Schweiger Paul, Gross Harald, Wesener Shane, Deppenmeier Uwe
Department of Biological Sciences, University of Wisconsin-Milwaukee, Milwaukee, WI 53211, USA.
Appl Microbiol Biotechnol. 2008 Oct;80(6):995-1006. doi: 10.1007/s00253-008-1600-5. Epub 2008 Jul 16.
Three cytosolic NADPH-dependent flavin-associated proteins (Gox2107, Gox0502, and Gox2684) from Gluconobacter oxydans 621H were overproduced in Escherichia coli, and the recombinant enzymes were purified and characterized. Apparent native molecular masses of 65.2, 78.2, and 78.4 kDa were observed for Gox2107, Gox0502, and Gox2684, corresponding to a trimeric structure for Gox2107 and dimers for Gox0502 and Gox2684. Analysis of flavin content revealed Gox2107 was flavin adenine dinucleotide dependent, whereas Gox0502 and Gox2684 contained flavin mononucleotide. The enzymes were able to reduce vinyl ketones and quinones, reducing the olefinic bond of vinyl ketones as shown by (1)H nuclear magnetic resonance. Additionally, Gox0502 and Gox2684 stereospecifically reduced 5S-(+)-carvone to 2R,5S-dihydrocarvone. All enzymes displayed highest activities with 3-butene-2-one and 1,4-naphthoquinone. Gox0502 and Gox2684 displayed a broader substrate spectrum also reducing short-chain alpha-diketones, whereas Gox2107 was most catalytically efficient.
从氧化葡萄糖酸杆菌621H中提取的三种胞质NADPH依赖性黄素相关蛋白（Gox2107、Gox0502和Gox2684）在大肠杆菌中过量表达，对重组酶进行了纯化和表征。观察到Gox2107、Gox0502和Gox2684的表观天然分子量分别为65.2、78.2和78.4 kDa，这表明Gox2107为三聚体结构，Gox0502和Gox2684为二聚体结构。黄素含量分析表明，Gox2107依赖黄素腺嘌呤二核苷酸，而Gox0502和Gox2684含有黄素单核苷酸。这些酶能够还原乙烯基酮和醌，如氢核磁共振所示，可还原乙烯基酮的烯键。此外，Gox0502和Gox2684能立体特异性地将5S-（+）-香芹酮还原为2R，5S-二氢香芹酮。所有酶对3-丁烯-2-酮和1,4-萘醌的活性最高。Gox0502和Gox2684的底物谱更广，还能还原短链α-二酮，而Gox2107的催化效率最高。
