Global analysis of adenylate-forming enzymes reveals β-lactone biosynthesis pathway in pathogenic Nocardia

Abstract

Enzymes that cleave ATP to activate carboxylic acids play essential roles in primary and secondary metabolism in all domains of life. Class I adenylate-forming enzymes share a conserved structural fold but act on a wide range of substrates to catalyze reactions involved in bioluminescence, nonribosomal peptide biosynthesis, fatty acid activation, and {beta}-lactone formation. Despite their metabolic importance, the substrates and catalytic functions of the vast majority of adenylate-forming enzymes are unknown without tools available to accurately predict them. Given the crucial roles of adenylate-forming enzymes in biosynthesis, this also severely limits our ability to predict natural product structures from biosynthetic gene clusters. Here we used machine learning to predict adenylate-forming enzyme function and substrate specificity from protein sequence. We built a web-based predictive tool and used it to comprehensively map the biochemical diversity of adenylate-forming enzymes across >50,000 candidate biosynthetic gene clusters in bacterial, fungal, and plant genomes. Ancestral enzyme reconstruction and sequence similarity networking revealed a hub topology suggesting radial divergence of the adenylate-forming superfamily from a core enzyme scaffold most related to contemporary aryl-CoA ligases. Our classifier also predicted {beta}-lactone synthetases in novel biosynthetic gene clusters conserved across >90 different strains of Nocardia. To test our computational predictions, we purified a candidate {beta}-lactone synthetase from Nocardia brasiliensis and reconstituted the biosynthetic pathway in vitro to link the gene cluster to the {beta}-lactone natural product, nocardiolactone. We anticipate our machine learning approach will aid in functional classification of enzymes and advance natural product discovery.