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Steven E Ealick - One of the best experts on this subject based on the ideXlab platform.
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the crystal structure of streptococcus pyogenes uridine Phosphorylase reveals a distinct subfamily of nucleoside Phosphorylases
Biochemistry, 2011Co-Authors: Timothy H Tran, Stig Christoffersen, Paula W Allan, William B Parker, Jure Piskur, Immacolata Serra, Marco Terreni, Steven E EalickAbstract:Uridine Phosphorylase (UP), a key enzyme in the pyrimidine salvage pathway, catalyzes the reversible phosphorolysis of uridine or 2'-deoxyuridine to uracil and ribose 1-phosphate or 2'-deoxyribose 1-phosphate. This enzyme belongs to the nucleoside Phosphorylase I superfamily whose members show diverse specificity for nucleoside substrates. Phylogenetic analysis shows Streptococcus pyogenes uridine Phosphorylase (SpUP) is found in a distinct branch of the pyrimidine subfamily of nucleoside Phosphorylases. To further characterize SpUP, we determined the crystal structure in complex with the products, ribose 1-phosphate and uracil, at 1.8 A resolution. Like Escherichia coli UP (EcUP), the biological unit of SpUP is a hexamer with an α/β monomeric fold. A novel feature of the active site is the presence of His169, which structurally aligns with Arg168 of the EcUP structure. A second active site residue, Lys162, is not present in previously determined UP structures and interacts with O2 of uracil. Biochemical studies of wild-type SpUP showed that its substrate specificity is similar to that of EcUP, while EcUP is ∼7-fold more efficient than SpUP. Biochemical studies of SpUP mutants showed that mutations of His169 reduced activity, while mutation of Lys162 abolished all activity, suggesting that the negative charge in the transition state resides mostly on uracil O2. This is in contrast to EcUP for which transition state stabilization occurs mostly at O4.
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structural basis for inhibition of escherichia coli uridine Phosphorylase by 5 substituted acyclouridines
Acta Crystallographica Section D-biological Crystallography, 2005Co-Authors: Ethan C. Settembre, Mahmoud H. El Kouni, Steven E EalickAbstract:Uridine Phosphorylase (UP) catalyzes the reversible phosphorolysis of uridine to uracil and ribose 1-phosphate and is a key enzyme in the pyrimidine-salvage pathway. Escherichia coli UP is structurally homologous to E. coli purine nucleoside Phosphorylase and other members of the type I family of nucleoside Phosphorylases. The structures of 5-benzylacyclouridine, 5-phenylthioacyclouridine, 5-phenylselenenylacyclouridine, 5-m-benzyloxybenzyl acyclouridine and 5-m-benzyloxybenzyl barbituric acid acyclonucleoside bound to the active site of E. coli UP have been determined, with resolutions ranging from 1.95 to 2.3 A. For all five complexes the acyclo sugar moiety binds to the active site in a conformation that mimics the ribose ring of the natural substrates. Surprisingly, the terminal hydroxyl group occupies the position of the nonessential 5′-hydroxyl substituent of the substrate rather than the 3′-hydroxyl group, which is normally required for catalytic activity. Until recently, inhibitors of UP were designed with limited structural knowledge of the active-site residues. These structures explain the basis of inhibition for this series of acyclouridine analogs and suggest possible additional avenues for future drug-design efforts. Furthermore, the studies can be extended to design inhibitors of human UP, for which no X-ray structure is available.
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Purine nucleoside Phosphorylase. 2. Catalytic mechanism.
Biochemistry, 1997Co-Authors: Mark D. Erion, Johanna D. Stoeckler, Wayne C. Guida, Richard L. Walter, Steven E EalickAbstract:X-ray crystallography, molecular modeling, and site-directed mutagenesis were used to delineate the catalytic mechanism of purine nucleoside Phosphorylase (PNP). PNP catalyzes the reversible phosphorolysis of purine nucleosides to the corresponding purine base and ribose 1-phosphate using a substrate-assisted catalytic mechanism. The proposed transition state (TS) features an oxocarbenium ion that is stabilized by the cosubstrate phosphate dianion which itself functions as part of a catalytic triad (Glu89-His86-PO4). Participation of phosphate in the TS accounts for the poor hydrolytic activity of PNP and is likely to be the mechanistic feature that differentiates Phosphorylases from glycosidases. The proposed PNP TS also entails a hydrogen bond between N7 and a highly conserved Asn. Hydrogen bond donation to N7 in the TS stabilizes the negative charge that accumulates on the purine ring during glycosidic bond cleavage. Kinetic studies using N7-modified analogs provided additional support for the hydrogen...
