Molecular cloning and biochemical characterization of a NAD-dependent sorbitol dehydrogenase from cold-adapted Pseudomonas mandelii

Abstract

ABSTRACT Sugar alcohols (polyols) have important roles as nutrients, anti-freezing agents and scavengers of free radicals in cold-adapted bacteria, but the characteristics of polyol dehydrogenases in cold-adapted bacteria remain largely unknown. In this study, based on the observation that a cold-adapted bacterium Pseudomonas mandelii JR-1 predominantly utilized d-sorbitol as its carbon source, among the four polyols examined (d-galactitol, d-mannitol, d-sorbitol and d-xylitol), we cloned and characterized a sorbitol dehydrogenase (SDH, EC 1.1.1.14) belonging to the short-chain dehydrogenase/reductase family from this bacterium (the SDH hereafter referred to as PmSDH). PmSDH contained Asn111, Ser140, Tyr153 and Lys157 as catalytic active site residues and existed as an ∼67-kDa dimer in size-exclusion chromatography. PmSDH converted d-sorbitol to d-fructose using nicotinamide adenine dinucleotide (NAD+) as a cofactor and, vice versa, d-fructose to d-sorbitol using nicotinamide adenine dinucleotide reduced (NADH) as a cofactor. PmSDH maintained its conformational flexibility, secondary and tertiary structures, and thermal stability at 4–25°C. These results indicate that PmSDH, which has a flexible structure and a high catalytic activity at colder temperatures, is well suited to sorbitol utilization in the cold-adapted bacterium P. mandelii JR-1.