Characterization of Sorbitol Dehydrogenase SmoS fromSinorhizobium meliloti1021

Abstract

AbstractSinorhizobium meliloti1021 is a Gram-negative alphaproteobacterium with a robust capacity for carbohydrate metabolism. The enzymes that facilitate these reactions assist in the survival of the bacterium across a range of environmental niches, and they may also be suitable for use in industrial processes. SmoS is a dehydrogenase that catalyzes the oxidation of the commonly occurring sugar alcohols sorbitol and galactitol into fructose and tagatose respectively using NAD+as a cofactor. The main objective of this study is to evaluate SmoS using biochemical techniques. The nucleotide sequence was codon optimized for heterologous expression inE. coliBL21 (DE3) GOLD cells, the protein was subsequently overexpressed and purified. Size exclusion chromatography and X-ray diffraction experiments suggest that SmoS is a tetrameric peptide. SmoS was crystallized to 2.1 Å in the absence of substrate and 2.0 Å in complex with sorbitol. SmoS was characterized kinetically and shown to have a preference for sorbitol despite a higher affinity for galactitol. Computational ligand docking experiments suggest that galactitol oxidation proceeds slowly because tagatose binds the protein in a more energetically favorable complex than fructose, and is retained in the active site for a longer time frame following oxidation which reduces the rate of the reaction. These results supplement the inventory of biomolecules with the potential for industrial applications and enhance our understanding of metabolism in the model organismS. meliloti.