Site-saturation mutagenesis of leucine 134 of Bacillus licheniformis nucleotide exchange factor GrpE reveals the importance of this residue to the co-chaperone activity.

To elucidate the role of leucine 134 of Bacillus licheniformis nucleotide exchange factor (BlGrpE), site-saturation mutagenesis was employed to generate all possible replacements for this residue. Wild-type and mutant proteins were purified by nickel-chelated chromatography and had a molecular mass of approximately 34.5 kDa. As compared with wild-type BlGrpE, the nucleotide exchange factor (NEF) activity of L134H, L134K, L134R, L134D, L134E, L134N, L134Q, L134S, L134G and L134P was reduced by more than 96%. In vitro binding assay revealed that wild-type BlGrpE and the functional variants mainly interacted with the monomer of BlDnaK, but no such interaction was observed for the remaining mutant proteins. BlGrpE and 9 mutant proteins synergistically stimulated the ATPase activity of B. licheniformis DnaK (BlDnaK), whereas the NEF-defective variants had no synergistic stimulation. Comparative analysis of the far-UV CD spectra showed that the alpha-helical content of the inactive mutant BlGrpEs was reduced significantly with respect to wild-type protein. Moreover, the inactive mutant proteins also exhibited a more sensitivity towards the temperature-induced denaturation. Taken together, these results indicate that Leu134 might play a structural role for the proper function of BlGrpE.

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 (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.

Significance of the conserved Tyr352 and Asp380 residues in the catalytic activity of Bacillus stearothermophilus aminopeptidase II as evaluated by site-directed mutagenesis.

The importance of the conserved Tyr352 and Asp380 residues of Bacillus stearothermophilus aminopeptidase II (AP-II) was investigated by site-directed mutagenesis. The wild-type and mutant enzymes were expressed in recombinant Escherichia coli M15 cells and the 45-kD proteins were purified from the cell-free extracts by Ni(2+)-NTA resin. The specific activity for Tyr352 and Asp380 replacements was decreased by more than 3.5-fold. Detailed analysis of the kinetic consequences in the mutant proteins revealed that the K (m) values were increased 1.9- to 2.6-fold with respect to wild-type enzyme. Catalytic efficiencies (k (cat)/K (m)) of mutant proteins were between 3.5- and 31-fold lower than the corresponding value of the wild-type enzyme. Tryptophan emission fluorescence and circular dichroism spectra were nearly identical for wild-type and mutant enzymes. These results indicate that residues Tyr352 and Asp380 are essential for the proper function of AP-II.

Histidines 345 and 378 of Bacillus stearothermophilus leucine aminopeptidase II are essential for the catalytic activity of the enzyme.

The conserved histidine residues, His-191, His-227, His-345, and His-378, in Bacillus stearothermophilus leucine aminopeptidase II (LAPII) were replaced with leucine by site-directed mutagenesis. The overexpressed wild-type and mutant enzymes have been purified by nickel-chelate chromatography and their molecular masses were approximately 44.5 kDa. Under assay conditions, no LAP activity was detected in H345L and H378L. Although the Km value for H191L increased more than 30% with respect to the wild-type LAPII, alteration in this residue did not lead to a significant change on the catalytic efficiency. The 39% decrease in Kcat/Km for H227L was partly caused by a 3.9-fold increase in Km value. Based on these results, it is suggested that His-345 and His-378 play a crucial role in the catalytic reaction of B. stearothermophilus LAPII.

Inactivation of Bacillus stearothermophilus leucine aminopeptidase II by hydrogen peroxide and site-directed mutagenesis of methionine residues on the enzyme.

Leucine aminopeptidases (LAPs) are exopeptidases that remove the N-terminal L-leucine from peptide substrates. Oxidative stability assay showed that the recombinant Bacillus stearothermophilus LAP II (rLAPII) was sensitive to oxidative damage by hydrogen peroxide at the elevated temperature. The H2O2-treated enzyme experienced obvious changes in the secondary structure when the oxidant concentration increased to 300 mM. To investigate the role of methionine residues on the oxidative inactivation, each of the five methionine residues in the rLAPII was replaced with leucine by site-directed mutagenesis. The mutant enzymes with an apparent Mr of approximately 44.5 kDa were overexpressed in Escherichia coli and were purified to homogeneity by nickel-chelate chromatography. The specific activities for Met82Leu, Met88Leu, Met254Leu, and Met382Leu were similar to that of the wild-type enzyme, whereas a reduced activity was observed in Met136Leu. The 50% decrease in the catalytic efficiency (kcat/Km) for Met136Leu was caused by 47% decrease in kcat value. As compared with the wild-type enzyme, all mutant proteins were more sensitive to the oxidant, implying that the methionine residues of B. stearothermophilus LAP II are important for the protection of the enzyme from oxidative inactivation.

Overexpression, purification, and characterization of the recombinant leucine aminopeptidase II of Bacillus stearothermophilus.

For expression of Bacillus stearothermophilus NCIB 8924 leucine aminopeptidase II (LAP II) in Escherichia coli regulated by a T5 promoter, the gene was amplified by polymerase chain reaction and cloned into expression vector pQE-32 to generate pQE-LAPII. The His(6)-tagged enzyme was overexpressed in IPTG-induced E. coli M15 (pQE-LAPII) as a soluble protein and was purified to homogeneity by nickel-chelate chromatography to a specific activity of 425 U/mg protein with a final yield of 76%. The subunit molecular mass of the purified protein was estimated to be 44.5 kDa by SDS-PAGE. The temperature and pH optima for the purified protein were 60 degrees C and 8.0, respectively. Under optimal condition, the purified enzyme showed a marked preference for Leu- p-nitroanilide, followed by Arg- and Lys-derivatives. The His(6)-tagged enzyme was stimulated by Co(2+) ions, but was strongly inhibited by Cu(2+) and Hg(2+) and by the chelating agents, DTT and EDTA. The EDTA-treated enzyme could be reactivated with Co(2+) ions, indicating that it is a cobalt-dependent exopeptidase. Taking the biochemical characteristics together, we found that the recombinant LAP II exhibits no important differences from those properties described for the native enzyme.