Characterisation of a catalytic triad and reaction selectivity in the dual mechanism of the catalyse hydride transfer in xylitol phosphate dehydrogenase.

Xylitol is a high-value low-calorie sweetener used as sugar substitute in food and pharmaceutical industry. Xylitol phosphate dehydrogenase (XPDH) catalyses the conversion of d-xylulose 5-phosphate (XU5P) and d-ribulose 5-phosphate (RU5P) to xylitol and ribitol respectively in the presence of nicotinamide adenine dinucleotide hydride (NADH). Although these enzymes have been shown to produce xylitol and ribitol, there is an incomplete understanding of the mechanism of the catalytic events of these reactions and the detailed mechanism has yet to be elucidated. The main goal of this work is to analyse the conformational changes of XPDH-bound ligands such as zinc NADH, XU5P, and RU5P to elucidate the key amino acids involved in the substrate binding. In silico modelling, comparative molecular dynamics simulations, interaction analysis and conformational study were carried out on three XPDH enzymes of the Medium-chain dehydrogenase (MDR) family in order to elucidate the atomistic details of conformational transition, especially on the open and closed state of XPDH. The analysis also revealed the possible mechanism of substrate specificity that are responsible in the catalyse hydride transfer are the residues His58 and Ser39 which would act as the proton donor for reduction of XU5P and RU5P respectively. The structural comparison and MD simulations displayed a significant difference in the conformational dynamics of the catalytic and coenzyme loops between Apo and XPDH-complexes and highlight the contribution of newly found triad residues. This study would assist future mutagenesis study and enzyme modification work to increase the catalysis efficiency of xylitol production in the industry.