K-091. Transcriptional and Functional Characterization of a Gene Encoding a Predicted Azoreductase in Shewanella oneidensis MR-1

I. Mugerfeld1, B. Law1, X-F. Wan2, S. Brown3, D. K. Thompson1;
1Purdue Univ., West Lafayette, IN, 2CDC, Atlanta, GA, 3Oak Ridge Natl. Lab., Oak Ridge, TN.

Azoreductases are enzymes that catalyze the reductive cleavage of azo dyes to aromatic amines. Azo dyes are of significant concern as xenobiotic pollutants that are released into the environment as industrial effluents from textile and dye manufacturing. Shewanella oneidensis MR-1 is a facultatively anaerobic gamma-proteobacterium that can respire anaerobically by reducing a wide variety of alternative electron acceptors. The gene SO3585 from MR-1 is annotated as a putative azoreductase and was shown in our previous studies to be upregulated at both the mRNA and protein levels in response to acute chromate exposure. The gene SO3585 encodes for a 204 amino acid residue protein (with a calculated molecular mass of 23 kDa), which displays 28% and 31% identity to the azoreductases from Bacillus subtilis and Bacillus cereus, respectively, and 28% identity to an NADPH-dependent FMN reductase from Pseudomonas putida GB-1. Regions of conservation in the deduced amino acid sequence of SO3585 include the molecular signature characteristic of the NADH_dh2 family of NAD(P)H oxidoreductases. In addition, SO3585 orthologs are present and conserved among other Shewanella species sequenced to date. RT-PCR analysis demonstrated that the SO3585 gene is co-transcribed with two downstream open-reading frames: SO3586 (glyoxalase family protein) and SO3587 (hypothetical protein). The transcriptional start of the polycistronic transcript was localized using 5' RACE. A mutant strain defective in SO3585 was comparable to the parental MR-1 strain in its ability to decolorize the azo dyes Orange II, Direct Blue 15, and Nitro Red under aerobic conditions. To characterize the encoded product of the SO3585 gene from a biochemical perspective, SO3585 was heterologously expressed in Escherichia coli as a recombinant fusion protein carrying an enterokinase-cleavable His-Patch thioredoxin at its 5' terminus. Studies are underway to purify the recombinant SO3585 protein and define its substrate specificity and coenzyme requirement for activity.