Nadia Kadi

AcsD catalyzes enantioselective citrate desymmetrization in siderophore biosynthesis

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.

Identification of a gene cluster that directs putrebactin biosynthesis in Shewanella species: PubC catalyzes cyclodimerization of N-hydroxy-N-succinylputrescine.

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.

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.

Chapter 17 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.

Petrobactin biosynthesis: AsbB catalyzes condensation of spermidine with N8-citryl-spermidine and its N1-(3,4-dihydroxybenzoyl) derivative

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.

Structural Basis for Acyl Acceptor Specificity in the Achromobactin Biosynthetic Enzyme AcsD

Siderophores are known virulence factors, and their biosynthesis is a target for new antibacterial agents. A non-ribosomal peptide synthetase-independent siderophore biosynthetic pathway in Dickeya dadantii is responsible for production of the siderophore achromobactin. The D. dadantii achromobactin biosynthesis protein D (AcsD) enzyme has been shown to enantioselectively esterify citric acid with l-serine in the first committed step of achromobactin biosynthesis. The reaction occurs in two steps: stereospecific activation of citric acid by adenylation, followed by attack of the enzyme-bound citryl adenylate by l-serine to produce the homochiral ester. We now report a detailed characterization of the substrate profile and mechanism of the second (acyl transfer) step of AcsD enzyme. We demonstrate that the enzyme catalyzes formation of not only esters but also amides from the citryl-adenylate intermediate. We have rationalized the substrate utilization profile for the acylation reaction by determining the first X-ray crystal structure of a product complex for this enzyme class. We have identified the residues that are important for both recognition of l-serine and catalysis of ester formation. Our hypotheses were tested by biochemical analysis of various mutants, one of which shows a reversal of specificity from the wild type with respect to non-natural substrates. This change can be rationalized on the basis of our structural data. That this change in specificity is accompanied by no loss in activity suggests that AcsD and other members of the non-ribosomal peptide synthetase-independent siderophore superfamily may have biotransformation potential.

Purification, crystallization and data collection of Pectobacterium chrysanthemi AcsD, a type A siderophore synthetase

AcsD, a type A siderophore synthetase with a molecular weight of 71 140 Da from Pectobacterium chrysanthemi, has been expressed, purified and crystallized at 293 K. The protein crystallized in the primitive orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 80.3, b = 95.7, c = 161.1 A, alpha = beta = gamma = 90 degrees . Systematic absences were consistent with space group P2(1)2(1)2(1). A complete data set has been collected to 2.25 A resolution on BM14 at the ESRF. Consideration of the likely solvent content suggested that the asymmetric unit contained two molecules. Gel-filtration experiments indicated that the protein was a dimer, although self-rotation analyses did not detect a convincing twofold symmetry axis in the asymmetric unit. The protein has no convincing sequence match to any known structure and thus solution is likely to require experimental phasing.