Department of Genetics and Microbiology, University of Murcia, Murcia, 30100, Spain.
Appl Microbiol Biotechnol. 2014 Apr;98(7):2981-9. doi: 10.1007/s00253-013-5168-3. Epub 2013 Aug 18.
Marinomonas mediterranea is a marine gamma-proteobacterium that synthesizes LodA, a novel L-lysine-ε-oxidase (E.C. 1.4.3.20). This enzyme oxidizes L-lysine generating 2-aminoadipate 6-semialdehyde, ammonium, and hydrogen peroxide. Unlike other L-amino acid oxidases, LodA is not a flavoprotein but contains a quinone cofactor. LodA is encoded by an operon with two genes, lodA and lodB. In the native system, LodB is required for the synthesis of a functional LodA. In this study, we report the recombinant expression of LodA in Escherichia coli using vectors that allow its expression and accumulation in the cytoplasm. To reveal the L-lysine-ε-oxidase activity using the Amplex Red method for hydrogen peroxide detection, it is necessary to first remove the E. coli cytoplasmic catalases. The flavoprotein LodB is the only M. mediterranea protein required in the recombinant system for the generation of the cofactor of LodA. In the absence of LodB, LodA does not contain the quinone cofactor and remains in an inactive form. The results presented indicate that LodB participates in the posttranslational modification of LodA that generates the quinone cofactor.
海洋γ-变形菌 Marinomonas mediterranea 能够合成 LodA，这是一种新型的 L-赖氨酸 ε-氧化酶（E.C. 1.4.3.20）。该酶能够氧化 L-赖氨酸，生成 2-氨基己二酸 6-半醛、铵和过氧化氢。与其他 L-氨基酸氧化酶不同，LodA 不是黄素蛋白，而是含有醌辅因子。LodA 由一个包含两个基因 lodA 和 lodB 的操纵子编码。在天然系统中，LodB 对于 LodA 功能的合成是必需的。在本研究中，我们使用允许 LodA 在细胞质中表达和积累的载体，在大肠杆菌中报告了 LodA 的重组表达。为了使用 Amplex Red 法检测过氧化氢来揭示 L-赖氨酸 ε-氧化酶的活性，首先需要去除大肠杆菌细胞质中的过氧化氢酶。黄素蛋白 LodB 是重组系统中生成 LodA 的辅因子所必需的唯一一种 M. mediterranea 蛋白。在没有 LodB 的情况下，LodA 不含醌辅因子，处于非活性状态。所呈现的结果表明，LodB 参与了 LodA 的翻译后修饰，从而生成了醌辅因子。
