Oligomerization and macrocyclization reactions are key steps in the biosynthesis of many bioactive natural products. Important macrocycles include the antibiotic daptomycin (1; ref. 1), the immunosuppressant FK-506 (2; ref. 2), the anthelmintic avermectin B1a (3; ref. 3) and the insecticide spinosyn A (4; ref. 4); important oligomeric macrocycles include the siderophores enterobactin (5; ref. 5) and desferrioxamine E (6; ref. 6). Biosynthetic oligomerization and macrocyclization reactions typically involve covalently tethered intermediates and are catalyzed by thioesterase domains of polyketide synthase and nonribosomal peptide synthetase multienzymes. Here we report that the purified recombinant desferrioxamine siderophore synthetase DesD from Streptomyces coelicolor M145 catalyzes ATP-dependent trimerization-macrocyclization of a chemically synthesized 10-aminocarboxylic acid substrate via noncovalently bound intermediates. DesD is dissimilar to other known synthetase families but is similar to other enzymes known or proposed to be required for the biosynthesis of omega-aminocarboxylic acid-derived cyclodimeric siderophores. This suggests that DesD is the first biochemically characterized member of a new family of oligomerizing and macrocyclizing synthetases.

Bacterial pathogens need to scavenge iron from their host for growth and proliferation during infection. They have evolved several strategies to do this, one being the biosynthesis and excretion of small, high-affinity iron chelators known as siderophores. The biosynthesis of siderophores is an important area of study, not only for potential therapeutic intervention, but also to illuminate new enzyme chemistries. Two general pathways for siderophore biosynthesis exist: the well-characterized nonribosomal peptide synthetase (NRPS)-dependent pathway and the NRPS-independent (NIS) pathway, which relies on a different family of sparsely-investigated synthetases. Here, we report structural and biochemical studies of AcsD from Pectobacterium (formerly Erwinia) chrysanthemi, a NIS synthetase involved in achromobactin biosynthesis. The structures of ATP and citrate complexes provide a mechanistic rationale for stereospecific formation of an enzyme-bound (3R)-citryl-adenylate, which reacts with L-serine to form a likely achromobactin precursor. AcsD is a novel acyl adenylate-forming enzyme with a new fold and chemical catalysis strategy.

The Scottish Structural Proteomics Facility was funded to develop a laboratory scale approach to high throughput structure determination. The effort was successful in that over 40 structures were determined. These structures and the methods harnessed to obtain them are reported here. This report reflects on the value of automation but also on the continued requirement for a high degree of scientific and technical expertise. The efficiency of the process poses challenges to the current paradigm of structural analysis and publication. In the 5 year period we published ten peer-reviewed papers reporting structural data arising from the pipeline. Nevertheless, the number of structures solved exceeded our ability to analyse and publish each new finding. By reporting the experimental details and depositing the structures we hope to maximize the impact of the project by allowing others to follow up the relevant biology.Electronic supplementary materialThe online version of this article (doi:10.1007/s10969-010-9090-y) contains supplementary material, which is available to authorized users.

Siderophores are high-affinity ferric iron chelators biosynthesised and excreted by most microorganisms that play an important role in iron acquisition. Siderophore-mediated scavenging of ferric iron from hosts contributes significantly to the virulence of pathogenic microbes. As a consequence siderophore biosynthesis is an attractive target for chemotherapeutic intervention. Two main pathways for siderophore biosynthesis exist in microbes. One pathway involves nonribosomal peptide synthetase (NRPS) multienzymes while the other is NRPS-independent. The enzymology of NRPS-mediated siderophore biosynthesis has been extensively studied for more than a decade. In contrast, the enzymology of NRPS-independent siderophore (NIS) biosynthesis was overlooked for almost thirty years since the first genetic characterisation of the NIS biosynthetic pathway to aerobactin. However, the past three years have witnessed an explosion of interest in the enzymology of NIS synthetases, the key enzymes in the assembly of siderophores via the NIS pathway. The biochemical characterisation of ten purified recombinant synthetases has been reported since 2007, along with the first structural characterisation of a synthetase by X-ray crystallography in 2009. In this feature article we summarise the recent progress that has been made in understanding the long-overlooked enzymology of NRPS-independent siderophore biosynthesis, highlight important remaining questions, and suggest likely directions for future research.

