Department of Biological Sciences, Duquesne University, Pittsburgh, Pennsylvania, USA.
Department of Chemistry and Chemical Biology, Indiana University-Purdue University Indianapolis, Indianapolis, Indiana, USA.
J Bacteriol. 2019 Mar 13;201(7). doi: 10.1128/JB.00614-18. Print 2019 Apr 1.
The putative respiratory selenite [Se(IV)] reductase (Srr) from MLS10 has been identified through a polyphasic approach involving genomics, proteomics, and enzymology. Nondenaturing gel assays were used to identify Srr in cell fractions, and the active band was shown to contain a single protein of 80 kDa. The protein was identified through liquid chromatography-tandem mass spectrometry (LC-MS/MS) as a homolog of the catalytic subunit of polysulfide reductase (PsrA). It was found to be encoded as part of an operon that contains six genes that we designated , , s, , , and SrrA is the catalytic subunit (80 kDa), with a twin-arginine translocation (TAT) leader sequence indicative of a periplasmic protein and one putative 4Fe-4S binding site. SrrB is a small subunit (17 kDa) with four putative 4Fe-4S binding sites, SrrC (43 kDa) is an anchoring subunit, and SrrD (24 kDa) is a chaperon protein. Both SrrE (38 kDa) and SrrF (45 kDa) were annotated as rhodanese domain-containing proteins. Phylogenetic analysis revealed that SrrA belonged to the PsrA/PhsA clade but that it did not define a distinct subgroup, based on the putative homologs that were subsequently identified from other known selenite-respiring bacteria (e.g., and ). The enzyme appeared to be specific for Se(IV), showing no activity with selenate, arsenate, or thiosulfate, with a of 145 ± 53 μM, a of 23 ± 2.5 μM min, and a of 23 ± 2.68 s These results further our understanding of the mechanisms of selenium biotransformation and its biogeochemical cycle. Selenium is an essential element for life, with Se(IV) reduction a key step in its biogeochemical cycle. This report identifies for the first time a dissimilatory Se(IV) reductase, Srr, from a known selenite-respiring bacterium, the haloalkalophilic strain MLS10. The work extends the versatility of the complex iron-sulfur molybdoenzyme (CISM) superfamily in electron transfer involving chalcogen substrates with different redox potentials. Further, it underscores the importance of biochemical and enzymological approaches in establishing the functionality of these enzymes.
通过涉及基因组学、蛋白质组学和酶学的多相方法，从 MLS10 中鉴定出假定的呼吸亚硒酸盐 [Se(IV)] 还原酶 (Srr)。非变性凝胶分析用于鉴定细胞部分中的 Srr，显示活性带含有一种 80 kDa 的单一蛋白质。该蛋白质通过液相色谱-串联质谱 (LC-MS/MS) 鉴定为多硫化物还原酶 (PsrA) 催化亚基的同源物。它被发现作为包含六个基因的操纵子的一部分被编码，我们将其命名为 、 、s、 、 、 和 。SrrA 是催化亚基（80 kDa），具有双精氨酸易位（TAT）前导序列，表明是周质蛋白，并有一个假定的 4Fe-4S 结合位点。SrrB 是一个小亚基（17 kDa），有四个假定的 4Fe-4S 结合位点，SrrC（43 kDa）是一个锚定亚基，SrrD（24 kDa）是一个伴侣蛋白。SrrE（38 kDa）和 SrrF（45 kDa）都被注释为硫代氨酸酶结构域蛋白。系统发育分析表明，SrrA 属于 PsrA/PhsA 分支，但根据随后从其他已知的亚硒酸盐呼吸细菌中鉴定出的假定同源物，它并没有定义一个独特的亚群（例如 和 ）。该酶似乎对 Se(IV)具有特异性，对硒酸盐、砷酸盐或硫代硫酸盐没有活性， 的值为 145 ± 53 μM， 的值为 23 ± 2.5 μM min， 的值为 23 ± 2.68 s。这些结果进一步加深了我们对硒生物转化及其生物地球化学循环机制的理解。硒是生命的必需元素，Se(IV)的还原是其生物地球化学循环中的关键步骤。本报告首次从已知的亚硒酸盐呼吸细菌、 haloalkalophilic 菌株 MLS10 中鉴定出一种异化 Se(IV)还原酶 Srr。这项工作扩展了具有不同氧化还原电位的含硫和硒底物的电子转移中复杂铁硫钼酶 (CISM) 超家族的多功能性。此外，它强调了生化和酶学方法在确定这些酶的功能方面的重要性。
