Abstract
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 siderophore (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, an 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)-citryladenylate, which reacts with L-serine to form a likely achromobactin precursor. AcsD is a unique acyladenylate-forming enzyme with a new fold and chemical catalysis strategy.
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Acknowledgements
We thank D. Expert (Université Paris 6) for kindly providing pL9G1 and P. Grice (University of Cambridge) for assistance with acquisition of 13C NMR of labeled and unlabeled N-citryl-L-serine. This work was supported by Biotechnology Biological Sciences Research Council (BBSRC) (grant reference BB/S/B14450) and the Scottish Funding Council (grant references SULSA and SSPF).
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S.S. purified, crystallized and determined structures of all co-complexes, developed fluorescence-based activity assay, made and assayed mutants, analyzed complexes and biochemical data and participated in the writing of the paper. N.K. cloned and overexpressed acsD in Escherichia coli, developed conditions for the purification and stabilization of recombinant AcsD, developed biochemical assays, isolated N-citryl-L-serine from incubations and structurally characterized it, carried out the experiments to determine the stereochemistry of the citric acid residue in N-citryl-L-serine and participated in data interpretation and writing of the paper. S.A.M. developed conditions for the purification of, stabilization of and crystallization of apo recombinant AcsD and refined the crystal structure of apo recombinant AcsD. L.S. acquired and assisted with the interpretation of spectroscopic data. D.O.-C. developed the procedure for determination of the stereochemistry of the citric acid residue in N-citryl-L-serine and participated in interpretation of the data. K.A.J. traced and refined the first model of the apo structure. M.O., H.L. and L.G.C. assisted with the structural biology. C.H.B. assisted in the mass spectrometric analyses. M.F.W. participated in analyzing data and writing the paper. G.L.C. participated in experiment design, data interpretation and writing of the paper. J.H.N. participated in experiment design, data interpretation and writing of the paper.
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Schmelz, S., Kadi, N., McMahon, S. et al. AcsD catalyzes enantioselective citrate desymmetrization in siderophore biosynthesis. Nat Chem Biol 5, 174–182 (2009). https://doi.org/10.1038/nchembio.145
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DOI: https://doi.org/10.1038/nchembio.145
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