Cytosolic Pyridoxine-β-d-Glucoside Hydrolase from Porcine Jejunal Mucosa

Abstract

During studies of the nutritional utilization of pyridoxine 5-␤-D-glucoside, a major form of vitamin B6 in plants, we detected two cytosolic ␤-glucosidases in jejunal mucosa. As expected, one was broad specificity ␤-glucosidase that hydrolyzed aryl ␤-D-glycosides but not pyridoxine ␤-D-glucoside. We also found a previously unknown enzyme, designated pyridoxine-␤-D-glucoside hydrolase, that efficiently hydrolyzed pyridoxine ␤-Dglucoside. These were separated and purified as follows: broad specificity ␤-glucosidase 1460-fold and pyridoxine-␤-D-glucoside hydrolase 36,500-fold. Purified pyridoxine-␤-D-glucoside hydrolase did not hydrolyze any of the aryl glycosides tested but did hydrolyze cellobiose and lactose. Pyridoxine-␤-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 ␤-glucosidase. Glucono-␦-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 ␤-glucosidase, inhibited pyridoxine-␤-D-glucoside hydrolase but not broad specificity ␤-glucosidase, but both were inhibited by the mechanism-based inhibitor 2-deoxy-2-fluoro-␤-D-glucosyl fluoride. Our findings indicate major differences between these two cytosolic ␤-glucosidases. Studies addressing the role of vitamin B6 nutrition in regulating the activity and its consequences regarding pyridoxine glucoside bioavailability are in progress.A naturally occurring glycosylated derivative of vitamin B6, pyridoxine 5Ј-␤-D-glucoside (PNG), 1 was first isolated from rice bran (1). PNG is now known to exist in most fruits, vegetables, and cereal grains, in which it comprises from 5-75% of total vitamin B6 (2, 3). Because of the prevalence of PNG in plantderived foods, its bioavailability as a source of available vitamin B6 is a matter of nutritional concern. The bioavailability of this glycosylated form of vitamin B6, relative to pyridoxine (PN), is ϳ25% in rats (4, 5) and ϳ50% for humans (6, 7), as estimated from urinary excretion of 4-pyridoxic acid after oral administration of isotopically labeled PNG. The rate-limiting phase of PNG utilization in vitamin B6 metabolism is the hydrolysis of the ␤-glycosidic bond in both rats and humans. These findings indicate that the hydrolysis of PNG is a major factor governing its bioavailability.Glucosidases exist widely in nature. The primary ␤-glucosidases in mammalian tissues consist of a lysosomal membranebound acid ␤-glucosidase (EC 3.2.1.45; N-acylsphingosyl-␤-Dglucopyranoside glucohydrolase) which is responsible for the hydrolytic cleavage of glucosphingolipids (for review see Ref. 8), and a cytosolic ␤-glucosidase capable of hydrolyzing a variety of aryl ␤-D-glycosides (for review see Ref. 9). This cytosolic enzyme has been detected in a variety of mammalian tissues and has been designated broad specificity ␤-glucosidase. A...

β-Glucosidases from the FungusTrichoderma: An Efficient Cellulase Machinery in Biotechnological Applications

“…Studies on β -glucosidases have been carried out from different sources, namely, microbes, plants, and animals [7–14]. Amongst these, fungal sources are immensely explored due to their better prospects in commercial applications.…”

“…The specificity of β -glucosidases is variable towards different substrates depending on the enzyme source. The enzyme is ubiquitously present in nature and found in bacteria [7], fungi [8], yeasts [9], plants [10–13], and animals [14], respectively. …”

All-enzymatic HPLC method for determination of individual and total contents of vitamin B6 in foods

“…The published method shows separate quantification of PN, PL, PM, and PNG (3). The nutritional availability of PNG in the human body is controversial (4). …”

Enzymatic Hydrolysis of Pyridoxine-5′-β-d-glucoside Is Catalyzed by Intestinal Lactase-Phlorizin Hydrolase

“…Cytosolic PNG hydrolysis was also reported to be inversely related to vitamin B 6 nutritional status in rodents (13,14). To further study cytosolic PNG hydrolysis, this laboratory purified BS␤G from pig intestinal mucosa, which led to the identification and subsequent purification of a distinct and novel cytosolic ␤-glucosidase, designated pyridoxine-5Ј-␤-D-glucoside hydrolase (PNG hydrolase) (15). It was found that PNG hydrolase, not BS␤G, was responsible for the cytosolic cleavage of PNG.…”

“…It was found that PNG hydrolase, not BS␤G, was responsible for the cytosolic cleavage of PNG. Partial characterization of cytosolic PNG hydrolase revealed its ability to hydrolyze PNG as well as lactose and cellobiose but not sucrose (15). Partial amino acid analysis showed substantial sequence homology but revealed significant differences between PNG hydrolase and the brush border membrane enzyme lactase phlorizin hydrolase (LPH) (EC 3.2.1.23, EC 3.2.1.62, and EC 3.2.1.108) (16,17), the enzyme primarily responsible for the hydrolysis of dietary lactose.…”