Dataset Information

Structural Analysis of Glutamine Synthetase from Helicobacter pylori.

ABSTRACT: Glutamine synthetase (GS) is an enzyme that regulates nitrogen metabolism and synthesizes glutamine via glutamate, ATP, and ammonia. GS is a homo-oligomeric protein of eight, ten, or twelve subunits, and each subunit-subunit interface has its own active site. GS can be divided into GS I, GS II, and GS III. GS I and GS III form dodecamer in bacteria and archaea, whereas GS II form decamer in eukaryotes. GS I can be further subdivided into GS I-? and GS I-? according to its sequence and regulatory mechanism. GS is an essential protein for the survival of Helicobacter pylori which its infection could promote gastroduodenal diseases. Here, we determined the crystal structures of the GS from H. pylori (Hpy GS) in its apo- and substrate-bound forms at 2.8?Å and 2.9?Å resolution, respectively. Hpy GS formed a dodecamer composed of two hexameric rings stacked face-to-face. Hpy GS, which belongs to GS I, cannot be clearly classified as either GS I-? or GS I-? based on its sequence and regulatory mechanism. In this study, we propose that Hpy GS could be classified as a new GS-I subfamily and provide structural information on the apo- and substrate-bound forms of the protein.

SUBMITTER: Joo HK

PROVIDER: S-EPMC6076278 | biostudies-literature | 2018 Aug

REPOSITORIES: biostudies-literature

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Structural Analysis of Glutamine Synthetase from Helicobacter pylori.

Joo Hyun Kyu HK Park Young Woo YW Jang Young Yoon YY Lee Jae Young JY

Scientific reports 20180803 1

Glutamine synthetase (GS) is an enzyme that regulates nitrogen metabolism and synthesizes glutamine via glutamate, ATP, and ammonia. GS is a homo-oligomeric protein of eight, ten, or twelve subunits, and each subunit-subunit interface has its own active site. GS can be divided into GS I, GS II, and GS III. GS I and GS III form dodecamer in bacteria and archaea, whereas GS II form decamer in eukaryotes. GS I can be further subdivided into GS I-α and GS I-β according to its sequence and regulatory ...[more]

PMID: 30076387

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Project description:Helicobacter pylori urease, produced in abundance, is indispensable for the survival of H. pylori in animal hosts. Urea is hydrolyzed by the enzyme, resulting in the liberation of excess ammonia, some of which neutralizes gastric acid. The remaining ammonia is assimilated into protein by glutamine synthetase (EC 6.3.1.2), which catalyzes the reaction: NH3 + glutamate + ATP-->glutamine + ADP + Pi. We hypothesized that glutamine synthetase plays an unusually critical role in nitrogen assimilation by H. pylori. We developed a phenotypic screen to isolate genes that contribute to the synthesis of a catalytically active urease. Escherichia coli SE5000 transformed with plasmid pHP808 containing the entire H. pylori urease gene cluster was cotransformed with a pBluescript plasmid library of the H. pylori ATCC 43504 genome. A weakly urease-positive 9.4-kb clone, pUEF728, was subjected to nucleotide sequencing. Among other genes, the gene for glutamine synthetase was identified. The complete 1,443-bp glnA gene predicts a polypeptide of 481 amino acid residues with a molecular weight of 54,317; this was supported by maxicell analysis of cloned glnA expressed in E. coli. The top 10 homologs were all bacterial glutamine synthetases, including Salmonella typhimurium glnA. The ATP-binding motif GDNGSG (residues 272 to 277) of H. pylori GlnA exactly matched and aligned with the sequence in 8 of the 10 homologs. The adenylation site found in the top 10 homologs (consensus sequence, NLYDLP) is replaced in H. pylori by NLFKLT (residues 405 to 410). Since the Tyr (Y) residue is the target of adenylation and since the H. pylori glutamine synthetase lacks that residue in four strains examined, we conclude that no adenylation occurs within this motif. Cloned H. pylori glnA complemented a glnA mutation in E. coli, and GlnA enzyme activity could be measured spectrophotometrically. In an attempt to produce a GlnA-deficient mutant of H. pylori, a kanamycin resistance cassette was cloned into the Tth111I site of H. pylori glnA. By using the standard technique of allelic exchange mutagenesis, no verifiable glutamine synthetase double-crossover mutant of strain UMAB41 could be isolated, suggesting that the mutation is lethal. We conclude that glutamine synthetase is critical for nitrogen assimilation in H. pylori and is active under all physiologic conditions.

Dataset Information

Structural Analysis of Glutamine Synthetase from Helicobacter pylori.

Publications

Structural Analysis of Glutamine Synthetase from Helicobacter pylori.

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