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Hyperthermophilic flavin reductase from Sulfolobus solfataricus P2: Production and biochemical characterization.
Biotechnology and Applied Biochemistry ( IF 3.2 ) Pub Date : 2019-09-11 , DOI: 10.1002/bab.1801
Gokhan Gun 1 , Rahmi Imamoglu 1 , Ozge Tatli 1, 2 , Yuda Yurum 3 , Ahmet Tarik Baykal 4 , Gizem Dinler-Doganay 1
Affiliation  

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.

中文翻译:

来自Sulfolobus solfataricus P2的高嗜性黄素还原酶:生产和生化表征。

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NAD(P)H还原黄素,并将黄素单核苷酸(FMN)还原为黄素依赖性单加氧酶。基于生物信息学分析,我们在超嗜热古生菌Sulfolobus solfataricus P2的基因组中鉴定出推定的黄素还原酶基因sso2055,并将该靶序列进一步克隆到表达载体中。将克隆的黄素还原酶(EC。1.5.1.30)纯化至均质并进一步表征。纯化的酶以17.8 kDa的单体形式存在,不含生色辅因子。同源性建模显示该酶为TIM桶,圆形二向色性测量也支持该酶,从而显示出富含β-折叠的含量。SSO2055活性的最佳pH为pH 6。在磷酸盐缓冲液中为5,观察到的最高活性是在可测量温度下于120°C时。我们显示该酶可以使用FMN和黄素腺嘌呤二核苷酸(FAD)作为底物来生成其还原形式。预计纯化的酶是黄素依赖性单加氧酶的潜在黄素还原酶,其可能与S. solfataricus P2的生物脱硫过程有关。
更新日期:2020-01-09
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