Characterization of recombinant YakC of Schizosaccharomyces pombe showing YakC defines a new family of aldo-keto reductases

Abstract

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).