Functional and Structural Studies on the Neisseria gonorrhoeae GmhA, the First Enzyme in the glycero-manno-heptose Biosynthesis Pathways, Demonstrate a Critical Role in Lipooligosaccharide Synthesis and Gonococcal Viability
Sedoheptulose-7-phosphate isomerase, GmhA, is the first enzyme in the biosynthesis of nucleotide-activated-glycero-manno-heptoses and an attractive, yet underexploited, target for development of broad-spectrum antibiotics. We demonstrated that GmhA homologs in Neisseria gonorrhoeae and N. meningitidis (hereafter called GmhAGC and GmhANM, respectively) were interchangeable proteins essential for lipooligosaccharide (LOS) synthesis, and their depletion had adverse effects on neisserial viability. In contrast, the Escherichia coli ortholog failed to complement GmhAGC depletion. Furthermore, we showed that GmhAGC is a cytoplasmic enzyme with induced expression at mid-logarithmic phase, upon iron deprivation and anaerobiosis, and conserved in contemporary gonococcal clinical isolates including the 2016 WHO reference strains. The untagged GmhAGCcrystallized as a tetramer in the closed conformation with four zinc ions in the active site, supporting that this is most likely the catalytically active conformation of the enzyme. Finally, site-directed mutagenesis studies showed that the active site residues E65 and H183 were important for LOS synthesis but not for GmhAGC function in bacterial viability. Our studies bring insights into the importance and mechanism of action of GmhA and may ultimately facilitate targeting the enzyme with small molecule inhibitors.
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This research used resources of the Advanced Photon Source, a U.S. Department of Energy (DOE) Office of Science User Facility operated for the DOE Office of Science by Argonne National Laboratory under Contract No. DE-AC02-06CH11357.
We acknowledge the Protein Core at the Center for Molecular Medicine, University of Kentucky, that is supported by the National Institute of General Medical Sciences of the National Institutes of Health under grant number P30GM110787. Funding for this work was provided to AES by grant R01-AI117235 from the National Institute of Allergy and Infectious Diseases, National Institutes of Health, and was partially supported by an Institutional Development Award (IDeA) from the NIGMS/NIH under grant number P20GM103486 to KVK.
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Wierzbicki, Igor H.; Zielke, Ryszard A.; Korotkov, Konstantin V.; and Sikora, Aleksandra E., "Functional and Structural Studies on the Neisseria gonorrhoeae GmhA, the First Enzyme in the glycero-manno-heptose Biosynthesis Pathways, Demonstrate a Critical Role in Lipooligosaccharide Synthesis and Gonococcal Viability" (2017). Molecular and Cellular Biochemistry Faculty Publications. 118.