Yutaka Tanaka - One of the best experts on this subject based on the ideXlab platform.
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preferential inhibition of bone metastases by 5 deoxy 5 fluorouridine and capecitabine in the 4t1 luc mouse breast cancer model
Oncology Reports, 2005Co-Authors: Toru Hiraga, Kenji Hata, Fumiyo Ikeda, Jirota Kitagaki, Kaori Fujimotoouchi, Yutaka Tanaka, Toshiyuki YonedaAbstract:5'-deoxy-5-fluorouridine (5'-DFUR) and capecitabine are oral anti-cancer agents, which are enzymatically converted to 5-fluorouracil (5-FU) by thymidine Phosphorylase in humans and uridine Phosphorylase in mice. Since the activity of these Phosphorylases is higher in cancerous tissue than in normal tissue, systemic administration of 5'-DFUR and capecitabine achieves high intratumoral 5-FU levels and low adverse effects on non-tumoral tissue. Accordingly, 5'-DFUR and capecitabine are widely used for the treatment of cancer patients. In the present study, we examined the effects of 5'-DFUR and capecitabine on bone metastases, one of the most common complications of breast cancer, using an animal model in which inoculation of 4T1/luc mouse breast cancer cells into the mammary fat pads of female BALB/c mice developed spontaneous metastases in distant organs including bone, lung and liver. Mice received 4T1/luc cell inoculation in the mammary fat pad at day 0 and oral 5'-DFUR (31, 62, 123 or 246 mg/kg) or capecitabine (90, 180 or 359 mg/kg) daily from day 7 to day 21. Both 5'-DFUR and capecitabine significantly inhibited orthotopic tumor formation and distant metastases to bone, lung and liver in a dose-dependent manner. Of note, the lowest dose of 5'-DFUR (31 mg/kg) and capecitabine (90 mg/kg), which failed to inhibit orthotopic tumor development and the lung and liver metastases, significantly reduced the bone metastases. In conclusion, our results suggest that oral 5'-DFUR and capecitabine are effective for the treatment of primary and secondary breast tumors. Most notably, they also suggest that these agents are preferentially beneficial for bone metastases.
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induction of thymidine Phosphorylase expression and enhancement of efficacy of capecitabine or 5 deoxy 5 fluorouridine by cyclophosphamide in mammary tumor models
International Journal of Cancer, 1999Co-Authors: Mika Endo, Hideo Ishitsuka, Noriko Shinbori, Yu Fukase, Noriaki Sawada, Tohru Ishikawa, Yutaka TanakaAbstract:Thymidine Phosphorylase (dThdPase) is an essential enzyme for the activation of the oral cytostatic drugs capecitabine (N4-pentyloxycarbonyl-5′-deoxy-5-fluorocytidine, Xeloda™) and its intermediate metabolite doxifluridine [5′-deoxy-5-fluorouridine (5′-dFUrd, Furtulon®)] to 5-fluorouracil (5-FUra) in tumors. In a previous study, we found that several cytostatics were able to up-regulate tumor levels of dThdPase in a human colon cancer xenograft model. In the present study, we confirmed that the administration of cytostatics used for breast cancer treatment, such as taxanes and cyclophosphamide (CPA), up-regulated the tumor level of dThdPase in mammary tumor models as well. Because the dThdPase up-regulation was observed even when CPA was given orally, we investigated further the usefulness of combination therapy with the 2 oral drugs, 5′-dFUrd/capecitabine and CPA in mammary tumor models. Daily oral administration of CPA up-regulated human dThdPase levels in the tumor tissue of mice bearing a human mammary tumor xenograft, MX-1, whereas in the small intestine and liver, it did not affect levels of pyrimidine nucleoside Phosphorylases (PyNPase) including dThdPase and uridine Phosphorylase. The preferential up-regulation of PyNPase activity in the tumor by CPA administration was also confirmed in mice bearing a syngeneic murine mammary adenocarcinoma, A755. In both models, combination therapy of 5′-dFUrd/capecitabine with CPA showed synergistic antitumor activity, without significant potentiation of toxicity. In contrast, treatment with CPA and either 5-FUra or UFT (a mixture of tegafur and uracil) in combination showed only additive activity. Our results suggest that CPA and capecitabine/5′-dFUrd, both available for oral administration, would be good partners, and that clinical trials with this drug combination against breast cancer are warranted. Int. J. Cancer 83:127–134, 1999. © 1999 Wiley-Liss, Inc.
