Institute of Biosciences, Environmental Microbiology, TU Bergakademie Freiberg, Leipziger Str. 29, 09599 Freiberg, Germany.
Microbial Biotechnology, Ruhr University Bochum, Universitätsstr. 150, 44780 Bochum, Germany.
Molecules. 2018 Apr 2;23(4):809. doi: 10.3390/molecules23040809.
Herein we describe the first representative of an E2-type two-component styrene monooxygenase of proteobacteria. It comprises a single epoxidase protein (StyA1) and a two domain protein (StyA2B) harboring an epoxidase (A2) and a FAD-reductase (B) domain. It was annotated as StyA1/StyA2B of EPS. StyA2B serves mainly as NADH:FAD-oxidoreductase. A of 33.6 ± 4.0 µM for FAD and a of 22.3 ± 1.1 s were determined and resulted in a catalytic efficiency () of 0.64 s μM. To investigate its NADH:FAD-oxidoreductase function the linker between A2- and B-domain (AREAV) was mutated. One mutant (AAAAA) showed 18.7-fold higher affinity for FAD ( of 5.21 s μM) while keeping wildtype NADH-affinity and -oxidation activity. Both components, StyA2B and StyA1, showed monooxygenase activity on styrene of 0.14 U mg and 0.46 U mg, as well as on benzyl methyl sulfide of 1.62 U mg and 3.11 U mg, respectively. The high sulfoxidase activity was the reason to test several thioanisole-like substrates in biotransformations. StyA1 showed high substrate conversions (up to 95% in 2 h) and produced dominantly ()-enantiomeric sulfoxides of all tested substrates. The AAAAA-mutant showed a 1.6-fold increased monooxygenase activity. In comparison, the GQWCSQY-mutant did neither show monooxygenase nor efficient FAD-reductase activity. Hence, the linker between the two domains of StyA2B has effects on the reductase as well as on the monooxygenase performance. Overall, this monooxygenase represents a promising candidate for biocatalyst development and studying natural fusion proteins.
我们在此描述了一种新型的革兰氏阴性菌 E2 型双组分苯乙烯单加氧酶。它由一个单加氧酶蛋白（StyA1）和一个含有单加氧酶（A2）和黄素腺嘌呤二核苷酸（FAD）还原酶（B）结构域的双结构域蛋白（StyA2B）组成。它被注释为 EPS 的 StyA1/StyA2B。StyA2B 主要作为 NADH：FAD-氧化还原酶。确定了 FAD 的 33.6 ± 4.0 µM 和 22.3 ± 1.1 s 的，并得到 0.64 s μM 的催化效率（）。为了研究其 NADH：FAD-氧化还原酶功能，对 A2-和 B-结构域之间的连接区（AREAV）进行了突变。一个突变体（AAAAA）对 FAD 的亲和力提高了 18.7 倍（的 5.21 s μM），同时保持了野生型 NADH 的亲和力和氧化活性。StyA2B 和 StyA1 都表现出对苯乙烯的单加氧酶活性，分别为 0.14 U mg 和 0.46 U mg，以及对苄基甲基硫醚的 1.62 U mg 和 3.11 U mg。高的亚砜氧化酶活性是在生物转化中测试几种硫醚类似物底物的原因。StyA1 表现出高的底物转化率（2 小时内高达 95%），并主要产生（）-对映体硫氧化物。AAAAA 突变体显示出 1.6 倍的单加氧酶活性增加。相比之下，GQWCSQY 突变体既没有表现出单加氧酶活性，也没有表现出有效的 FAD 还原酶活性。因此，StyA2B 两个结构域之间的连接区对还原酶以及单加氧酶的性能都有影响。总的来说，这种单加氧酶代表了生物催化剂开发和研究天然融合蛋白的有前途的候选物。
