Morita Tomotake, Huruta Tomoko, Ashiuchi Makoto, Yagi Toshiharu
Department of Bioresources Science, Faculty of Agriculture, Kochi University, Monobe Otsu Nankoku, Kochi 783-8502, Japan.
J Biochem. 2002 Oct;132(4):635-41. doi: 10.1093/oxfordjournals.jbchem.a003267.
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).
粟酒裂殖酵母中的yakC基因被克隆并在大肠杆菌细胞中表达，该基因编码醛酮还原酶（AKR）家族的潜在成员yakC蛋白（YakC）。纯化至同质的重组YakC催化2-硝基苯甲醛的还原反应（催化常数k(cat)为44.1 s⁻¹，米氏常数K(m)为0.185±0.018 mM）、2-邻苯二甲醛（19.8，0.333±0.032）和吡啶-2-甲醛（7.64，0.302±0.028）。吡哆醛和其他检测的化合物均不能作为底物。NADPH是氢供体，而NADH不是。该酶是一种单体，分子量为38,900±6,600（SDS-PAGE）。从yakC推导的氨基酸序列与已鉴定的AKR中吡哆醛还原酶（AKR8A1）的序列具有最高的（34%）同一性。21种功能未知的蛋白质与推导的氨基酸序列具有40%或更高的同一性：耐辐射球菌的DR2261蛋白具有最高的（50%）同一性。YakC的预测二级结构与人醛糖还原酶（一种代表性的AKR）相似。这些结果确立了YakC作为新的AKR家族AKR13的首个成员。酵母细胞中除YakC和吡哆醛还原酶外还含有其他能够还原2-硝基苯甲醛的酶：粗提物中的总（100%）活性约23%由YakC贡献，约44%由吡哆醛还原酶贡献，约33%由其他酶贡献。