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induction of thymidine Phosphorylase expression and enhancement of efficacy of capecitabine or 5 deoxy 5 fluorouridine by cyclophosphamide in mammary tumor models
International Journal of Cancer, 1999Co-Authors: Mika Endo, Hideo Ishitsuka, Noriko Shinbori, Yu Fukase, Noriaki Sawada, Tohru Ishikawa, Yutaka TanakaAbstract:Thymidine Phosphorylase (dThdPase) is an essential enzyme for the activation of the oral cytostatic drugs capecitabine (N(4)-pentyloxycarbonyl-5'-deoxy-5-fluorocytidine, Xeloda(trade mark)) and its intermediate metabolite doxifluridine [5'-deoxy-5-fluorouridine (5'-dFUrd, Furtulon((R)))] to 5-fluorouracil (5-FUra) in tumors. In a previous study, we found that several cytostatics were able to up-regulate tumor levels of dThdPase in a human colon cancer xenograft model. In the present study, we confirmed that the administration of cytostatics used for breast cancer treatment, such as taxanes and cyclophosphamide (CPA), up-regulated the tumor level of dThdPase in mammary tumor models as well. Because the dThdPase up-regulation was observed even when CPA was given orally, we investigated further the usefulness of combination therapy with the 2 oral drugs, 5'-dFUrd/capecitabine and CPA in mammary tumor models. Daily oral administration of CPA up-regulated human dThdPase levels in the tumor tissue of mice bearing a human mammary tumor xenograft, MX-1, whereas in the small intestine and liver, it did not affect levels of pyrimidine nucleoside Phosphorylases (PyNPase) including dThdPase and uridine Phosphorylase. The preferential up-regulation of PyNPase activity in the tumor by CPA administration was also confirmed in mice bearing a syngeneic murine mammary adenocarcinoma, A755. In both models, combination therapy of 5'-dFUrd/capecitabine with CPA showed synergistic antitumor activity, without significant potentiation of toxicity. In contrast, treatment with CPA and either 5-FUra or UFT (a mixture of tegafur and uracil) in combination showed only additive activity. Our results suggest that CPA and capecitabine/5'-dFUrd, both available for oral administration, would be good partners, and that clinical trials with this drug combination against breast cancer are warranted.
Hiroyuki Nakai - One of the best experts on this subject based on the ideXlab platform.
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discovery of two β 1 2 mannoside Phosphorylases showing different chain length specificities from thermoanaerobacter sp x 514
PLOS ONE, 2014Co-Authors: Kazuhiro Chiku, Takanori Nihira, Erika Suzuki, Ken'ichi Ohtsubo, Mamoru Nishimoto, Motomitsu Kitaoka, Hiroyuki NakaiAbstract:We characterized Teth514_1788 and Teth514_1789, belonging to glycoside hydrolase family 130, from Thermoanaerobacter sp. X-514. These two enzymes catalyzed the synthesis of 1,2-β-oligomannan using β-1,2-mannobiose and d-mannose as the optimal acceptors, respectively, in the presence of the donor α-d-mannose 1-phosphate. Kinetic analysis of the phosphorolytic reaction toward 1,2-β-oligomannan revealed that these enzymes followed a typical sequential Bi Bi mechanism. The kinetic parameters of the phosphorolysis of 1,2-β-oligomannan indicate that Teth514_1788 and Teth514_1789 prefer 1,2-β-oligomannans containing a DP ≥3 and β-1,2-Man2, respectively. These results indicate that the two enzymes are novel inverting Phosphorylases that exhibit distinct chain-length specificities toward 1,2-β-oligomannan. Here, we propose 1,2-β-oligomannan:phosphate α-d-mannosyltransferase as the systematic name and 1,2-β-oligomannan Phosphorylase as the short name for Teth514_1788 and β-1,2-mannobiose:phosphate α-d-mannosyltransferase as the systematic name and β-1,2-mannobiose Phosphorylase as the short name for Teth514_1789.
