Structural and biochemical characterization of an atypical short-chain dehydrogenase/reductase reveals an unusual cofactor preference.

Short-chain dehydrogenases/reductases (SDRs) encompass a large and functionally diverse family of enzymes with representative members in all kingdoms of life. Despite the wealth of reactions catalyzed by SDRs, they operate through a well-conserved and efficient reaction mechanism centered in a conserved catalytic tetrad (Asn-Ser-Tyr-Lys) and the employment of an appropriate cofactor. In recent years, SDRs that lack the signature catalytic tetrad have been identified, thus adding a perplexing twist to SDR functionality. In the present study, we report the crystal structure of SDRvv, an atypical SDR from Vibrio vulnificus devoid of the catalytic tetrad, thereby defining the structural signature of this apparent SDR family outlier. Further structural analysis of SDRvv in complex with its putative cofactor NADPH, site-directed mutagenesis and binding studies via isothermal titration calorimetry, and further biochemical characterization have allowed us to dissect the cofactor preferences of SDRvv. The retained capacity to bind the NADPH cofactor, the conceivable existence of a proton relay and the conservation of the coordination distances between the key residues in the cofactor binding pocket define a first set of rules towards catalytic activity for SDRvv. The findings of the present study set the stage for deriving the identity of the natural substrate of SDRvv and add a new twist to the structure-function landscape for Rossmann-fold-dependent cofactor discrimination.

Crystallization of an atypical short-chain dehydrogenase from Vibrio vulnificus lacking the conserved catalytic tetrad.

Short-chain dehydrogenases/reductases (SDRs) are a rapidly expanding superfamily of enzymes that are found in all kingdoms of life. Hallmarked by a highly conserved Asn-Ser-Tyr-Lys catalytic tetrad, SDRs have a broad substrate spectrum and play diverse roles in key metabolic processes. Locus tag VVA1599 in Vibrio vulnificus encodes a short-chain dehydrogenase (hereafter referred to as SDRvv) which lacks the signature catalytic tetrad of SDR members. Structure-based protein sequence alignments have suggested that SDRvv may harbour a unique binding site for its nicotinamide cofactor. To date, structural studies of SDRs with altered catalytic centres are underrepresented in the scientific literature, thus limiting understanding of their spectrum of substrate and cofactor preferences. Here, the expression, purification and crystallization of recombinant SDRvv are presented. Two well diffracting crystal forms could be obtained by cocrystallization in the presence of the reduced form of the phosphorylated nicotinamide cofactor NADPH. The collected data were of sufficient quality for successful structure determination by molecular replacement and subsequent refinement. This work sets the stage for deriving the identity of the natural substrate of SDRvv and the structure-function landscape of typical and atypical SDRs.

Bacterial decolorization of textile dyes is an extracellular process requiring a multicomponent electron transfer pathway.

Many studies have reported microorganisms as efficient biocatalysts for colour removal of dye-containing industrial wastewaters. We present the first comprehensive study to identify all molecular components involved in decolorization by bacterial cells. Mutants from the model organism Shewanella oneidensis MR-1, generated by random transposon and targeted insertional mutagenesis, were screened for defects in decolorization of an oxazine and diazo dye. We demonstrate that decolorization is an extracellular reduction process requiring a multicomponent electron transfer pathway that consists of cytoplasmic membrane, periplasmic and outer membrane components. The presence of melanin, a redox-active molecule excreted by S. oneidensis, was shown to enhance the dye reduction rates. Menaquinones and the cytochrome CymA are the crucial cytoplasmic membrane components of the pathway, which then branches off via a network of periplasmic cytochromes to three outer membrane cytochromes. The key proteins of this network are MtrA and OmcB in the periplasm and outer membrane respectively. A model of the complete dye reduction pathway is proposed in which the dye molecules are reduced by the outer membrane cytochromes either directly or indirectly via melanin.