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

Abstract

An obligatory step in the mammalian nutritional utilization of pyridoxine-5-␤-D-glucoside (PNG) is the intestinal hydrolysis of its ␤-glucosidic bond that releases pyridoxine (PN). This laboratory previously reported the purification and partial characterization of a novel cytosolic enzyme, designated PNG hydrolase, which hydrolyzed PNG. An investigation of the subcellular distribution of intestinal PNG hydrolysis found substantial hydrolytic activity in the total membrane fraction, of which 40 -50% was localized to brush border membrane. To investigate the possible role of a brush border ␤-glucosidase in the hydrolysis of PNG, lactase phlorizin hydrolase (LPH) was purified from rat small intestinal mucosa. LPH hydrolyzed PNG with a K m of 1.0 ؎ 0.1 mM, a V max of 0.11 ؎ 0.01 mol/min⅐mg protein, and a k cat of 1.0 s ؊1 . LPH-catalyzed PNG hydrolysis was inhibited by glucose, lactose, and cellobiose but not by PN. Specific blockage of the phlorizin hydrolase site of LPH using 2,4-dintrophenyl-2-fluoro-2-deoxy-␤-D-glucopyranoside did not reduce PNG hydrolysis. Evidence of transferase activity was also obtained. Reaction mixtures containing LPH, PNG, and lactose yielded the formation of another PN derivative that was identified as a pyridoxine disaccharide. These results indicate that LPH may play an important role in the bioavailability of PNG, but further characterization is needed to assess its physiological function.A significant dietary source of vitamin B 6 for humans is provided by a glycosylated form of the vitamin, pyridoxine-5Ј-␤-D-glucoside (PNG).1 PNG, found predominantly in foods of plant origin, provides 15% of total vitamin B 6 in a mixed diet, depending on food selection (1, 2). PNG exhibits ϳ50% bioavailability in humans (3, 4) and 25-30% in rodents (5-8) in comparison to pyridoxine, the metabolically usable form of vitamin B 6 . The rate-limiting step in the utilization of PNG is not its intestinal absorption but rather the enzymatic hydrolysis of the ␤-glucosidic linkage to release glucose and pyridoxine in the intestine (6 -8). Although PNG can be absorbed intact, this form is not metabolized or retained by the liver (6 -8) and can antagonize the metabolism of nonglycosylated forms of vitamin B 6 (9, 10). The remainder of PNG that is not absorbed is likely to be accounted for through fecal losses.This laboratory has examined the intracellular (i.e. cytosolic) hydrolysis of PNG in mammalian small intestinal mucosa. Intestinal cytosolic PNG hydrolysis was initially thought to be catalyzed by a broad specificity ␤-glucosidase (BS␤G) (EC 3.2.1.21) (11), a cytosolic enzyme found in the intestine and liver (12-14). 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 hydrolas...

Dietary Reference Values for vitamin B6

“…Pyridoxine-5 0 -b-D-glucoside (PNG) (Section 2.1) is hydrolysed enzymatically by the PNG hydrolase (Nakano et al, 1997) and the brush border lactase-phlorizin hydrolase (Mackey et al, 2002) before its absorption in the small intestine. However, PNG can also be absorbed unchanged (IOM, 1998).…”

Systemic lactose intolerance: a new perspective on an old problem

“…Lactase (lactase phlorizinhydrolase-LPH, EC 3.2.1.62/108; note b galactosidase is EC 3.2.1.23, mistakenly used for lactase in some publications) is highly unusual, having two active sites within one polypeptide chain, one hydrolysing lactose, the other aryl and aliphatic glycosides such as phlorizin into glucose and phloretin, the latter being a potent diabetic agent. Two important natural substrates for this latter site are cerebrosides, a crucial source of sphingosine, and glycosyl-pyridoxal, 27 a vital source of vitamin B6. Lactase has no sequence similarity to its bacterial counterpart b galactosidase.…”

Partial amino acid sequence and mRNA analysis of cytosolic pyridoxine-beta-d-glucoside hydrolase from porcine intestinal mucosa: proposed derivation from the lactase-phlorizin hydrolase gene

“…We assumed that the activity of cytosolic PNG hydrolase is physiologically relevant in releasing pyridoxine from the fraction of dietary PNG taken up by enterocytes without prior hydrolysis. We have shown that purified LPH of the brush-border membrane also catalyses PNG hydrolysis [12]. This indicates that PNG is a secondary substrate for LPH and that either partial hydrolysis of dietary PNG in the mammalian small intestine can occur at the brush-border membrane prior to absorption or PNG can be absorbed in intact form and hydrolysed by cytosolic PNGH.…”