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Discovery of β-1,4-d-Mannosyl-N-acetyl-d-glucosamine Phosphorylase Involved in the Metabolism of N-Glycans
Journal of Biological Chemistry, 2013Co-Authors: Takanori Nihira, Erika Suzuki, Ken'ichi Ohtsubo, Mamoru Nishimoto, Motomitsu Kitaoka, Hiroyuki NakaiAbstract:Abstract A gene cluster involved in N-glycan metabolism was identified in the genome of Bacteroides thetaiotaomicron VPI-5482. This gene cluster encodes a major facilitator superfamily transporter, a starch utilization system-like transporter consisting of a TonB-dependent oligosaccharide transporter and an outer membrane lipoprotein, four glycoside hydrolases (α-mannosidase, β-N-acetylhexosaminidase, exo-α-sialidase, and endo-β-N-acetylglucosaminidase), and a Phosphorylase (BT1033) with unknown function. It was demonstrated that BT1033 catalyzed the reversible phosphorolysis of β-1,4-d-mannosyl-N-acetyl-d-glucosamine in a typical sequential Bi Bi mechanism. These results indicate that BT1033 plays a crucial role as a key enzyme in the N-glycan catabolism where β-1,4-d-mannosyl-N-acetyl-d-glucosamine is liberated from N-glycans by sequential glycoside hydrolase-catalyzed reactions, transported into the cell, and intracellularly converted into α-d-mannose 1-phosphate and N-acetyl-d-glucosamine. In addition, intestinal anaerobic bacteria such as Bacteroides fragilis, Bacteroides helcogenes, Bacteroides salanitronis, Bacteroides vulgatus, Prevotella denticola, Prevotella dentalis, Prevotella melaninogenica, Parabacteroides distasonis, and Alistipes finegoldii were also suggested to possess the similar metabolic pathway for N-glycans. A notable feature of the new metabolic pathway for N-glycans is the more efficient use of ATP-stored energy, in comparison with the conventional pathway where β-mannosidase and ATP-dependent hexokinase participate, because it is possible to directly phosphorylate the d-mannose residue of β-1,4-d-mannosyl-N-acetyl-d-glucosamine to enter glycolysis. This is the first report of a metabolic pathway for N-glycans that includes a Phosphorylase. We propose 4-O-β-d-mannopyranosyl-N-acetyl-d-glucosamine:phosphate α-d-mannosyltransferase as the systematic name and β-1,4-d-mannosyl-N-acetyl-d-glucosamine Phosphorylase as the short name for BT1033.
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characterization of a laminaribiose Phosphorylase from acholeplasma laidlawii pg 8a and production of 1 3 β d glucosyl disaccharides
Carbohydrate Research, 2012Co-Authors: Takanori Nihira, Mamoru Nishimoto, Motomitsu Kitaoka, Yuka Saito, Kenichi Otsubo, Hiroyuki NakaiAbstract:We identified a glycoside hydrolase family 94 homolog (ACL0729) from Acholeplasma laidlawii PG-8A as a laminaribiose (1,3-β-D-glucobiose) Phosphorylase (EC 2.4.1.31). The recombinant ACL0729 produced in Escherichia coli catalyzed phosphorolysis of laminaribiose with inversion of the anomeric configuration in a typical sequential bi bi mechanism releasing α-D-glucose 1-phosphate and D-glucose. Laminaritriose (1,3-β-D-glucotriose) was not an efficient substrate for ACL0729. The phosphorolysis is reversible, enabling synthesis of 1,3-β-D-glucosyl disaccharides by reverse phosphorolysis with strict regioselectivity from α-D-glucose 1-phosphate as the donor and suitable monosaccharide acceptors (D-glucose, 2-deoxy-D-arabino-hexopyranose, D-xylose, D-glucuronic acid, 1,5-anhydro-D-glucitol, and D-mannose) with C-3 and C-4 equatorial hydroxyl groups. The D-glucose and 2-deoxy-D-arabino-hexopyranose caused significantly strong competitive substrate inhibition compared with other glucobiose Phosphorylases reported, in which the acceptor competitively inhibited the binding of the donor substrate. By contrast, none of the examined disaccharides served as acceptor in the synthetic reaction.
Mamoru Nishimoto - One of the best experts on this subject based on the ideXlab platform.
