Amino acid residues involved in substrate recognition of the Escherichia coli Orf135 protein.
The Escherichia coli Orf135 protein, a MutT-type enzyme, hydrolyzes mutagenic 2-hydroxy-dATP (2-OH-dATP) and 8-hydroxy-dGTP, in addition to dCTP and 5-methyl-dCTP, and its deficiency causes increases in both the spontaneous and H(2)O(2)-induced mutation frequencies. To identify the amino acid residues that interact with these nucleotides, the Glu-33, Arg-72, Arg-77, and Asp-118 residues of Orf135, which are candidates for residues interacting with the base, were substituted, and the enzymatic activities of these mutant proteins were examined. The mutant proteins with a substitution at the 33rd, 72nd, and 118th amino acid residues displayed activities affected to various degrees for each substrate, suggesting the involvement of these residues in substrate binding. On the other hand, the mutant protein with a substitution at the 77th Arg residue had activitiy similar to that of the wild-type protein, excluding the possibility that this Arg side chain is involved in base recognition. In addition, the expression of some Orf135 mutants in orf135(-) E. coli reduced the level of formation of rpoB mutants elicited by H(2)O(2). These results reveal the residues involved in the substrate binding of the E. coli Orf135 protein.
Iida
Emiko
E
Graduate School of Pharmaceutical Sciences, Hokkaido University, Kita-12, Nishi-6, Kita-ku, Sapporo 060-0812, Japan.
Satou
Kazuya
K
Mishima
Masaki
M
Kojima
Chojiro
C
Harashima
Hideyoshi
H
Kamiya
Hiroyuki
H
D019943:Amino Acid Substitution; D001483:Base Sequence; D020134:Catalytic Domain / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D010744:Phosphoric Monoester Hydrolases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011487:Protein Conformation; D011755:Pyrophosphatases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity; D000096964:Nudix Hydrolases
D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; D010744:Phosphoric Monoester Hydrolases; D011755:Pyrophosphatases; D011994:Recombinant Proteins; C052211:mutT protein, E coli; C422942:nudix hydrolase Orf135; D000096964:Nudix Hydrolases