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Role of the conserved Thr399 and Thr417 residues of Bacillus licheniformis gamma-Glutamyltranspeptidase as evaluated by mutational analysis

Role of the conserved Thr399 and Thr417 residues of Bacillus licheniformis gamma-Glutamyltranspeptidase as evaluated by mutational analysis

Current Microbiology 59(2): 101-106

Role of the conserved Thr399 and Thr417 residues of Bacillus licheniformis gamma-glutamyltranspeptidase (BlGGT) was investigated by site-directed mutagenesis. Substitutions of Thr399 and Thr417 of BlGGT with Ser resulted in a dramatic reduction in enzymatic activity. A complete loss of the GGT activity was observed in T399A, T399C, T417A, and T417K mutant enzymes. Furthermore, mutations on these two residues impaired the capability of autocatalytic processing of the enzyme. In vitro maturation experiments showed that BlGGT mutant precursors, pro-T399S, pro-T417S, and pro-T417A, could precede a time-dependent autocatalytic process to generate the 44.9- and 21.7-kDa subunits; however, the processed T417A had no enzymatic activity. Measurement of intrinsic tryptophan fluorescence revealed alteration of the microenvironment of aromatic amino acid residues, while Far-UV circular dichroism spectra were nearly identical for wild-type and mutant enzymes. These results suggest that residues Thr399 and Thr417 are important for BlGGT in the enzymatic maturation and reaction.

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Accession: 055613540

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PMID: 19340483

DOI: 10.1007/s00284-009-9403-1

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