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RESEARCH ARTICLE Discovery of Two b-1,2-Mannoside Phosphorylases Showing Different
2016Co-Authors: Chain-length Specificities From, Kazuhiro Chiku, Takanori Nihira, Erika Suzuki, Thermoanaerobacter Sp. X, Mamoru NishimotoAbstract:. These authors contributed equally to this work. We characterized Teth514_1788 and Teth514_1789, belonging to glycoside hydrolase family 130, from Thermoanaerobacter sp. X-514. These two enzymes catalyzed the synthesis of 1,2-b-oligomannan using b-1,2-mannobiose and D-mannose as the optimal acceptors, respectively, in the presence of the donor a-D-mannose 1-phosphate. Kinetic analysis of the phosphorolytic reaction toward 1,2-b-oligomannan revealed that these enzymes followed a typical sequential Bi Bi mechanism. The kinetic parameters of the phosphorolysis of 1,2-b-oligomannan indicate that Teth514_1788 and Teth514_1789 prefer 1,2-b-oligomannans containing a DP $3 and b-1,2-Man2, respectively. These results indicate that the two enzymes are novel inverting Phosphorylases that exhibit distinct chain-length specificities toward 1,2-b-oligomannan. Here, we propose 1,2-b-oligomannan:phosphate a-D-mannosyltransferase as the systematic name and 1,2-b-oligomannan Phosphorylase as the short name for Teth514_1788 and b-1,2-mannobiose:phosphate a-D-mannosyltransferase as the systematic name and b-1,2-mannobiose Phosphorylase as the short name for Teth514_1789
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discovery of two β 1 2 mannoside Phosphorylases showing different chain length specificities from thermoanaerobacter sp x 514
PLOS ONE, 2014Co-Authors: Kazuhiro Chiku, Takanori Nihira, Erika Suzuki, Ken'ichi Ohtsubo, Mamoru Nishimoto, Motomitsu Kitaoka, Hiroyuki NakaiAbstract:We characterized Teth514_1788 and Teth514_1789, belonging to glycoside hydrolase family 130, from Thermoanaerobacter sp. X-514. These two enzymes catalyzed the synthesis of 1,2-β-oligomannan using β-1,2-mannobiose and d-mannose as the optimal acceptors, respectively, in the presence of the donor α-d-mannose 1-phosphate. Kinetic analysis of the phosphorolytic reaction toward 1,2-β-oligomannan revealed that these enzymes followed a typical sequential Bi Bi mechanism. The kinetic parameters of the phosphorolysis of 1,2-β-oligomannan indicate that Teth514_1788 and Teth514_1789 prefer 1,2-β-oligomannans containing a DP ≥3 and β-1,2-Man2, respectively. These results indicate that the two enzymes are novel inverting Phosphorylases that exhibit distinct chain-length specificities toward 1,2-β-oligomannan. Here, we propose 1,2-β-oligomannan:phosphate α-d-mannosyltransferase as the systematic name and 1,2-β-oligomannan Phosphorylase as the short name for Teth514_1788 and β-1,2-mannobiose:phosphate α-d-mannosyltransferase as the systematic name and β-1,2-mannobiose Phosphorylase as the short name for Teth514_1789.
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Discovery of β-1,4-d-Mannosyl-N-acetyl-d-glucosamine Phosphorylase Involved in the Metabolism of N-Glycans
Journal of Biological Chemistry, 2013Co-Authors: Takanori Nihira, Erika Suzuki, Ken'ichi Ohtsubo, Mamoru Nishimoto, Motomitsu Kitaoka, Hiroyuki NakaiAbstract:Abstract A gene cluster involved in N-glycan metabolism was identified in the genome of Bacteroides thetaiotaomicron VPI-5482. This gene cluster encodes a major facilitator superfamily transporter, a starch utilization system-like transporter consisting of a TonB-dependent oligosaccharide transporter and an outer membrane lipoprotein, four glycoside hydrolases (α-mannosidase, β-N-acetylhexosaminidase, exo-α-sialidase, and endo-β-N-acetylglucosaminidase), and a Phosphorylase (BT1033) with unknown function. It was demonstrated that BT1033 catalyzed the reversible phosphorolysis of β-1,4-d-mannosyl-N-acetyl-d-glucosamine in a typical sequential Bi Bi mechanism. These results indicate that BT1033 plays a crucial role as a key enzyme in the N-glycan catabolism where β-1,4-d-mannosyl-N-acetyl-d-glucosamine is liberated from N-glycans by sequential glycoside hydrolase-catalyzed reactions, transported into the cell, and intracellularly converted into α-d-mannose 1-phosphate and N-acetyl-d-glucosamine. In addition, intestinal anaerobic bacteria such as Bacteroides fragilis, Bacteroides helcogenes, Bacteroides salanitronis, Bacteroides vulgatus, Prevotella denticola, Prevotella dentalis, Prevotella melaninogenica, Parabacteroides distasonis, and Alistipes finegoldii were also suggested to possess the similar metabolic pathway for N-glycans. A notable feature of the new metabolic pathway for N-glycans is the more efficient use of ATP-stored energy, in comparison with the conventional pathway where β-mannosidase and ATP-dependent hexokinase participate, because it is possible to directly phosphorylate the d-mannose residue of β-1,4-d-mannosyl-N-acetyl-d-glucosamine to enter glycolysis. This is the first report of a metabolic pathway for N-glycans that includes a Phosphorylase. We propose 4-O-β-d-mannopyranosyl-N-acetyl-d-glucosamine:phosphate α-d-mannosyltransferase as the systematic name and β-1,4-d-mannosyl-N-acetyl-d-glucosamine Phosphorylase as the short name for BT1033.
