Husain Nor Asyikin Che, Jamaluddin Haryati, Jonet Mohd Anuar
Department of Biosciences, Faculty of Science, Universiti Teknologi Malaysia, 81310 Johor Bahru, Johor, Malaysia; Structural Biology & Functional Omics, Malaysian Genome and Vaccine Institute, 43000 Kajang, Selangor, Malaysia.
Department of Biosciences, Faculty of Science, Universiti Teknologi Malaysia, 81310 Johor Bahru, Johor, Malaysia.
Int J Biol Macromol. 2024 Aug;275(Pt 2):133721. doi: 10.1016/j.ijbiomac.2024.133721. Epub 2024 Jul 8.
Flavin reductases play a vital role in catalyzing the reduction of flavin through NADH or NADPH oxidation. The gene encoding flavin reductase from the thermophilic bacterium Geobacillus mahadii Geo-05 (GMHpaC) was cloned, overexpressed in Escherichia coli BL21 (DE3) pLysS, and purified to homogeneity. The purified recombinant GMHpaC (Class II) contains chromogenic cofactors, evidenced by maximal absorbance peaks at 370 nm and 460 nm. GMHpaC stands out as the most thermostable and pH-tolerant flavin reductase reported to date, retaining up to 95 % catalytic activity after incubation at 70 °C for 30 min and maintaining over 80 % activity within a pH range of 2-12 for 30 min. Furthermore, GMHpaC's catalytic activity increases by 52 % with FMN as a co-factor compared to FAD and riboflavin. GMHpaC, coupled with 4-hydroxyphenylacetate-3-monooxygenase (GMHpaB) from G. mahadii Geo-05, enhances the hydroxylation of 4-hydroxyphenylacetate (HPA) by 85 %. The modeled structure of GMHpaC reveals relatively conserved flavin and NADH binding sites. Modeling and docking studies shed light on structural features and amino acid substitutions that determine GMHpaC's co-factor specificity. The remarkable thermostability, high catalytic activity, and general stability exhibited by GMHpaC position it as a promising enzyme candidate for various industrial applications.
黄素还原酶在通过NADH或NADPH氧化催化黄素还原过程中起着至关重要的作用。克隆了嗜热细菌马氏嗜热栖热菌Geo-05（GMHpaC）中编码黄素还原酶的基因，在大肠杆菌BL21（DE3）pLysS中进行了过表达，并纯化至均一。纯化后的重组GMHpaC（II类）含有发色辅因子，在370nm和460nm处有最大吸收峰可证明。GMHpaC是迄今为止报道的最耐热和耐pH的黄素还原酶，在70°C孵育30分钟后仍保留高达95%的催化活性，在2-12的pH范围内保持30分钟以上80%以上的活性。此外，与FAD和核黄素相比，以FMN作为辅因子时，GMHpaC的催化活性提高了52%。GMHpaC与来自马氏嗜热栖热菌Geo-05的4-羟基苯乙酸-3-单加氧酶（GMHpaB）结合，使4-羟基苯乙酸（HPA）的羟基化增强了85%。GMHpaC的模拟结构揭示了相对保守的黄素和NADH结合位点。建模和对接研究揭示了决定GMHpaC辅因子特异性的结构特征和氨基酸取代。GMHpaC表现出的卓越热稳定性、高催化活性和总体稳定性使其成为各种工业应用中有前景的酶候选物。
