Identification and functional characterization of a novel CaSrpA enzyme for selenite reduction and selenium nanoparticle formation

Abstract

Selenite reductases are widely distributed across various oxidoreductase families (e.g., ThxR, OYE, and FccA enzymes) [1]. The ability of short-chain dehydrogenase/reductase (SDR) family enzymes for selenite reduction remains unknown. Using metagenomic and metatranscriptomic analyses, 40 putative selenite reductases were identified from selenium-rich regions based on catalytic domain homology and transcriptional upregulation. These enzymes mainly belong to the SDR family and metalloenzymes. Enzyme activity analysis indicated that CaSrpA possessed the ability (V_max, 18.85 μM/min/g) to reduce selenite to selenium nanoparticles (SeNPs). Phylogenetic analysis showed that CaSrpA was clustered in the clade of SDR enzymes, with the typical Rossmann fold. CaSrpA also oxidized S-1-phenylethanol to phenylacetone (V_max, 15.4 μM/min/mg), sharing 53 % sequence similarity with the alcohol dehydrogenase RasADH. Molecular docking and structural superposition identified sixteen key residues associated with CaSrpA activity. Site-directed mutagenesis revealed that over 14 mutants exhibited a 30–90 % reduction in relative activity. Mutant M206A enhanced catalytic efficiency towards selenite by 2.4-fold and S-1-phenylethanol by 5.4-fold via a lid-opening mechanism. Molecular dynamics simulation elucidated that the mutant M206A utilized lid opening mechanism to accommodate more substrate and co-factor for catalysis via altering the conformation of the α7-α8 loop. This study helps understand the intrinsic connection between the SDR family and selenite-reducing capability, broadening the repertoire of selenite reductases.