Characterization of recombinant YakC of Schizosaccharomyces pombe showing YakC defines a new family of aldo-keto reductases.
The yakC gene in Schizosaccharomyces pombe, which encodes yakC protein (YakC), a potential member of an aldo-keto reductase (AKR) family, was cloned and expressed in Escherichia coli cells. The recombinant YakC purified to homogeneity catalyzed the reduction of 2-nitrobenzaldehyde (k(cat), 44.1 s(-1), K(m), 0.185 +/- 0.018 mM), 2-phthalaldehyde (19.8, 0.333 +/- 0.032), and pyridine-2-aldehyde (7.64, 0.302 +/- 0.028). Neither pyridoxal nor other compounds examined acted as substrates. NADPH, but not NADH, was a hydrogen donor. The enzyme is a monomer with a molecular weight of 38,900 +/- 6,600 (SDS-PAGE). The amino acid sequence deduced from yakC showed the highest (34%) identity with that of pyridoxal reductase (AKR8A1) among the identified AKRs. Twenty-one function-unknown proteins showed 40% or higher identity to the deduced amino acid sequence: DR2261 protein of Deionococcus radiodurans showed the highest (50%) identity. The predicted secondary structure of YakC is similar to that of human aldose reductase, a representative AKR. The results establish YakC as the first member of a new AKR family, AKR13. The yeast cells contained enzyme(s) other than YakC and pyridoxal reductase with the ability to reduce 2-nitrobenzaldehyde: total (100%) activity in the crude extract consisted of about 23% YakC, about 44% pyridoxal reductase, and about 33% other enzyme(s).
D000429:Alcohol Oxidoreductases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D000449:Aldehyde Reductase; D000074408:Aldo-Keto Reductases; D000595:Amino Acid Sequence; D001483:Base Sequence; D002384:Catalysis; D003001:Cloning, Molecular; D004274:DNA, Recombinant; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D010802:Phylogeny; D010957:Plasmids; D011233:Precipitin Tests; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012568:Schizosaccharomyces / Q000201:enzymology* / Q000235:genetics; D029702:Schizosaccharomyces pombe Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D017186:Titrimetry / Q000379:methods
D004274:DNA, Recombinant; D011994:Recombinant Proteins; D029702:Schizosaccharomyces pombe Proteins; D000429:Alcohol Oxidoreductases; D000074408:Aldo-Keto Reductases; D000449:Aldehyde Reductase