Cytosolic Pyridoxine-b-D-Glucoside Hydrolase from Porcine Jejunal Mucosa

During studies of the nutritional utilization of pyridoxine 5*-b-D-glucoside, a major form of vitamin B6 in plants, we detected two cytosolic b-glucosidases in jejunal mucosa. As expected, one was broad specificity b-glucosidase that hydrolyzed aryl b-D-glycosides but not pyridoxine b-D-glucoside. We also found a previously unknown enzyme, designated pyridoxine-b-D-glucoside hydrolase, that efficiently hydrolyzed pyridoxine b-Dglucoside. These were separated and purified as follows: broad specificity b-glucosidase 1460-fold and pyridoxine-b-D-glucoside hydrolase 36,500-fold. Purified pyridoxine-b-D-glucoside hydrolase did not hydrolyze any of the aryl glycosides tested but did hydrolyze cellobiose and lactose. Pyridoxine-b-D-glucoside hydrolase exhibited a pH optimum of 5.5 and apparent molecular mass of 130 kDa by SDS-polyacrylamide gel electrophoresis and 160 kDa by nondenaturing gel filtration, in contrast to 60 kDa for native and denatured broad specificity b-glucosidase. Glucono-d-lactone was a strong inhibitor of both enzymes. Ionic and nonionic detergents were inhibitory for each enzyme. Conduritol B epoxide, a potent inhibitor of lysosomal acid b-glucosidase, inhibited pyridoxine-b-D-glucoside hydrolase but not broad specificity b-glucosidase, but both were inhibited by the mechanism-based inhibitor 2-deoxy-2-fluoro-b-D-glucosyl fluoride. Our findings indicate major differences between these two cytosolic b-glucosidases. Studies addressing the role of vitamin B6 nutrition in regulating the activity and its consequences regarding pyridoxine glucoside bioavailability are in progress.