29 research outputs found
Desferrioxamine biosynthesis : diverse hydroxamate assembly by substrate-tolerant acyl transferase DesC
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 Streptomyces species involves the DesD-catalysed condensation of various N-acylated derivatives of N-hydroxycadaverine with two molecules of N-succinyl-N-hydroxycadaverine to form a range of linear and macrocyclic tris-hydroxamates. However, the mechanism for assembly of the various N-acyl-N-hydroxycadaverine substrates of DesD from N-hydroxycadaverine has until now been unclear. Here we show that the desC gene of Streptomyces coelicolor encodes the acyl transferase responsible for this process. DesC catalyses the N-acylation of N-hydroxycadaverine with acetyl, succinyl and myristoyl-CoA, accounting for the diverse array of desferrioxamines produced by S. coelicolor. The X-ray crystal structure of DesE, the ferrioxamine lipoprotein receptor, in complex with ferrioxamine B (which is derived from two units of N-succinyl-N-hydroxycadaverine and one of N-acetyl-N-hydroxycadaverine) was also determined. This shows 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 functio
Synthèse enzymatique de glycosides de composés d'arôme
MONTPELLIER-BU Sciences (341722106) / SudocSudocFranceF
Transglycosylation reaction of endoxylanase from Trichoderma longibrachiatum
The study of hydrolytic activities of several enzymatic preparations showed that the Glucanase GL-200 and Xylanase XL-200 enzymatic preparations from Trichoderma longibrachiatum and the Xylanase from Trichoderma viride, possessed an endoxylanase activity useful for the transfer reaction. The enzymatic synthesis of hexylxyloside and hexylxylobioside were achieved by xylose transfer, catalysed by T longibrachiatum XL-200 xylanase crude preparation, from xylan (donor) to hexanol, with (50%, v:v) or without n-hexane used as co-solvent. Benzyl alcohol was also used as acceptor for the synthesis reaction of benzylxyloside, benzylxylobioside, and benzylxylotrioside, with the T longibrachiatum XL-200 xylanase crude preparation and partially pure T longibrachiatum endoxylanase. The transfer reaction due to the T longibrachiatum endoxylanase was confirmed by the enzymatic synthesis, catalysed by T longibrachiattum partially pure endoxylanase, of phenyl primeveroside performed from phenyl glucoside and xylan, in the presence of n-hexane. We showed that the T longibrachiatum endoxylanase was a good tool for the synthesis of xylosyl derivatives (homo- and hetero-xylosides), by transfer reaction. (C) 2007 Elsevier Ltd. All rights reserved
Siderophore biosynthesis: a substrate specificity assay for nonribosomal peptide synthetase-independent siderophore synthetases involving trapping of acyl-adenylate intermediates with hydroxylamine
Siderophores are an important group of structurally diverse natural products that play key roles in ferric iron acquisition in most microorganisms. Two major pathways exist for siderophore biosynthesis. One is dependent on nonribosomal peptide synthetase (NRPS) multienzymes. The enzymology of several NRPS-dependent pathways to structurally diverse siderophores has been intensively studied for more than 10 years and is generally well understood. The other major pathway is NRPS-independent. It relies on a novel family of synthetase enzymes that until recently has received very little attention. Over the last 2 years, these enzymes have begun to be intensively investigated and several examples have now been characterized. In this article, we give an overview of the enzymology of NRPS-dependent and NRPS-independent pathways for siderophore biosynthesis, using selected examples to highlight key features.
An important facet of many studies of the enzymology of siderophore biosynthesis has been to investigate the substrate specificity of the synthetase enzymes involved. For NRPS-dependent pathways, the ATP-pyrophophate exchange assay has been widely used to investigate the substrate specificity of adenylation domains within the synthetase multienzymes. This assay is ineffective for NRPS-independent siderophore (NIS) synthetases, probably because pyrophosphate is not released from the enzyme after the carboxylic acid substrate and ATP react to form an acyl adenylate. An alternative assay for enzymes that form acyl adenylates involves trapping of the activated carboxyl group with hydroxylamine to form a hydroxamic acid that can be converted to its ferric complex and detected spectrophotometrically. This assay has not been widely used for NRPS adenylation domains. Here, we show that it is an effective assay for examining the carboxylic acid substrate specificity of NIS synthetases. Application of the assay to the type B NIS synthetase AcsA shows that it is selective for alpha-ketoglutaric acid, confirming a bioinformatics-based prediction of the substrate specificity of this enzyme
Bisucaberin biosynthesis : an adenylating domain of the BibC multi-enzyme catalyzes cyclodimerization of N-hydroxy-N-succinylcadaverine
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
The long-overlooked enzymology of a nonribosomal peptide synthetase-independent pathway for virulence-conferring siderophore biosynthesis
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
Enzymatic Synthesis of Aroma Compound Xylosides Using Transfer Reaction by Trichoderma longibrachiatum
Erratum : a new family of ATP-dependent oligomerization-macrocyclization biocatalysts
In the version of this article initially published, an extra methyl group was inadvertently added to the structure of desferrioxamine G1 and related compounds. The error has been corrected in the HTML and PDF versions of the article
Identification of a gene cluster that directs putrebactin biosynthesis in Shewanella species : PubC catalyzes cyclodimerization of N-hydroxy-N-succinyiputrescine
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 similar to 65% similarity to DesD. We overexpressed pubC from Shewanella species MR-4 and MR-7 in E. coli. The resulting His(6)-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