title: 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.
abstract: 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 GmhAGCand 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 GmhAGCdepletion. Furthermore, we showed that GmhAGCis 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 GmhAGCfunction 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.
authors.affiliation: Department of Pharmaceutical Sciences, College of Pharmacy, Oregon State University, Corvallis, OR, USA.
authors.forename: Igor H
authors.initials: IH
authors.lastname: Wierzbicki
authors.affiliation: Department of Pharmaceutical Sciences, College of Pharmacy, Oregon State University, Corvallis, OR, USA.
authors.forename: Ryszard A
authors.initials: RA
authors.lastname: Zielke
authors.affiliation: Department of Molecular & Cellular Biochemistry, College of Medicine, University of Kentucky, Lexington, KY, USA.
authors.forename: Konstantin V
authors.initials: KV
authors.lastname: Korotkov
authors.affiliation: Department of Pharmaceutical Sciences, College of Pharmacy, Oregon State University, Corvallis, OR, USA.
authors.forename: Aleksandra E
authors.identifier: 0000-0001-7977-2596
authors.initials: AE
authors.lastname: Sikora
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics / Q000648:ultrastructure*; D002238:Carbohydrate Epimerases / Q000235:genetics / Q000648:ultrastructure*; D020134:Catalytic Domain / Q000235:genetics; D006539:Heptoses / Q000096:biosynthesis*; D008070:Lipopolysaccharides / Q000096:biosynthesis*; D016297:Mutagenesis, Site-Directed; D009344:Neisseria gonorrhoeae / Q000201:enzymology* / Q000235:genetics; D009345:Neisseria meningitidis / Q000201:enzymology* / Q000235:genetics; D013403:Sugar Phosphates / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D006539:Heptoses; D008070:Lipopolysaccharides; D013403:Sugar Phosphates; C023023:lipid-linked oligosaccharides; C020495:sedoheptulose 7-phosphate; C026312:glycero-manno-heptose; D002238:Carbohydrate Epimerases; C000621162:sedoheptulose-7-phosphate isomerase, Neisseria gonorrhoeae
keywords: Neisseria gonorrhoeae; crystal structure; drug target; sedoheptulose-7-phosphate isomerase GmhA