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

Serina L. Robinson, Barbara R. Terlouw, Megan D. Smith, Sacha J. Pidot, Timothy P. Stinear, Marnix H. Medema, Lawrence P. Wackett

Research output: Contribution to journalArticlepeer-review

19 Scopus citations


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 β-lactone formation. Despite their metabolic importance, the substrates and 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 sequences. 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 phylogenetic reconstruction and sequence similarity networking of enzymes from these clusters suggested divergent evolution of the adenylate-forming superfamily from a core enzyme scaffold most related to contemporary CoA ligases toward more specialized functions including β-lactone synthetases. Our classifier predicted β-lactone synthetases in uncharacterized biosynthetic gene clusters conserved in >90 different strains of Nocardia. To test our prediction, we purified a candidate β-lactone synthetase from Nocardia brasiliensis and reconstituted the biosynthetic pathway in vitro to link the gene cluster to the β-lactone natural product, nocardiolactone. We anticipate that our machine learning approach will aid in functional classification of enzymes and advance natural product discovery.

Original languageEnglish (US)
Pages (from-to)14826-14839
Number of pages14
JournalJournal of Biological Chemistry
Issue number44
StatePublished - Oct 30 2020

Bibliographical note

Funding Information:
Funding and additional information—S.L.R. is supported by the National Science Foundation Graduate Research Fellowship under NSF grant number 00039202 and a Graduate Research Opportunities Worldwide (GROW) fellowship to the Netherlands supported by the NSF and the Netherlands Organization for Scientific Research (NWO) grant number 040.15.054/6097 (to S. L. R. and M. H. M.).

Publisher Copyright:
© 2020 Robinson et al. Published under exclusive license by The American Society for Biochemistry and Molecular Biology, Inc.


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