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Staphylopine and pseudopaline dehydrogenase from bacterial pathogens catalyze reversible reactions and produce stereospecific metallophores
McFarlane, Jeffrey S. ; Zhang, Jian ; Wang, Sanshan ; Lei, Xiaoguang ; Moran, Graham R. ; Lamb, Audrey L.
McFarlane, Jeffrey S.
Zhang, Jian
Wang, Sanshan
Lei, Xiaoguang
Moran, Graham R.
Lamb, Audrey L.
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Abstract
Pseudopaline and staphylopine are opine metallophores biosynthesized by Pseudomonas aeruginosa and Staphylococcus aureus, respectively. The final step in opine metallophore biosynthesis is the condensation of the product of a nicotianamine (NA) synthase reaction (i.e. l-HisNA for pseudopaline and d-HisNA for staphylopine) with an α-keto acid (α-ketoglutarate for pseudopaline and pyruvate for staphylopine), which is performed by an opine dehydrogenase. We hypothesized that the opine dehydrogenase reaction would be reversible only for the opine metallophore product with (R)-stereochemistry at carbon C2 of the α-keto acid (prochiral prior to catalysis). A kinetic analysis using stopped-flow spectrometry with (R)- or (S)-staphylopine and kinetic and structural analysis with (R)- and (S)-pseudopaline confirmed catalysis in the reverse direction for only (R)-staphylopine and (R)-pseudopaline, verifying the stereochemistry of these two opine metallophores. Structural analysis at 1.57–1.85 Å resolution captured the hydrolysis of (R)-pseudopaline and allowed identification of a binding pocket for the l-histidine moiety of pseudopaline formed through a repositioning of Phe-340 and Tyr-289 during the catalytic cycle. Transient-state kinetic analysis revealed an ordered release of NADP+ followed by staphylopine, with staphylopine release being the rate-limiting step in catalysis. Knowledge of the stereochemistry for opine metallophores has implications for future studies involving kinetic analysis, as well as opine metallophore transport, metal coordination, and the generation of chiral amines for pharmaceutical development.
Description
This research was originally published in the Journal of Biological Chemistry. McFarlane, Jeffrey S. et al. Staphylopine and pseudopaline dehydrogenase from bacterial pathogens catalyze reversible reactions and produce stereospecific metallophores. J Biol Chem. 2019; Vol294:17988-18001. © the American Society for Biochemistry and Molecular Biology.
This work is licensed under a Creative Commons Attribution 4.0 International License.
Date
2019-11-22
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American Society for Biochemistry and Molecular Biology
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Keywords
Pseudomonas aeruginosa (P. aeruginosa), Staphylococcus aureus (S. aureus), Dehydrogenase, Siderophore, Kinetics, Crystal structure, Metallophore, Opine, Pseudopaline, Staphylopine
Citation
McFarlane, Jeffrey S. et al., Staphylopine and pseudopaline dehydrogenase from bacterial pathogens catalyze reversible reactions and produce stereospecific metallophores, Journal of Biological Chemistry, Volume 294, Issue 47, 17988 - 18001