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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 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 GmhAGC crystallized 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.

SUBMITTER: Wierzbicki IH

PROVIDER: S-EPMC5387315 | biostudies-literature | 2017 Apr

REPOSITORIES: biostudies-literature

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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.

Wierzbicki Igor H IH Zielke Ryszard A RA Korotkov Konstantin V KV Sikora Aleksandra E AE

MicrobiologyOpen 20170107 2

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 GmhA<sub>GC</sub> and GmhA<sub>NM</sub> , respectively) were interchangeable proteins essential for lipooligosaccharide (LOS) synthesis, and their depletion had adverse effects on ...[more]

PMID: 28063198

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Project description:D-Glycero-d-manno-heptose-1,7-bisphosphate phosphatase (GmhB) is a member of the histidinol-phosphate phosphatase (HisB) subfamily of the haloalkanoic acid dehalogenase (HAD) enzyme superfamily. GmhB supports two divergent biochemical pathways in bacteria: the d-glycero-d-manno-heptose-1alpha-GDP pathway (in S-layer glycoprotein biosynthesis) and the l-glycero-d-manno-heptose-1beta-ADP pathway (in lipid A biosynthesis). Herein, we report the comparative analysis of substrate recognition in selected GmhB orthologs. The substrate specificity of the l-glycero-d-manno-heptose-1beta-ADP pathway GmhB from Escherichia coli K-12 was evaluated using hexose and heptose bisphosphates, histidinol phosphate, and common organophosphate metabolites. Only d-glycero-d-manno-heptose 1beta,7-bisphosphate (k(cat)/K(m) = 7 x 10(6) M(-1) s(-1)) and d-glycero-d-manno-heptose 1alpha,7-bisphosphate (k(cat)/K(m) = 7 x 10(4) M(-1) s(-1)) displayed physiologically significant substrate activity. (31)P NMR analysis demonstrated that E. coli GmhB selectively removes the C(7) phosphate. Steady-state kinetic inhibition studies showed that d-glycero-d-manno-heptose 1beta-phosphate (K(is) = 60 microM, and K(ii) = 150 microM) and histidinol phosphate (K(is) = 1 mM, and K(ii) = 6 mM), while not hydrolyzed, do in fact bind to E. coli GmhB, which leads to the conclusion that nonproductive binding contributes to substrate discrimination. High catalytic efficiency and a narrow substrate range are characteristic of a well-evolved metabolic enzyme, and as such, E. coli GmhB is set apart from most HAD phosphatases (which are typically inefficient and promiscuous). The specialization of the biochemical function of GmhB was examined by measuring the kinetic constants for hydrolysis of the alpha- and beta-anomers of d-glycero-d-manno-heptose 1beta,7-bisphosphate catalyzed by the GmhB orthologs of the l-glycero-d-manno-heptose 1beta-ADP pathways operative in Bordetella bronchiseptica and Mesorhizobium loti and by the GmhB of the d-glycero-d-manno-heptose 1alpha-GDP pathway operative in Bacteroides thetaiotaomicron. The results show that although each of these representatives possesses physiologically significant catalytic activity toward both anomers, each displays substantial anomeric specificity. Like E. coli GmhB, B. bronchiseptica GmhB and M. loti GmhB prefer the beta-anomer, whereas B. thetaiotaomicron GmhB is selective for the alpha-anomer. By determining the anomeric configuration of the physiological substrate (d-glycero-d-manno-heptose 1,7-bisphosphate) for each of the four GmhB orthologs, we discovered that the anomeric specificity of GmhB correlates with that of the pathway kinase. The conclusion drawn from this finding is that the evolution of the ancestor to GmhB in the HisB subfamily provided for specialization toward two distinct biochemical functions.

Dataset Information

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.

Publications

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.

Similar Datasets

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