Abstract
Short-chain dehydrogenase reductases (SDRs) have been utilized for catalyzing the reduction of many aromatic/aliphatic prochiral ketones to their respective alcohols. However, there is a paucity of data that elucidates their innate biological role and diverse substrate space. In this study, we executed an in-depth biochemical characterization and substrate space mapping (with 278 prochiral ketones) of an unannotated SDR (DHK) from Debaryomyces hansenii and compared it with structurally and functionally characterized SDR Synechococcus elongatus. PCC 7942 FabG to delineate its industrial significance. It was observed that DHK was significantly more efficient than FabG, reducing a diverse set of ketones albeit at higher conversion rates. Comparison of the FabG structure with a homology model of DHK and a docking of substrate to both structures revealed the presence of additional flexible loops near the substrate binding site of DHK. The comparative elasticity of the cofactor and substrate binding site of FabG and DHK was experimentally substantiated using differential scanning fluorimetry. It is postulated that the loop flexibility may account for the superior catalytic efficiency of DHK although the positioning of the catalytic triad is conserved.
Highlights
Short-chain dehydrogenase reductases (SDRs) were first characterized in Drosophila melanogaster and were found to have similar catalytic properties like alcohol dehydrogenases (Medium Chain Dehydrogenase Reductase) albeit having lesser length
NADPH, NADH, Ethyl 4-chloro acetoacetate, DMSO were purchased from Sigma-Aldrich, USA, Sodium Phosphate monobasic, Sodium Phosphate dibasic, Sodium Chloride, Imidazole were purchased from Amresco, USA and was the finest grade available
Differential scanning fluorimetry showed that FabG is more rigid compared to DHK, wherein after binding with NADPH both enzymes display a 7 ̊C shift which shows the protein with cofactor to be more stable
Summary
Short-chain dehydrogenase reductases (SDRs) were first characterized in Drosophila melanogaster and were found to have similar catalytic properties like alcohol dehydrogenases (Medium Chain Dehydrogenase Reductase) albeit having lesser length. They comprise a large superfamily of lyases, isomerases, epimerases, dehydrogenases, etc. Showing similar architecture viz. Rossmann folds and a conserved catalytic triad. SDRs are divided into seven major classes namely classical, extended, intermediate, complex, atypical, divergent and unknown. Most of the dehydrogenases and reductases catalyze the same type of reaction, they are grouped in either classical or extended families and are ubiquitous in plants, fungi, and bacteria. Classical and extended SDRs include almost 30,000 members across 150 subfamilies.
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