An acyl-adenylate mimic reveals the structural basis for substrate recognition by the iterative siderophore synthetase DesD

Yang, Jinping; Banas, Victoria S.; Patel, Ketan D.; Rivera, Gerry S. M.; Mydy, Lisa S.; Gulick, Andrew M.; Wencewicz, Timothy A.

doi:10.1016/j.jbc.2022.102166

Cited by 5 publications

(10 citation statements)

References 71 publications

(155 reference statements)

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“…The NIS biosynthesis pathway for schizokinen involves the proteins IucA and IucC ( Figure 1 A). IucA catalyses the attachment of the first N-acetyl-N-hydroxylysine to a carboxylic group of citric acid to yield N-citryl-N-acetyl-N-hydroxylysine, whereas IucC catalyses the attachment of the second N-acetyl-N-hydroxylysine to the carboxylic group of N-citryl-N-acetyl-N-hydroxylysine to yield schizokinen [ 37 ] ( Figure 1 A).…”

Section: Resultsmentioning

confidence: 99%

A Four-Step Platform to Optimize Growth Conditions for High-Yield Production of Siderophores in Cyanobacteria

et al. 2023

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In response to Iron deprivation and in specific environmental conditions, the cyanobacteria Anabaena flos aquae produce siderophores, iron-chelating molecules that in virtue of their interesting environmental and clinical applications, are recently gaining the interest of the pharmaceutical industry. Yields of siderophore recovery from in vitro producing cyanobacterial cultures are, unfortunately, very low and reach most of the times only analytical quantities. We here propose a four-step experimental pipeline for a rapid and inexpensive identification and optimization of growth parameters influencing, at the transcriptional level, siderophore production in Anabaena flos aquae. The four-steps pipeline consists of: (1) identification of the promoter region of the operon of interest in the genome of Anabaena flos aquae; (2) cloning of the promoter in a recombinant DNA vector, upstream the cDNA coding for the Green Fluorescent Protein (GFP) followed by its stable transformation in Escherichia Coli; (3) identification of the environmental parameters affecting expression of the gene in Escherichia coli and their application to the cultivation of the Anabaena strain; (4) identification of siderophores by the combined use of high-resolution tandem mass spectrometry and molecular networking. This multidisciplinary, sustainable, and green pipeline is amenable to automation and is virtually applicable to any cyanobacteria, or more in general, to any microorganisms.

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Section: Resultsmentioning

confidence: 99%

A Four-Step Platform to Optimize Growth Conditions for High-Yield Production of Siderophores in Cyanobacteria

et al. 2023

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“…Most siderophores are peptide-based structures assembled via two primary biosynthetic enzyme types: (1) nonribosomal peptide synthetases (NRPSs); (2) NRPS-independent siderophore (NISs) synthetases. NRPS and NIS siderophore biosynthetic pathways have been targeted for inhibition by small molecules and antibodies as antivirulence agents. − Detailed characterization of siderophore biosynthesis and utilization in target pathogens is needed to guide the development of inhibitors, vaccines, and drug delivery systems as therapeutic strategies.…”

Section: Introductionmentioning

confidence: 99%

In Vitro Reconstitution of Fimsbactin Biosynthesis from Acinetobacter baumannii

Yang

Wencewicz

2022

ACS Chem. Biol.

