Crystal Structure of Acivicin-Inhibited γ-Glutamyltranspeptidase Reveals Critical Roles for Its C-Terminus in Autoprocessing and Catalysis

Abstract

Helicobacter pylori γ-glutamyltranspeptidase (HpGT) is a general γ-glutamyl hydrolase and a demonstrated virulence factor. The enzyme confers a growth advantage to the bacterium, providing essential amino acid precursors by initiating the degradation of extracellular glutathione and glutamine. HpGT is a member of the N-terminal nucleophile (Ntn) hydrolase superfamily and undergoes autoprocessing to generate the active form of the enzyme. Acivicin is a widely used γ-glutamyltranspeptidase inhibitor that covalently modifies the enzyme, but its precise mechanism of action remains unclear. The time-dependent inactivation of HpGT exhibits a hyperbolic dependence on acivicin concentration with k max = 0.033 ± 0.006 sec −1 and K I = 19.7 ± 7.2 μM. Structure determination of acivicin-modified HpGT (1.7 Å; R factor =17.9%; R free =20.8%) demonstrates that acivicin is accommodated within the γ-glutamyl binding pocket of the enzyme. The hydroxyl group of Thr 380, the catalytic nucleophile in the autoprocessing and enzymatic reactions, displaces chloride from the acivicin ring to form the covalently linked complex. Within the acivicin-modified HpGT structure, the C-terminus of the protein becomes ordered with Phe 567 positioned over the active site. Substitution or deletion of Phe 567 leads to a >10-fold reduction in enzymatic activity, underscoring its importance in catalysis. The mobile C-terminus is positioned by several electrostatic interactions within the C-terminal region, most notably a salt bridge between Arg 475 and Glu 566. Mutational analysis reveals that Arg 475 is critical for the proper placement of the C-terminal region, the Tyr 433 containing loop, and the proposed oxyanion hole. Keywordsγ-glutamyltranspeptidase; Ntn-hydrolase; glutathione; acivicin; x-ray crystallography H. pylori γ-glutamyltranspeptidase (HpGT) is a γ-glutamyl hydrolase with broad substrate specificity (1,2), and is a member of the N-terminal nucleophile (Ntn) hydrolase superfamily (3,4). The inactive precursor undergoes an intramolecular autoprocessing event, generating the mature and catalytically active heterotetramer. A conserved threonine residue, Thr 380, serves as the N-terminal nucleophile and is required for both maturation and enzymatic activity (1).HpGT has been shown to degrade extracellular glutathione and glutamine, providing a growth advantage to the bacterium within its microenvironment (2,5,6). Similarly, upregulation of human γ-glutamyltranspeptidase in cancer is thought to help supplement these rapidly dividing cells with essential amino acid precursors for glutathione and protein biosynthesis (7,8). In mammalian systems, γ-glutamyltranspeptidase has been shown to be critical for the transport of cysteine for use in protein and glutathione biosynthesis (9,10). The enzyme is required for normal glutathione metabolism, initiating extracellular glutathione degradation. Subsequent steps lead to the cellular uptake of the composite amino acids of glutathione: glutamate, cysteine, and glycine (11,12).Acivicin is a com...

Biophysical characterization of Bacillus licheniformis and Escherichia coli γ-glutamyltranspeptidases: A comparative analysis

“…The structure of acyl-enzyme intermediate of EcGGT confirms the functional role of Thr391 and the location of the donor substrate-binding site [8]. Further studies on these two enzymes have provided the structural evidence for autocatalytic processing [9,10] and critical roles of the C-terminal region in autoprocessing and catalysis [11].…”

Structure ofBacillus subtilisγ-glutamyltranspeptidase in complex with acivicin: diversity of the binding mode of a classical and electrophilic active-site-directed glutamate analogue

“…(a) The structure of acivicin. Previously reported acivicin-binding configurations in (b) E. coli GGT at 1.65 Å resolution (Wada et al, 2008) and (c) H. pylori GGT at 1.70 Å (Williams et al, 2009). An OMIT F o À F c map for the acivicin adduct contoured at 2.0 (blue) is overlaid on its stick model of each GGT and bound acivicin.…”

“…However, in a subsequent study with Helicobacter pylori GGT, acivicin was reported to bind to the catalytic Thr380 through the C3 atom but with a planar and perhaps sp 2 hybridization ( Fig. 1c; Williams et al, 2009). This is apparently the result of a simple and conventional nucleophilic substitution of Cl at the imidoyl C atom by Thr380 O .…”

“…human, pig and rat GGTs share $80% sequence identity), but in contrast bacterial GGTs share limited sequence homology ($30%) to one another and have insertion/deletion regions in their sequence. However, the residues involved in the catalysis, substrate/inhibitor recognition and binding of the -glutamyl moiety are highly conserved among bacterial GGT orthologues (Okada et al, 2006Boanca et al, 2007;Wada et al, 2008Wada et al, , 2010Williams et al, 2009). Hence, the catalytic/inhibitor-binding reactions by bacterial GGTs are believed to proceed with a common mechanism.…”

The N-terminal Nucleophile Serine of Cephalosporin Acylase Executes the Second Autoproteolytic Cleavage and Acylpeptide Hydrolysis

“…Ntn hydrolases feature an ␣␤␤␣ sandwich and an N-terminal, catalytically active, nucleophile that is released by the first proteolytic step (5)(6)(7). Examples of Ntn hydrolases are glutamine 5-phosphoribosyl-1-pyrophosphate amidotransferase (8), proteasome ␤-subunits (9), penicillin acylases (7), glucosamine-6-phosphate synthase (10), isoaspartyl aminopeptidase (11), taspase 1 (12), glycosylasparaginase (GA) (13,14), the human asparaginase-like protein 1 (hASRGL1) (15), Helicobacter pylori ␥-glutamyltranspeptidase (16), N-acylhomoserine lactone acylase PvdQ (PvdQ) (17), and cephalosporin acylases (18) whose crystal structures are available.…”