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 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 towards 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 our machine learning approach will aid in functional classification of enzymes and advance natural product discovery.

Aryl Coenzyme A Ligases, a Subfamily of the Adenylate-Forming Enzyme Superfamily

“…Five NRPS A domains were bifunctional, showing CoA ligase activity ( 53 ), similar to luciferases ( 54 ). Moreover, the fact that single point mutations were able to restore CoA ligase activity in ANL superfamily members strengthens the hypothesis of an ancestral CoA ligase-like scaffold ( 51 ). Nonetheless, the alternative of CoA ligase activity developing independently at several times throughout ANL superfamily evolution, as shown with other superfamilies, cannot be conclusively excluded at this point.…”

“…Recently, Robinson et al ( 51 ) used a machine-learning approach to identify enzymes of the ANL superfamily and construct a maximum likelihood phylogenetic tree of the characterized proteins. This tree shows that β-lactone synthetases, NRPSs, as well as luciferases form very distinct monophyletic clades.…”

Terpenoid Biosynthesis Dominates among Secondary Metabolite Clusters in Mucoromycotina Genomes

“…All representatives of candidate families representing nonribosomal peptide synthases (NRPS), putative adenylate-forming reductases (called also non-(nonribosomal)peptide synthase-NPS, i.e., NPS_1, NPS_2, LYS2), luciferases (LUC) and PKS/NRPS hybrids (PKS–NRPS) were further annotated in AdenylPred [ 31 ]. Notably, this prediction was initially only possible for 380 sequences (out of 562 in total) where the AMP-binding domain matched the underlying HMMer model with sufficient quality.…”

Discovery of Cyanobacterial Natural Products Containing Fatty Acid Residues**

“…[22] Such FA-incorporating enzymes are abundant in cyanobacterial genomes (Figure S1), often within orphan BGCs [18] and represent opportunities for NP discovery.N evertheless,i ti s difficult to predict the structure of NPs produced by most cyanobacterial FA-incorporating BGCs.F or one,t he exact type and size of fatty acyl moiety loaded by these enzymes cannot be accurately predicted. [23] Adding to this,d ifferent biosynthetic logics,t ailoring enzymes and hypothetical proteins are found associated with these FA-incorporating enzymes. [18,22] Cyanobacterial FA metabolism has another particular aspect:a pparently,afunctional b-oxidation pathway is not found in these organisms,a sb riefly noted by von Berlepsch and co-workers.…”