Molecular characterization of three mutations in katG affecting the activity of hydroperoxidase I of Escherichia coli.
Hydroperoxidase I (HPI) of Escherichia coli is a bifunctional enzyme exhibiting both catalase and peroxidase activities. Mutants lacking appreciable HPI have been generated using nitrosoguanidine and the gene encoding HPI, katG, has been cloned from three of these mutants using either classical probing methods or polymerase chain reaction amplification. The mutant genes were sequenced and the changes from wild-type sequence identified. Two mutants contained G to A changes in the coding strand, resulting in glycine to aspartate changes at residues 119 (katG15) and 314 (katG16) in the deduced amino acid sequence of the protein. A third mutant contained a C to T change resulting in a leucine to phenylalanine change at residue 139 (katG14). The Phe139-, Asp119-, and Asp314-containing mutants exhibited 13, less than 1, and 18%, respectively, of the wild-type catalase specific activity and 43, 4, and 45% of the wild-type peroxidase specific activity. All mutant enzymes bound less protoheme IX than the wild-type enzyme. The sensitivities of the mutant enzymes to the inhibitors hydroxylamine, azide, and cyanide and the activators imidazole and Tris were similar to those of the wild-type enzyme. The mutant enzymes were more sensitive to high temperature and to beta-mercaptoethanol than the wild-type enzyme. The pH profiles of the mutant catalases were unchanged from the wild-type enzyme.
Loewen
P C
PC
Department of Microbiology, University of Manitoba, Winnipeg, Canada.
Switala
J
J
Smolenski
M
M
Triggs-Raine
B L
BL
D001483:Base Sequence; D002374:Catalase / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism; D002874:Chromosome Mapping; D003001:Cloning, Molecular; D004789:Enzyme Activation; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D029968:Escherichia coli Proteins; D006861:Hydrogen Peroxide; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D009154:Mutation; D009604:Nitrosoguanidines; D013057:Spectrum Analysis; D013329:Structure-Activity Relationship
D029968:Escherichia coli Proteins; D009604:Nitrosoguanidines; D006861:Hydrogen Peroxide; D002374:Catalase; C051253:katG protein, E coli