Genomic analysis of siderophore β-hydroxylases reveals divergent stereocontrol and expands the condensation domain family

Genome mining of biosynthetic pathways streamlines discovery of secondary metabolites but can leave ambiguities in the predicted structures, which must be rectified experimentally. Through coupling the reactivity predicted by biosynthetic gene clusters with verified structures, the origin of the beta-hydroxyaspartic acid diastereomers in siderophores is reported herein. Two functional subtypes of nonheme Fe(II)/alpha-ketoglutarate-dependent aspartyl beta-hydroxylases are identified in siderophore biosynthetic gene clusters, which differ in genomic organization-existing either as fused domains (I beta H-Asp) at the carboxyl terminus of a nonribosomal peptide synthetase (NRPS) or as stand-alone enzymes (T beta H-Asp)-and each directs opposite stereoselectivity of Asp beta-hydroxylation. The predictive power of this subtype delineation is confirmed by the stereochemical characterization of beta-OHAsp residues in pyoverdine GB-1, delftibactin, histicorrugatin, and cupriachelin. The L-threo (2S, 3S) beta-OHAsp residues of alterobactin arise from hydroxylation by the beta-hydroxylase domain integrated into NRPS AltH, while L-erythro (2S, 3R) beta-OHAsp in delftibactin arises from the stand-alone beta-hydroxylase DelD. Cupriachelin contains both L-threo and L- erythro beta-OHAsp, consistent with the presence of both types of beta-hydroxylases in the biosynthetic gene cluster. A third subtype of nonheme Fe(II)/alpha-ketoglutarate-dependent enzymes (I beta HHis) hydroxylates histidyl residues with L-threo stereospecificity. A previously undescribed, noncanonical member of the NRPS condensation domain superfamily is identified, named the interface domain, which is proposed to position the beta-hydroxylase and the NRPS-bound amino acid prior to hydroxylation. Through mapping characterized beta-OHAsp diastereomers to the phylogenetic tree of siderophore beta-hydroxylases, methods to predict beta-OHAsp stereochemistry in silico are realized.