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Crystal Structure of Helicobacter Pylori Gamma-Glutamyltranspeptidase T380A Mutant
Structural highlights
FunctionEvolutionary Conservation Check, as determined by ConSurfDB. You may read the explanation of the method and the full data available from ConSurf. Publication Abstract from PubMedHelicobacter pylori gamma-glutamyltranspeptidase (HpGT) is a member of the N-terminal nucleophile hydrolase superfamily. It is translated as an inactive 60 kDa polypeptide precursor that undergoes intramolecular autocatalytic cleavage to generate a fully active heterodimer composed of a 40 kDa and a 20 kDa subunit. The resultant N-terminus, Thr 380, has been shown to be the catalytic nucleophile in both autoprocessing and enzymatic reactions. Once processed, HpGT catalyzes the hydrolysis of the gamma-glutamyl bond in glutathione and its conjugates. To facilitate the determination of physiologically relevant substrates for the enzyme, crystal structures of HpGT in complex with glutamate (1.6 A, Rfactor = 16.7%, Rfree = 19.0%) and an inactive HpGT mutant, T380A, in complex with S-(nitrobenzyl)glutathione (1.55 A, Rfactor = 18.7%, Rfree = 21.8%) have been determined. Residues that comprise the gamma-glutamyl binding site are primarily located in the 20 kDa subunit and make numerous hydrogen bonds with the alpha-amino and alpha-carboxylate groups of the substrate. In contrast, a single hydrogen bond occurs between the T380A mutant and the remainder of the ligand. Lack of specific coordination beyond the gamma-glutamyl moiety may account for the substrate binding permissiveness of the enzyme. Structural analysis was combined with site-directed mutagenesis of residues involved in maintaining the conformation of a loop region that covers the gamma-glutamyl binding site. Results provide evidence that access to this buried site may occur through conformational changes in the Tyr 433-containing loop, as disruption of the intricate hydrogen-bond network responsible for optimal placement of Tyr 433 significantly diminishes catalytic activity. Characterization of Helicobacter pylori gamma-glutamyltranspeptidase reveals the molecular basis for substrate specificity and a critical role for the tyrosine 433-containing loop in catalysis.,Morrow AL, Williams K, Sand A, Boanca G, Barycki JJ Biochemistry. 2007 Nov 20;46(46):13407-14. Epub 2007 Oct 26. PMID:17960917[1] From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. References
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