College of Biotechnology and Bioengineering, Zhejiang University of Technology, Hangzhou-310014, China.
Protein Pept Lett. 2021;28(2):229-239. doi: 10.2174/0929866527666200708151327.
Flavin adenine dinucleotide (FAD) is a redox-active coenzyme that regulates several important enzymatic reactions during metabolism. FAD is used in the medicinal and food industries and FAD supplements have been used to treat some inheritable diseases. FAD can be biosynthesized from flavin mononucleotide (FMN) and adenosine triphosphate (ATP), catalyzed by FAD synthetase (FADS).
The aim of this study was to heterologously express the gene encoding FADS from the flavinogenic yeast Candida famata (FADS) for biosynthesis of FAD.
The sequence encoding FADS was retrieved and heterologously expressed in Escherichia coli. The structure and enzymatic properties of recombinant FADS were characterized.
FADS (279 amino acids) was successfully expressed in E. coli BL21 (DE3), with a theoretical molecular weight of 32299.79 Da and an isoelectric point of 6.09. Secondary structural analysis showed that the number of α-helices was 2-fold higher than the number of β-sheets, indicating that the protein was highly hydrophilic. Under fixed ATP concentration, FADS had a Km of 0.04737±0.03158 mM and a V of 3.271±0.79 μM/min/mg. Under fixed FMN concentration, FADSCf had a Km of 0.1214±0.07464 mM and a V of 2.6695±0.3715 μM/min/mg. Enzymatic reactions in vitro showed that expressed FADS could form 80 mM of FAD per mg of enzyme after 21 hours under the following conditions: 0.5 mM FMN, 5 mM ATP and 10 mM Mg.
Under optimized conditions (0.5 mM FMN, 5 mM ATP and 10 mM Mg), the production of FAD reached 80 mM per mg of FADS after a 21-hour reaction. Our results indicate that purified recombinant FADS can be used for the biosynthesis of FAD.
黄素腺嘌呤二核苷酸（FAD）是一种氧化还原活性辅酶，可调节代谢过程中几种重要的酶促反应。FAD 用于医药和食品工业，FAD 补充剂已用于治疗某些遗传性疾病。FAD 可以由黄素单核苷酸（FMN）和三磷酸腺苷（ATP）生物合成，由 FAD 合成酶（FADS）催化。
本研究旨在异源表达产黄素酵母 Candida famata（FADS）的 FADS 基因，用于 FAD 的生物合成。
检索编码 FADS 的序列并在大肠杆菌中异源表达。对重组 FADS 的结构和酶学性质进行了表征。
FADS（279 个氨基酸）在大肠杆菌 BL21（DE3）中成功表达，理论分子量为 32299.79 Da，等电点为 6.09。二级结构分析表明，α-螺旋数是β-折叠数的两倍，表明该蛋白具有高度的亲水性。在固定的 ATP 浓度下，FADS 的 Km 为 0.04737±0.03158 mM，V 为 3.271±0.79 μM/min/mg。在固定的 FMN 浓度下，FADSCf 的 Km 为 0.1214±0.07464 mM，V 为 2.6695±0.3715 μM/min/mg。体外酶反应表明，在以下条件下，表达的 FADS 在 21 小时内每毫克酶可形成 80 mM 的 FAD：0.5 mM FMN、5 mM ATP 和 10 mM Mg。
在优化条件下（0.5 mM FMN、5 mM ATP 和 10 mM Mg），FADS 反应 21 小时后，FAD 的产量达到每毫克 FADS 80 mM。我们的结果表明，纯化的重组 FADS 可用于 FAD 的生物合成。
