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Desferrioxamine siderophores are assembled by the nonribosomal-peptide-synthetase-independent-siderophore (NIS) synthetase enzyme DesD via ATP-dependent iterative condensation of three N1-hydroxy-N1-succinyl-cadaverine (HSC) units. Current knowledge of NIS enzymology and the desferrioxamine bio-synthetic pathway does not account for the existence of most known members of this natural product family which differ in substitution patterns of the N- and C-termini. The directionality of desferrioxamine biosyn-thetic assembly, N-to-C vs C-to-N, is a longstanding knowledge gap that is limiting further progress in un-derstanding the origins of natural products in this structural family. Here, we establish the directionality of desferrioxamine biosynthesis using a chemoenzymatic approach with stable isotope incorporation and di-meric substrates. We propose a mechanism where DesD catalyzes the N-to-C condensation of HSC units to establish a unifying biosynthetic paradigm for desferrioxamine natural products in Streptomyces.
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Chemoenzymatic Synthesis of Select Intermediates and Natural Products of the Desferrioxamine E Siderophore Pathway
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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- Award ID(s):
- 1716986
- PAR ID:
- 10382422
- Date Published:
- Journal Name:
- Molecules
- Volume:
- 27
- Issue:
- 19
- ISSN:
- 1420-3049
- Page Range / eLocation ID:
- 6144
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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- Free Publicly Accessible Full Text
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Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.3390/molecules27196144