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characterization of a laminaribiose Phosphorylase from acholeplasma laidlawii pg 8a and production of 1 3 β d glucosyl disaccharides
Carbohydrate Research, 2012Co-Authors: Takanori Nihira, Mamoru Nishimoto, Motomitsu Kitaoka, Yuka Saito, Kenichi Otsubo, Hiroyuki NakaiAbstract:We identified a glycoside hydrolase family 94 homolog (ACL0729) from Acholeplasma laidlawii PG-8A as a laminaribiose (1,3-β-D-glucobiose) Phosphorylase (EC 2.4.1.31). The recombinant ACL0729 produced in Escherichia coli catalyzed phosphorolysis of laminaribiose with inversion of the anomeric configuration in a typical sequential bi bi mechanism releasing α-D-glucose 1-phosphate and D-glucose. Laminaritriose (1,3-β-D-glucotriose) was not an efficient substrate for ACL0729. The phosphorolysis is reversible, enabling synthesis of 1,3-β-D-glucosyl disaccharides by reverse phosphorolysis with strict regioselectivity from α-D-glucose 1-phosphate as the donor and suitable monosaccharide acceptors (D-glucose, 2-deoxy-D-arabino-hexopyranose, D-xylose, D-glucuronic acid, 1,5-anhydro-D-glucitol, and D-mannose) with C-3 and C-4 equatorial hydroxyl groups. The D-glucose and 2-deoxy-D-arabino-hexopyranose caused significantly strong competitive substrate inhibition compared with other glucobiose Phosphorylases reported, in which the acceptor competitively inhibited the binding of the donor substrate. By contrast, none of the examined disaccharides served as acceptor in the synthetic reaction.
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characterization of three β galactoside Phosphorylases from clostridium phytofermentans discovery of d galactosyl β1 4 l rhamnose Phosphorylase
Journal of Biological Chemistry, 2009Co-Authors: Masahiro Nakajima, Mamoru Nishimoto, Motomitsu KitaokaAbstract:We characterized three d-galactosyl-β1→3-N-acetyl-d-hexosamine Phosphorylase (EC 2.4.1.211) homologs from Clostridium phytofermentans (Cphy0577, Cphy1920, and Cphy3030 proteins). Cphy0577 and Cphy3030 proteins exhibited similar activity on galacto-N-biose (GNB; d-Gal-β1→3-d-GalNAc) and lacto-N-biose I (LNB; d-Gal-β1→3-d-GlcNAc), thus indicating that they are d-galactosyl-β1→3-N-acetyl-d-hexosamine Phosphorylases, subclassified as GNB/LNB Phosphorylase. In contrast, Cphy1920 protein phosphorolyzed neither GNB nor LNB. It showed the highest activity with l-rhamnose as the acceptor in the reverse reaction using α-d-galactose 1-phosphate as the donor. The reaction product was d-galactosyl-β1→4-l-rhamnose. The enzyme also showed activity on l-mannose, l-lyxose, d-glucose, 2-deoxy-d-glucose, and d-galactose in this order. When d-glucose derivatives were used as acceptors, reaction products were β-1,3-galactosides. Kinetic parameters of phosphorolytic activity on d-galactosyl-β1→4-l-rhamnose were kcat = 45 s−1 and Km = 7.9 mm, thus indicating that these values are common among other Phosphorylases. We propose d-galactosyl-β1→4-l-rhamnose Phosphorylase as the name for Cphy1920 protein.
