title: Mutational analysis of NADH-binding residues in triphenylmethane reductase from Citrobacter sp. strain KCTC 18061P.
abstract: Triphenylmethane reductase (TMR) catalyzes the NADH-dependent reduction of triphenylmethane dyes. Sequence alignment revealed a region with a conserved GXXGXXG 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.
authors.affiliation: College of Natural Resources and Life Science, Dong-A University, Busan, Korea.
authors.forename: Moon-Sun
authors.initials: MS
authors.lastname: Jang
authors.forename: Nam-Young
authors.initials: NY
authors.lastname: Kang
authors.forename: Kyoung-Sook
authors.initials: KS
authors.lastname: Kim
authors.forename: Cheorl-Ho
authors.initials: CH
authors.lastname: Kim
authors.forename: Jai-Heon
authors.initials: JH
authors.lastname: Lee
authors.forename: Young-Choon
authors.initials: YC
authors.lastname: Lee
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D002942:Circular Dichroism; D002954:Citrobacter / Q000201:enzymology* / Q000235:genetics; D005998:Glycine / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009243:NAD / Q000378:metabolism*; D010088:Oxidoreductases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D011485:Protein Binding / Q000235:genetics; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D012394:Rosaniline Dyes / Q000378:metabolism; D017385:Sequence Homology
chemical_list: D012394:Rosaniline Dyes; D009243:NAD; C025485:basic fuchsin; D010088:Oxidoreductases; C507510:triphenylmethane reductase, Citrobacter; D005998:Glycine