Enhancement of the enzymatic activity of ribonuclease HI from Thermus thermophilus HB8 with a suppressor mutation method.
A genetic method for isolating a mutant enzyme of ribonuclease HI (RNase HI) from Thermus thermophilus HB8 with enhanced activity at moderate temperatures was developed. T. thermophilus RNase HI has an ability to complement the RNase H-dependent temperature-sensitive (ts) growth phenotype of Escherichia coli MIC3001. However, this complementation ability was greatly reduced by replacing Asp(134), which is one of the active site residues, with His, probably due to a reduction in the catalytic activity. Random mutagenesis of the gene encoding the resultant D134H enzyme, followed by screening for second-site revertants, allowed us to isolate three single mutations (Ala(12) --> Ser, Lys(75) --> Met, and Ala(77) --> Pro) that restore the normal complementation ability to the D134H enzyme. These mutations were individually or simultaneously introduced into the wild-type enzyme, and the kinetic parameters of the resultant mutant enzymes for the hydrolysis of a DNA-RNA-DNA/DNA substrate were determined at 30 degrees C. Each mutation increased the k(cat)/K(m) value of the wild-type enzyme by 2.1-4.8-fold. The effects of the mutations on the enzymatic activity were roughly cumulative, and the combination of these three mutations increased the k(cat)/K(m) value of the wild-type enzyme by 40-fold (5.5-fold in k(cat)). Measurement of thermal stability of the mutant enzymes with circular dichroism spectroscopy in the presence of 1 M guanidine hydrochloride and 1 mM dithiothreitol showed that the T(m) value of the triple mutant enzyme, in which all three mutations were combined, was comparable to that of the wild-type enzyme (75.0 vs 77.4 degrees C). These results demonstrate that the activity of a thermophilic enzyme can be improved without a cost of protein stability.
Hirano
N
N
Department of Material and Life Science, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan.
Haruki
M
M
Morikawa
M
M
Kanaya
S
S
D019943:Amino Acid Substitution / Q000235:genetics; D001224:Aspartic Acid / Q000235:genetics; D002942:Circular Dichroism; D004252:DNA Mutational Analysis; D004789:Enzyme Activation / Q000235:genetics; D004795:Enzyme Stability / Q000235:genetics; D005798:Genes, Bacterial; D016153:Genes, Suppressor; D005816:Genetic Complementation Test; D006639:Histidine / Q000235:genetics; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D016914:Ribonuclease H / Q000235:genetics* / Q000378:metabolism*; D013816:Thermodynamics; D016963:Thermus thermophilus / Q000201:enzymology* / Q000235:genetics*
D001224:Aspartic Acid; D006639:Histidine; D016914:Ribonuclease H; C078856:ribonuclease HI