Sakonwan Kuhaudomlarp - One of the best experts on this subject based on the ideXlab platform.
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Unraveling the subtleties of β-(1→3)-glucan Phosphorylase specificity in the GH94, GH149, and GH161 glycoside hydrolase families
Journal of Biological Chemistry, 2019Co-Authors: Sakonwan Kuhaudomlarp, Bernard Henrissat, Giulia Pergolizzi, Nicola Patron, Robert FieldAbstract:Glycoside Phosphorylases (GPs) catalyze the phosphorolysis of glycans into the corresponding sugar 1-phosphates and shortened glycan chains. Given the diversity of natural β-(1→3)-glucans and their wide range of biotechnological applications, the identification of enzymatic tools that can act on β-(1→3)-glucooligosaccharides is an attractive area of research. GP activities acting on β-(1→3)-glucooligosaccharides have been described in bacteria, the photosynthetic excavate Euglena gracilis, and the heterokont Ochromonas spp. Previously, we characterized β-(1→3)-glucan GPs from bacteria and E. gracilis, leading to their classification in glycoside hydrolase family GH149. Here, we characterized GPs from Gram-positive bacteria and heterokont algae acting on β-(1→3)-glucooligosaccharides. We identified a Phosphorylase sequence from Ochromonas spp. (OcP1) together with its orthologs from other species, leading us to propose the establishment of a new GH family, designated GH161. To establish the activity of GH161 members, we recombinantly expressed a bacterial GH161 gene sequence (PapP) from the Gram-positive bacterium Paenibacillus polymyxa ATCC 842 in Escherichia coli. We found that PapP acts on β-(1→3)-glucooligosaccharide acceptors with a degree of polymerization (DP) ≥ 2. This activity was distinct from that of characterized GH149 β-(1→3)-glucan Phosphorylases, which operate on acceptors with DP ≥ 1. We also found that bacterial GH161 genes co-localize with genes encoding β-glucosidases and ATP-binding cassette transporters, highlighting a probable involvement of GH161 enzymes in carbohydrate degradation. Importantly, in some species, GH161 and GH94 genes were present in tandem, providing evidence that GPs from different CAZy families may work sequentially to degrade oligosaccharides.
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Identification of Euglena gracilis β-1,3-glucan Phosphorylase and establishment of a new glycoside hydrolase (GH) family GH149
The Journal of biological chemistry, 2018Co-Authors: Sakonwan Kuhaudomlarp, Bernard Henrissat, Nicola J. Patron, Martin Rejzek, Gerhard Saalbach, Robert A. FieldAbstract:Glycoside Phosphorylases (EC 2.4.x.x) carry out the reversible phosphorolysis of glucan polymers, producing the corresponding sugar 1-phosphate and a shortened glycan chain. β-1,3-Glucan Phosphorylase activities have been reported in the photosynthetic euglenozoan Euglena gracilis, but the cognate protein sequences have not been identified to date. Continuing our efforts to understand the glycobiology of E. gracilis, we identified a candidate Phosphorylase sequence, designated EgP1, by proteomic analysis of an enriched cellular protein lysate. We expressed recombinant EgP1 in Escherichia coli and characterized it in vitro as a β-1,3-glucan Phosphorylase. BLASTP identified several hundred EgP1 orthologs, most of which were from Gram-negative bacteria and had 37-91% sequence identity to EgP1. We heterologously expressed a bacterial metagenomic sequence, Pro_7066 in E. coli and confirmed it as a β-1,3-glucan Phosphorylase, albeit with kinetics parameters distinct from those of EgP1. EgP1, Pro_7066, and their orthologs are classified as a new glycoside hydrolase (GH) family, designated GH149. Comparisons between GH94, EgP1, and Pro_7066 sequences revealed conservation of key amino acids required for the Phosphorylase activity, suggesting a Phosphorylase mechanism that is conserved between GH94 and GH149. We found bacterial GH149 genes in gene clusters containing sugar transporter and several other GH family genes, suggesting that bacterial GH149 proteins have roles in the degradation of complex carbohydrates. The Bacteroidetes GH149 genes located to previously identified polysaccharide utilization loci, implicated in the degradation of complex carbohydrates. In summary, we have identified a eukaryotic and a bacterial β-1,3-glucan Phosphorylase and uncovered a new family of Phosphorylases that we name GH149.
