In silico analysis of chromium-reducing OXR genes derived from tannery effluent-contaminated soil metagenome

Tannery effluent waste comprises various potentially toxic metals, including chromium (Cr) with varying acute or chronic toxicity. Cr(VI) is known to be a category-A carcinogen. Reduction of toxic Cr(VI) to Cr(III), which has lesser bioavailability, is one of the mechanisms used by many microbes to withstand Cr(VI) toxicity in the contaminated effluents. Oxidoreductase (OXRs) reduces toxic Cr(VI) to Cr(III); hence a thorough understanding of the OXRs is important for developing a suitable strategy to minimize Cr(VI) toxicity. Therefore, the OXR-encoding genes were sequenced using metagenomic DNA shotgun sequencing from the tannery effluent-contaminated soil. Six OXR-encoding genes were expressed in Escherichia coli, and OXR activity was confirmed by in situ quantitative assays. The six proteins were subjected to phylogenetic and evolutionary analysis. Further, detailed structural analysis of the two OXRs, namely, OXR3 and OXR8 with lowest and highest activity respectively, were investigated in silico for structural characteristics. The results revealed that both the proteins were soluble FMN-linked oxidoreductases. Eight conserved active site residues (Pro24, Thr26, Ala59, Tyr139, His178, Tyr180, His219, Tyr221, Arg269, and Lys360) in the enzyme OXR3 were predicted. Similarly, nine conserved active site residues (Pro20, Thr22, Ala55, Glu97, His191, Tyr193, Arg241, Cys334, and Arg335) were predicted in OXR8. The tertiary structure of OXR8 was an aldolase TIM barrel structure, like Thermus scotoductus chromate reductase. Docking with FMN revealed the involvement of all the nine predicted active site residues in FMN binding with Pro20, Thr22, and Cys334 as the most important ones. The study investigated OXR-encoding genes that were sequenced using metagenomic DNA shotgun sequencing. In silico analysis revealed both the proteins were soluble FMN-linked oxidoreductases. Eight conserved active site residues were found in the enzyme OXR3 while nine were predicted in OXR8. The tertiary structure of OXR8 was an aldolase TIM barrel structure. P20, T22, and C334 active sites were confirmed by docking. image