Open AccessMutational analysis of NADH‐binding residues in triphenylmethane reductase from Citrobacter sp. strain KCTC 18061P
Author(s) -
Jang MoonSun,
Kang NamYoung,
Kim KyoungSook,
Kim CheorlHo,
Lee JaiHeon,
Lee YoungChoon
Publication year - 2007
Publication title -
fems microbiology letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.899
H-Index - 151
eISSN - 1574-6968
pISSN - 0378-1097
DOI - 10.1111/j.1574-6968.2007.00709.x
Subject(s) - cofactor , triphenylmethane , alanine , stereochemistry , mutant , site directed mutagenesis , chemistry , biochemistry , circular dichroism , reductase , mutagenesis , enzyme , amino acid , gene , organic chemistry
Triphenylmethane reductase (TMR) catalyzes the NADH‐dependent reduction of triphenylmethane dyes. Sequence alignment revealed a region with a conserved G XX G XX G motif near its N‐terminus, which corresponds to a conserved structural motif of known dinucleotide‐binding proteins. To verify whether some of these glycine residues are important for the enzyme catalysis, these three glycine residues (Gly‐7, Gly‐10 and Gly‐13) were individually replaced by alanine using site‐directed mutagenesis. The secondary structures of these mutants, as measured by circular dichroism spectroscopy, did not show remarkable differences as compared with the wild type. The V max / K m values of mutants G7A and G13A for both Basic fuchsin and NADH were increased about three and twofold over that of the wild type, respectively, whereas the V max / K m value of mutant G10A were decreased about sixfold. These results suggest that these three glycine residues are involved in the interaction with both substrate and cofactor for the catalytic activity of TMR.