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

Abstract

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 β-hydroxyaspartic acid diastereomers in siderophores is reported herein. Two functional subtypes of nonheme Fe(II)/α-ketoglutarate–dependent aspartyl β-hydroxylases are identified in siderophore biosynthetic gene clusters, which differ in genomic organization—existing either as fused domains (IβHAsp) at the carboxyl terminus of a nonribosomal peptide synthetase (NRPS) or as stand-alone enzymes (TβHAsp)—and each directs opposite stereoselectivity of Asp β-hydroxylation. The predictive power of this subtype delineation is confirmed by the stereochemical characterization of β-OHAsp residues in pyoverdine GB-1, delftibactin, histicorrugatin, and cupriachelin. The l-threo (2S, 3S) β-OHAsp residues of alterobactin arise from hydroxylation by the β-hydroxylase domain integrated into NRPS AltH, while l-erythro (2S, 3R) β-OHAsp in delftibactin arises from the stand-alone β-hydroxylase DelD. Cupriachelin contains both l-threo and l-erythro β-OHAsp, consistent with the presence of both types of β-hydroxylases in the biosynthetic gene cluster. A third subtype of nonheme Fe(II)/α-ketoglutarate–dependent enzymes (IβHHis) 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 β-hydroxylase and the NRPS-bound amino acid prior to hydroxylation. Through mapping characterized β-OHAsp diastereomers to the phylogenetic tree of siderophore β-hydroxylases, methods to predict β-OHAsp stereochemistry in silico are realized.

Understanding the early stages of peptide formation during the biosynthesis of teicoplanin and related glycopeptide antibiotics

“…Hence, whilst our results now strongly support epimerisation of the first amino acid via the actions of the initial C‐domain in GPA biosynthesis, the mechanism by which this is accomplished remains unclear and likely will require structural characterisation. This also adds to the rapidly growing range of roles in NRPS biosynthesis that C‐domains can play in peptide assembly beyond simple peptide bond formation [4,8,43,45–50.…”

Lipopeptide families at the interface between pathogenic and beneficialPseudomonas-plant interactions

“…The third gene, absent from some clusters (e.g. nunamycin) or present individually (P. syringae SM and P. cichorii strains), is encoding a member of the b-hydroxylase family (Pfam PF05118) for which no function has yet been assigned (Reitz et al 2019; Figure 3).…”

The Desotamide Family of Antibiotics

“…Siderophores produced by NRPS biosynthetic pathways very often contain β-hydroxylated Asp and His residues, which function as bidentate chelating groups for coordination of metal ions [ 61 , 62 ]. These hydroxyl groups are primarily added by the activity of standalone factors, a catalytic methodology also employed by numerous other NRPS systems.…”