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Exploitation of structural and regulatory diversity in glutamate racemases

Abstract

Glutamate racemase is an enzyme essential to the bacterial cell wall biosynthesis pathway, and has therefore been considered as a target for antibacterial drug discovery. We characterized the glutamate racemases of several pathogenic bacteria using structural and biochemical approaches. Here we describe three distinct mechanisms of regulation for the family of glutamate racemases: allosteric activation by metabolic precursors, kinetic regulation through substrate inhibition, and d-glutamate recycling using a d-amino acid transaminase. In a search for selective inhibitors, we identified a series of uncompetitive inhibitors specifically targeting Helicobacter pylori glutamate racemase that bind to a cryptic allosteric site, and used these inhibitors to probe the mechanistic and dynamic features of the enzyme. These structural, kinetic and mutational studies provide insight into the physiological regulation of these essential enzymes and provide a basis for designing narrow-spectrum antimicrobial agents.

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Figure 1: Crystal structures of MurI proteins.
Figure 2: Inhibitor characterization.
Figure 3: Inhibitor-binding site in H. pylori MurI.

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Acknowledgements

We thank M. Gowravaram for compound synthesis and characterization, and all members of the MurI Team and collaborators across AstraZeneca for support of this work. The atomic coordinates of the protein structures have been deposited in the European Bioinformatics Institute Macromolecular Structure Database: H. pylori MurI complexed with D-glutamate (Native I, accession code 2jfx; Native II, accession code 2jfy); H. pylori complex with D-glutamate and compound A, accession code 2jfz; E. coli MurI complexed with L-glutamate and UDP-MurNAc-Ala, accession code 2jfn; S. aureus MurI complexed with D-glutamate, accession code 2jfq; E. faecalis MurI complexed with D- or L-glutamate, accession code 2jfo; E. faecalis MurI complexed with D-glutamate, accession code 2jfp; E. faecium MurI complexed with phosphate, accession code 2jfu; E. faecium MurI complexed with citrate, accession code 2jfv; E. faecium MurI complexed with tartrate, accession code 2jfw.

Author Contributions S.L.F and T.L. contributed equally to this work. S.L.F., G.K., D.T.N. and T.A.K. performed the protein biochemical and kinetic characterizations, Y.X. solved the H. pylori MurI Native I structure and T.L. solved all other protein structures. R.H.A.F. performed the NMR studies, R.A.A. performed the phylogenetic, genomic and sequence analyses and B.L.M.dJ. performed the microbial physiology analyses. All authors discussed the results, contributed and commented on the manuscript.

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Correspondence to Stewart L. Fisher.

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Competing interests

The atomic coordinates of the protein structures have been deposited in the European Bioinformatics Institute Macromolecular Structure Database: H. pylori MurI complexed with d-glutamate (Native I, accession code 2jfx; Native II, accession code 2jfy); H. pylori complex with d-glutamate and compound A, accession code 2jfz; E. coli MurI complexed with l-glutamate and UDP-MurNAc-Ala, accession code 2jfn; S. aureus MurI complexed with d-glutamate, accession code 2jfq; E. faecalis MurI complexed with d- or l-glutamate, accession code 2jfo; E. faecalis MurI complexed with d-glutamate, accession code 2jfp; E. faecium MurI complexed with phosphate, accession code 2jfu; E. faecium MurI complexed with citrate, accession code 2jfv; E. faecium MurI complexed with tartrate, accession code 2jfw. Reprints and permissions information is available at www.nature.com/reprints. The authors declare no competing financial interests.

Supplementary information

Supplementary Information 1

This file contains Supplementary Methods, Supplementary Tables S1-S9, Supplementary Figures S1-S14 with Legends. (PDF 4935 kb)

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Lundqvist, T., Fisher, S., Kern, G. et al. Exploitation of structural and regulatory diversity in glutamate racemases. Nature 447, 817–822 (2007). https://doi.org/10.1038/nature05689

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