Single amino acid mutation altered substrate specificity for l-glucose and inositol in scyllo-inositol dehydrogenase isolated from Paracoccus laeviglucosivorans

Abstract

scyllo-inositol dehydrogenase, isolated from Paracoccus laeviglucosivorans (Pl-sIDH), exhibits a broad substrate specificity: it oxidizes scyllo- and myo-inositols as well as l-glucose, converting l-glucose to l-glucono-1,5-lactone. Based on the crystal structures previously reported, Arg178 residue, located at the entry port of the catalytic site, seemed to be important for accepting substrates. Here, we report the role of Arg178 by using an alanine-substituted mutant for kinetic analysis as well as to determine the crystal structures. The wild-type Pl-sIDH exhibits the activity for scyllo-inositol most preferably followed by myo-inositol and l-glucose. On the contrary, the R178A mutant abolished the activities for both inositols, but remained active for l-glucose to the same extent as its wild-type. Based on the crystal structures of the mutant, the side chain of Asp191 flipped out of the substrate binding site. Therefore, Arg178 is important in positioning Asp191 correctly to exert its catalytic activities. Abbreviations: IDH: inositol dehydrogenase; LB: Luria-Bertani; kcat: catalyst rate constant; Km: Michaelis constant; NAD: nicotinamide dinucleotide; NADH: nicotinamide dinucleotide reduced form; PDB; Protein Data Bank; PDB entry: 6KTJ, 6KTK, 6KTL

Preparation and Application of 13C-Labeled myo-Inositol to Identify New Catabolic Products in Inositol Metabolism in Lactobacillus casei

“…Recent reports indicate that the observed activity of sIDH family enzymes on mI might not be uncommon and that it can lead to previously unknown metabolites. Paracoccus laeviglucosivorans sIDH has been shown to be active on mI forming 1 l - epi -2-inosose, , also a previously unidentified metabolite. We suggest the use of our protocol to determine if 1 l - epi -2-inosose is the only metabolic product of the oxidation.…”