Putrebactin is a dihydroxamate iron chelator produced by the metabolically versatile marine bacterium Shewanella putrefaciens. It is a macrocyclic dimer of N-hydroxy-N-succinyl-putrescine (HSP) and is structurally related to desferrioxamine E, which is a macrocyclic trimer of N-hydroxy-N-succinyl-cadaverine (HSC). We recently showed that DesD, a member of the NIS synthetase superfamily, catalyzes the key step in desferrioxamine E biosynthesis: ATP-dependent trimerisation and macrocylization of HSC. Here we report identification of a conserved gene cluster in the sequenced genomes of several Shewanella species, including Shewanella putrefaciens, which is hypothesized to direct putrebactin biosynthesis from putrescine, succinyl-CoA and molecular oxygen. The pubC gene within this gene cluster encodes a protein with 65% similarity to DesD. We overexpressed pubC from Shewanella species MR-4 and MR-7 in E. coli. The resulting His6-PubC fusion proteins were purified by Ni-NTA affinity and gel filtration chromatography. The recombinant proteins were shown to catalyze ATP-dependent cyclodimerization of HSP to form putrebactin. The uncyclized dimer of HSP pre-putrebactin was shown to be an intermediate in the conversion of two molecules of HSP to putrebactin. The data indicate that pre-putrebactin is converted to putrebactin via PubC-catalyzed activation of the carboxyl group by adenylation, followed by PubC-catalyzed nucleophilic attack of the amino group on the carbonyl carbon of the acyl adenylate. This mechanism for macrocycle formation is very different from the mechanism involved in the biosynthesis of many other macrocyclic natural products, where already-activated acyl thioesters are converted by thioesterase domains of polyketide synthases and nonribosomal peptide synthetases to macrocycles via covalent enzyme bound intermediates. The results of this study demonstrate that two closely related enzymes, PubC and DesD, catalyze specific cyclodimerization and cyclotrimerization reactions, respectively, of structurally similar substrates, raising intriguing questions regarding the molecular mechanism of specificity.

The bisucaberin biosynthetic gene cluster has been identified in Vibrio salmonicida and a domain from within the BibC multienzyme encoded by the cluster has been shown to catalyse ATP-dependent dimerisation and macrocyclisation of N-hydroxy-N-succinylcadaverine to form bisucaberin.

Hydroxamate groups play key roles in the biological function of diverse natural products. Important examples include trichostatin A, which inhibits histone deacetylases via coordination of the active site zinc(II) ion with a hydroxamate group, and the desferrioxamines, which use three hydroxamate groups to chelate ferric iron. Desferrioxamine biosynthesis in species involves the DesD-catalysed condensation of various-acylated derivatives of -hydroxycadaverine with two molecules of-succinyl--hydroxycadaverine to form a range of linear and macrocyclic tris-hydroxamates. However, the mechanism for assembly of the various -acyl--hydroxycadaverine substrates of DesD from -hydroxycadaverine has until now been unclear. Here we show that the gene of encodes the acyl transferase responsible for this process. DesC catalyses the-acylation of -hydroxycadaverine with acetyl, succinyl and myristoyl-CoA, accounting for the diverse array of desferrioxamines produced by The X-ray crystal structure of DesE, the ferrioxamine lipoprotein receptor, in complex with ferrioxamine B (which is derived from two units of -succinyl--hydroxycadaverine and one of -acetyl--hydroxycadaverine) was also determined. This showed that the acetyl group of ferrioxamine B is solvent exposed, suggesting that the corresponding acyl group in longer chain congeners can protrude from the binding pocket, providing insights into their likely function. This article is part of a discussion meeting issue 'Frontiers in epigenetic chemical biology'.This article is part of a discussion meeting issue 'Frontiers in epigenetic chemical biology'.

The AsbB enzyme, which is involved in the biosynthesis of the virulence-conferring siderophore petrobactin in Bacillus anthracis, is shown to catalyze efficient ATP-dependent condensation of spermidine, but not N1-(3,4-dihydroxbenzoyl)-spermidine, with N8-citryl-spermidine or N1-(3,4-dihydroxbenzoyl)-N8-citryl-spermidine, suggesting that N1-(3,4-dihydroxbenzoyl)-spermidine is very unlikely to be a significant intermediate in petrobactin biosynthesis, contrary to previous suggestions.