Biochemical and structural properties of gamma-glutamyl transpeptidase from Geobacillus thermodenitrificans: an enzyme specialized in hydrolase activity.
Gamma-glutamyltranspeptidases (gamma-GTs) catalyze the transfer of the gamma-glutamyl moiety of glutathione and related gamma-glutamyl amides to water (hydrolysis) or to amino acids and peptides (transpeptidation) and play a key role in glutathione metabolism. Recently, gamma-GTs have been considered attractive pharmaceutical targets for cancer and useful tools to produce gamma-glutamyl compounds. To find out gamma-GTs with special properties we have chosen microorganisms belonging to Geobacillus species which are source of several thermostable enzymes of potential interest for biotechnology. gamma-GT from Geobacillus thermodenitrificans (GthGT) was cloned, expressed in Escherichia coli, purified to homogeneity and characterized. The enzyme, synthesized as a precursor homotetrameric protein of 61-kDa per subunit, undergoes an internal post-translational cleavage of the 61 kDa monomer into 40- and 21-kDa shorter subunits, which are then assembled into an active heterotetramer composed of two 40- and two 21-kDa subunits. The kinetic characterization of the hydrolysis reaction using L-glutamic acid gamma-(4-nitroanilide) as the substrate reveals that the active enzyme has K(m) 7.6 microM and V(max) 0.36 micromol min/mg. The optimum pH and temperature for the hydrolysis activity are 7.8 and 52 degrees C, respectively. GthGT hydrolyses the physiological antioxidant glutathione, suggesting an involvement of the enzyme in the cellular defense mechanism against oxidative stress. Unlike other gamma-GTs, the mutation of the highly conserved catalytic nucleophile, Thr353, abolishes the post-translational cleavage of the pro-enzyme, but does not completely block the hydrolytic action. Furthermore, GthGT does not show any transpeptidase activity, suggesting that the enzyme is a specialized gamma-glutamyl hydrolase. The GthGT homology-model structure reveals peculiar structural features, which should be responsible for the different functional properties of the enzyme and suggests the structural bases of protein thermostability.
CNR, Institute of Protein Biochemistry, via Pietro Castellino 111, I-80131 Naples, Italy. i.castellano@ibp.cnr.it
Immacolata
I
Castellano
Antonello
A
Merlino
Mosè
M
Rossi
Francesco
F
La Cara
D000595:Amino Acid Sequence; D001483:Base Sequence; D055162:Biocatalysis; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D056506:Geobacillus / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed; D017386:Sequence Homology, Amino Acid; D013696:Temperature; D011623:gamma-Glutamyl Hydrolase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D005723:gamma-Glutamyltransferase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D017931:DNA Primers; D005723:gamma-Glutamyltransferase; D011623:gamma-Glutamyl Hydrolase