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

Ambiguity of NRPS Structure Predictions: Four Bidentate Chelating Groups in the Siderophore Pacifibactin.

Identified through a bioinformatics approach, a nonribosomal peptide synthetase gene cluster in Alcanivorax pacificus encodes the biosynthesis of the new siderophore pacifibactin. The structure of pacifibactin differs markedly from the bioinformatic prediction and contains four bidentate metal chelation sites, atypical for siderophores. Genome mining and structural characterization of pacifibactin is reported herein, as well as characterization of pacifibactin variants accessible due to a lack of adenylation domain fidelity during biosynthesis. A spectrophotometric titration of pacifibactin with Fe(III) and 13C NMR spectroscopy of the Ga(III)-pacifibactin complex establish 1:1 metal:pacifibactin coordination and reveal which of the bidentate binding groups are coordinated to the metal. The photoreaction of Fe(III)-pacifibactin, resulting from Fe(III) coordination of the ß-hydroxyaspartic acid ligands, is reported.

ß-Hydroxyaspartic acid in siderophores: biosynthesis and reactivity.

A growing number of siderophores are found to contain ß-hydroxyaspartic acid (ß-OH-Asp) as a functional group for Fe(III) coordination, along with the more common catechol and hydroxamic acid groups. This review covers the structures, biosynthesis, and reactions of peptidic ß-OH-Asp siderophores. Hydroxylation of Asp in siderophore biosynthesis is predicted to be carried out either through discrete aspartyl ß-hydroxylating enzymes or through hydroxylating domains within non-ribosomal peptide synthetases, both of which display sequence homology to known non-heme iron(II), α-ketoglutarate-dependent dioxygenases. Ferric complexes of ß-OH-Asp siderophores are photoreactive, resulting in reduction of Fe(III) and oxidative cleavage of the siderophore to yield distinct types of photoproducts. Probing the photoreactivity of synthetic Fe(III)-α-hydroxycarboxylate clusters yields mechanistic insights into the different photoproducts observed for ß-OH-Asp and other α-hydroxycarboxylate siderophore Fe(III) complexes.

High interfacial activity of polymers "grafted through" functionalized iron oxide nanoparticle clusters.

The mechanism by which polymers, when grafted to inorganic nanoparticles, lower the interfacial tension at the oil-water interface is not well understood, despite the great interest in particle stabilized emulsions and foams. A simple and highly versatile free radical "grafting through" technique was used to bond high organic fractions (by weight) of poly(oligo(ethylene oxide) monomethyl ether methacrylate) onto iron oxide clusters, without the need for catalysts. In the resulting ∼1 µm hybrid particles, the inorganic cores and grafting architecture contribute to the high local concentration of grafted polymer chains to the dodecane/water interface to produce low interfacial tensions of only 0.003 w/v % (polymer and particle core). This "critical particle concentration" (CPC) for these hybrid inorganic/polymer amphiphilic particles to lower the interfacial tension by 36 mN/m was over 30-fold lower than the critical micelle concentration of the free polymer (without inorganic cores) to produce nearly the same interfacial tension. The low CPC is favored by the high adsorption energy (∼10(6) kBT) for the large ∼1 µm hybrid particles, the high local polymer concentration on the particles surfaces, and the ability of the deformable hybrid nanocluster cores as well as the polymer chains to conform to the interface. The nanocluster cores also increased the entanglement of the polymer chains in bulk DI water or synthetic seawater, producing a viscosity up to 35,000 cP at 0.01 s(-1), in contrast with only 600 cP for the free polymer. As a consequence of these interfacial and rheological properties, the hybrid particles stabilized oil-in-water emulsions at concentrations as low as 0.01 w/v %, with average drop sizes down to 30 µm. In contrast, the bulk viscosity was low for the free polymer, and it did not stabilize the emulsions. The ability to influence the interfacial activity and rheology of polymers upon grafting them to inorganic particles, including clusters, may be expected to be broadly applicable to stabilization of emulsions and foams.