Department of Molecular Biology & Genetics, Istanbul Technical University, Maslak, Istanbul, Turkey.
Department of Molecular Biology & Genetics, Istanbul Medeniyet University, Istanbul, Turkey.
Biotechnol Appl Biochem. 2019 Nov;66(6):915-923. doi: 10.1002/bab.1801. Epub 2019 Sep 11.
Nicotinamide adenine dinucleotide phosphate (NAD(P)H)-flavin oxidoreductases (flavin reductases) catalyze the reduction of flavin by NAD(P)H and provide the reduced form of flavin mononucleotide (FMN) to flavin-dependent monooxygenases. Based on bioinformatics analysis, we identified a putative flavin reductase gene, sso2055, in the genome of hyperthermophilic archaeon Sulfolobus solfataricus P2, and further cloned this target sequence into an expression vector. The cloned flavin reductase (EC. 1.5.1.30) was purified to homogeneity and characterized further. The purified enzyme exists as a monomer of 17.8 kDa, free of chromogenic cofactors. Homology modeling revealed this enzyme as a TIM barrel, which is also supported by circular dichroism measurements revealing a beta-sheet rich content. The optimal pH for SSO2055 activity was pH 6.5 in phosphate buffer and the highest activity observed was at 120 °C within the measurable temperature. We showed that this enzyme can use FMN and flavin adenine dinucleotide (FAD) as a substrate to generate their reduced forms. The purified enzyme is predicted to be a potential flavin reductase of flavin-dependent monooxygenases that could be involved in the biodesulfurization process of S. solfataricus P2.
烟酰胺腺嘌呤二核苷酸磷酸（NAD(P)H）-黄素氧化还原酶（黄素还原酶）催化黄素被 NAD(P)H 还原，并向黄素依赖的单加氧酶提供黄素单核苷酸（FMN）的还原形式。基于生物信息学分析，我们在嗜热古菌 Sulfolobus solfataricus P2 的基因组中鉴定出一个假定的黄素还原酶基因 sso2055，并进一步将该靶序列克隆到表达载体中。克隆的黄素还原酶（EC. 1.5.1.30）被纯化为均一状态，并进一步进行了表征。纯化的酶以 17.8 kDa 的单体形式存在，不含发色辅因子。同源建模表明该酶为 TIM 桶，圆二色性测量也支持这一结构，表明其富含β-折叠。SSO2055 活性的最佳 pH 值为磷酸盐缓冲液中的 pH 6.5，在可测量的温度范围内，观察到的最高活性为 120°C。我们表明，该酶可以使用 FMN 和黄素腺嘌呤二核苷酸（FAD）作为底物生成其还原形式。纯化的酶被预测为黄素依赖的单加氧酶的潜在黄素还原酶，可能参与 S. solfataricus P2 的生物脱硫过程。
