Investigation of Indigoidine Synthetase Reveals a Conserved Active-Site Base Residue of Nonribosomal Peptide Synthetase Oxidases

Nonribosomal peptide synthetase (NRPS)
oxidase (Ox) domains oxidize
protein-bound intermediates to install crucial structural motifs in
bioactive natural products. The mechanism of this domain remains elusive.
Here, by studying indigoidine synthetase, a single-module NRPS involved
in the biosynthesis of indigoidine and several other bacterial secondary
metabolites, we demonstrate that its Ox domain utilizes an active-site
base residue, tyrosine 665, to deprotonate a protein-bound l-glutaminyl residue. We further validate the generality of this active-site
residue among NRPS Ox domains. These findings not only resolve the
biosynthetic pathway mediated by indigoidine synthetase but enable
mechanistic insight into NRPS Ox domains.