Mutational analysis of NADH-binding residues in triphenylmethane reductase from Citrobacter sp. strain KCTC 18061P.
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.
Jang
Moon-Sun
MS
College of Natural Resources and Life Science, Dong-A University, Busan, Korea.
Kang
Nam-Young
NY
Kim
Kyoung-Sook
KS
Kim
Cheorl-Ho
CH
Lee
Jai-Heon
JH
Lee
Young-Choon
YC
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
D012394:Rosaniline Dyes; D009243:NAD; C025485:basic fuchsin; D010088:Oxidoreductases; C507510:triphenylmethane reductase, Citrobacter; D005998:Glycine