Triphenylmethane reductase from Citrobacter sp. strain KCTC 18061P: purification, characterization, gene cloning, and overexpression of a functional protein in Escherichia coli.
We purified to homogeneity an enzyme from Citrobacter sp. strain KCTC 18061P capable of decolorizing triphenylmethane dyes. The native form of the enzyme was identified as a homodimer with a subunit molecular mass of about 31 kDa. It catalyzes the NADH-dependent reduction of triphenylmethane dyes, with remarkable substrate specificity related to dye structure. Maximal enzyme activity occurred at pH 9.0 and 60 degrees C. The enzymatic reaction product of the triphenylmethane dye crystal violet was identified as its leuco form by UV-visible spectral changes and thin-layer chromatography. A gene encoding this enzyme was isolated based on its N-terminal and internal amino acid sequences. The nucleotide sequence of the gene has a single open reading frame encoding 287 amino acids with a predicted molecular mass of 30,954 Da. Although the deduced amino acid sequence displays 99% identity to the hypothetical protein from Listeria monocytogenes strain 4b H7858, it shows no overall functional similarity to any known protein in the public databases. At the N terminus, the amino acid sequence has high homology to sequences of NAD(P)H-dependent enzymes containing the dinucleotide-binding motif GXXGXXG. The enzyme was heterologously expressed in Escherichia coli, and the purified recombinant enzyme showed characteristics similar to those of the native enzyme. This is the first report of a triphenylmethane reductase characterized from any organism.
Jang
Moon-Sun
MS
Department of Biotechnology, College of Natural Resources and Life Science, Dong-A University, Busan 604-714, Korea.
Lee
Young-Mi
YM
Kim
Cheorl-Ho
CH
Lee
Jai-Heon
JH
Kang
Dong-Woo
DW
Kim
Seok-Jo
SJ
Lee
Young-Choon
YC
D000595:Amino Acid Sequence; D001483:Base Sequence; D002954:Citrobacter / Q000201:enzymology* / Q000235:genetics; D003001:Cloning, Molecular / Q000379:methods; D017931:DNA Primers; D004926:Escherichia coli / Q000235:genetics; D007700:Kinetics; D008969:Molecular Sequence Data; D010088:Oxidoreductases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry; D013379:Substrate Specificity
D017931:DNA Primers; D010446:Peptide Fragments; D010088:Oxidoreductases; C507510:triphenylmethane reductase, Citrobacter