemble bioactive peptides from various building blocks. The binding pocket residues governing building block specificity have allowed prediction of NRPS products but not design of specificity. A reason for this failure has been ignorance of NRPS multispecificity. Here, we employ a hydroxamate assay (HAMA) to determine multispecificity for mutant libraries of the adenylation (A-)domain in module SrfAC of surfactin synthetase. A multispecific version of SrfAC is developed and its functional flexibility probed by fully randomizing 15 residues around the active site. We identify mutations with profound impact on specificity revealing remarkable evolvability and explain the effect of a selected mutant by computational modelling. Statistical analysis of the specificity divergence caused by 285 point mutations has revealed an outstanding influence of three sequence positions on specificity, which provides a roadmap for NRPS engineering. Our results suggest that promiscuity drives neofunctionalization of A-domains and mimicking this process will help to design valuable peptides in the lab.

From specialist to generalist and back: probing active-site mutations defines a roadmap for redesigning substrate specificity of adenylation domains from nonribsomal peptide synthesis.