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Siderophores produced via nonribosomal peptide synthetase (NRPS) pathways serve as critical virulence factors for many pathogenic bacteria. Improved knowledge of siderophore biosynthesis guides the development of inhibitors, vaccines, and other therapeutic strategies. Fimsbactin A is a mixed ligand siderophore derived from human pathogenic Acinetobacter baumannii that contains phenolate−oxazoline, catechol, and hydroxamate metal chelating groups branching from a central L-Ser tetrahedral unit via amide and ester linkages. Fimsbactin A is derived from two molecules of L-Ser, two molecules of 2,3dihydroxybenzoic acid (DHB), and one molecule of L-Orn and is a product of the f bs biosynthetic operon. Here, we report the complete in vitro reconstitution of fimsbactin A biosynthesis in a cell-free system using purified enzymes. We demonstrate the conversion of L-Orn to N 1 -acetyl-N 1 -hydroxy-putrescine (ahPutr) via ordered action of FbsJ (decarboxylase), FbsI (flavin Nmonooxygenase), and FbsK (N-acetyltransferase). We achieve conversion of L-Ser, DHB, and L-Orn to fimsbactin A using FbsIJK in combination with the NRPS modules FbsEFGH. We also demonstrate chemoenzymatic conversion of synthetic ahPutr to fimsbactin A using FbsEFGH and establish the substrate selectivity for the NRPS adenylation domains in FbsH (DHB) and FbsF (L-Ser). We assign a role for the type II thioesterase FbsM in producing the shunt metabolite 2-(2,3-dihydroxyphenyl)-4,5-dihydrooxazole-4carboxylic acid (DHB-oxa) via cleavage of the corresponding thioester intermediate that is tethered to NRPS peptidyl carrier domains during biosynthetic assembly. We propose a mechanism for branching NRPS-derived peptides via amide and ester linkages via the dynamic equilibration of N-DHB-Ser and O-DHB-Ser thioester intermediates via hydrolysis of DHB-oxa thioester intermediates. We also propose a genetic signature for NRPS "branching" in the presence of a terminating C-T-C motif (FbsG).

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“…The iterative enzymes tend to display broader substrate specificity because of efficient recognition of progressively larger substrates [ 21 , 22 ], and they therefore show greater potential for developing chemotherapeutic agents. A scarcity of basic biochemical research has hampered drug design in this enzyme family, but recent crystal structures and assay developments have jump-started the field [ 23 , 24 , 25 , 26 , 27 , 28 , 29 , 30 , 31 , 32 ].…”

Section: Introductionmentioning

confidence: 99%

Chemoenzymatic Synthesis of Select Intermediates and Natural Products of the Desferrioxamine E Siderophore Pathway

et al. 2022

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The NIS synthetase family of enzymes responsible for the biosynthesis of siderophores is increasingly associated with bacterial virulence. Proteins in this class represent outstanding potential drug targets, assuming that basic biochemical and structural characterizations can be completed. Towards this goal, we have mated an improved synthesis of the non-commercial amino acid N-hydroxy-N-succinylcadaverine (HSC, 6) with an isothermal titration calorimetry (ITC) assay that profiles the iterative stages of HSC trimerization and macrocyclization by NIS synthetase DesD from Streptomyces coelicolor. HSC synthesis begins with multigram-scale Gabrielle and tert-butyl N-(benzyloxy)carbamate alkylations of 1-bromo-5-chloropentane following prior literature, but the end-game reported herein has two advantages for greater material throughput: (1) hydrogenolysis of benzyl ether and Cbz blocking groups is best accomplished with Pearlman’s catalyst at 40 psi of H2 and (2) purification of neutral (zwitterionic) HSC is effected by simple flash chromatography over silica gel in MeOH. HSC is subsequently shown to be a substrate for NIS synthetase DesD, which catalyzes three successive amide bond syntheses via adenyl monophosphate ester intermediates. We quantify and present the iterative and overall enzyme kinetic constants associated with formation of the cyclotrimeric siderophore desferrioxamine E (dfoE, 1).

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An acyl-adenylate mimic reveals the structural basis for substrate recognition by the iterative siderophore synthetase DesD

Cited by 5 publications

References 71 publications

A Four-Step Platform to Optimize Growth Conditions for High-Yield Production of Siderophores in Cyanobacteria

A Four-Step Platform to Optimize Growth Conditions for High-Yield Production of Siderophores in Cyanobacteria

In Vitro Reconstitution of Fimsbactin Biosynthesis from Acinetobacter baumannii

Chemoenzymatic Synthesis of Select Intermediates and Natural Products of the Desferrioxamine E Siderophore Pathway

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