Cloning and expression of a Clostridium kluyveri gene responsible for diaphorase activity

• Published in: Bioscience, biotechnology, and biochemistry Vol. 72; no. 3; pp. 735 - 741
• Main Authors: Chakraborty, S.(Mie Univ., Tsu (Japan)), Sakka, M, Kimura, T, Sakka, K
• Format: Journal Article
• Language: English
• Published: Tokyo Japan Society for Bioscience, Biotechnology, and Agrochemistry 01.03.2008; Japan Society for Bioscience Biotechnology and Agrochemistry; Oxford University Press
• Subjects: Amino Acid Sequence; AMINO ACID SEQUENCES; Bacterial Proteins; Biological and medical sciences; CHROMATOGRAPHIE EN COUCHE MINCE; CLONACION; CLONAGE; CLONING; Cloning, Molecular - methods; CLOSTRIDIO; CLOSTRIDIUM; Clostridium kluyveri; Clostridium kluyveri - enzymology; Clostridium kluyveri - genetics; CROMATOGRAFIA DE CAPA FINA; diaphorase; ESCHERICHIA COLI; EXPRESION GENICA; EXPRESSION DES GENES; FER; Flavin Mononucleotide; Fundamental and applied biological sciences. Psychology; GENE EXPRESSION; Genes, Bacterial; GENETICALLY MODIFIED MICROORGANISMS; HIERRO; IRON; MICRO-ORGANISME MODIFIE GENETIQUE; MICROORGANISMOS MODIFICADOS GENETIC; Molecular Sequence Data; NADH dehydrogenase; NADH Dehydrogenase - genetics; NADH Dehydrogenase - isolation & purification; NUCLEOTIDE SEQUENCE; rubredoxin; SECUENCIA NUCLEOTIDICA; SECUENCIAS DE AMINOACIDOS; SEQUENCE D'ACIDES AMINES; SEQUENCE NUCLEOTIDIQUE; THIN LAYER CHROMATOGRAPHY; Dihydrolipoamide dehydrogenase; Gene; Enzyme; Rubredoxin; Clostridiaceae; Clostridiales; NADH dehydrogenase; Bacteria; Oxidoreductases; Clostridium kluyveri; diaphorase
• ISSN: 0916-8451; 1347-6947
• DOI: 10.1271/bbb.70606

A small enzyme showing diaphorase activity was purified from culture supernatant of Clostridium kluyveri and its N-terminal amino acid sequence was determined. This sequence identified a gene (diaA) encoding a protein (DiaA) of 229 amino acids with a predicted molecular weight of 24,981 in the genomic DNA sequence database of C. kluyveri constructed by the Research Institute of Innovative Technology for the Earth. The predicted protein was composed of a flavin reductase-like domain and a rubredoxin-like domain from its N-terminus. The diaA gene was cloned into an expression vector and expressed in an Escherichia coli recombinant. Recombinant enzyme rDiaA showed NADH/NADPH diaphorase activity with 2,6-dichlorophenolindophenol and nitro blue tetrazolium. The enzyme was most active at pH 8.0 at 40 deg C. The UV-visible absorption spectrum and thin layer chromatography (TLC) analyses indicated that one rDiaA molecule contained a tightly bound FMN molecule as a prosthetic group. An iron molecule was also detected in an enzyme molecule.