Respiratory Selenite Reductase from Bacillus selenitireducens Strain MLS10

The putative respiratory selenite [Se(IV)] reductase (Srr) from MLS10 has been identified through a polyphasic approach involving genomics, proteomics, and enzymology. Non-denaturing 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 LC-MS-MS as a homolog of the catalytic subunit of polysulfide reductase (PsrA). It was found to be part of an operon that contains six genes that we designated srrE, srrA , s rrB, srrC, srrD, srrF . SrrA is the catalytic subunit (80 kDa), with a 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 4Fe4S binding sites, SrrC (43 kDa) is an anchoring subunit, and SrrD (24 kDa) a chaperon protein. Both SrrE (38 kDa) and SrrF (45 kDa) where 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., Desulfurispirillum indicum, Pyrobaculum aerophilum ). The enzyme appeared to be specific for Se(IV), showing no activity with selenate, arsenate, or thiosulfate, with a K m of 145 ± 53 μM, V max of 23 ± 2.5 μMmin -1 , and a kcat of 23 ± 2.68 sec -1 . These results further our understanding of the mechanisms of selenium biotransformation and its biogeochemical cycle. IMPORTANCE 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 Bacillus selenitireducens 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.