Characterization of Two α-1,3-Glucoside Phosphorylases from Clostridium phytofermentans (original) (raw)
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Characterization of Three β-Galactoside Phosphorylases from Clostridium phytofermentans
Journal of Biological Chemistry, 2009
We characterized three D-galactosyl-133-N-acetyl-Dhexosamine 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-133-D-GalNAc) and lacto-N-biose I (LNB; D-Gal-133-D-GlcNAc), thus indicating that they are D-galactosyl-133-Nacetyl-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 ␣-D-galactose 1-phosphate as the donor. The reaction product was D-galactosyl-134-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 -1,3-galactosides. Kinetic parameters of phosphorolytic activity on D-galactosyl-134-L-rhamnose were k cat ؍ 45 s ؊1 and K m ؍ 7.9 mM, thus indicating that these values are common among other phosphorylases. We propose D-galactosyl-134-L-rhamnose phosphorylase as the name for Cphy1920 protein.
Development and application of a screening assay for glycoside phosphorylases
Analytical Biochemistry, 2010
Glycoside phosphorylases (GPs) are interesting enzymes for the glycosylation of chemical molecules. They only require a glycosyl phosphate as sugar donor and an acceptor molecule with a free hydroxyl group. Their narrow substrate specificity, however, limits the application of GPs for general glycoside synthesis. Although an enzyme's substrate specificity can be altered and broadened by protein engineering and directed evolution, this requires a suitable screening assay. Such a screening assay has not yet been described for GPs. Here, we report a screening procedure for GPs based on the measurement of released inorganic phosphate in the direction of glycoside synthesis. It appeared necessary to inhibit endogenous phosphatase activity in crude Escherichia coli cell extracts with molybdate, and inorganic phosphate was measured with a modified phosphomolybdate method. The screening system is general and can be used to screen GP enzyme libraries for novel donor and acceptor specificities. It was successfully applied to screen an E649 saturation mutagenesis library of Cellulomonas uda cellobiose phosphorylase (CP) for novel acceptor specificity. An E649C enzyme variant was found with novel acceptor specificity towards alkyl β-glucosides and phenyl β-glucoside. This is the first report of a CP enzyme variant with modified acceptor specificity.
1999
Phosphorylases are key enzymes of carbohydrate metabolism. Structural studies have provided explanations for almost all features of control and substrate recognition of phosphorylase but one question remains unanswered. How does phosphorylase recognize and cleave an oligosaccharide substrate? To answer this question we turned to the Escherichia coli maltodextrin phosphorylase (MalP), a non-regulatory phosphorylase that shares similar kinetic and catalytic properties with the mammalian glycogen phosphorylase. The crystal structures of three MalP-oligosaccharide complexes are reported: the binary complex of MalP with the natural substrate, maltopentaose (G5); the binary complex with the thio-oligosaccharide, 4-S-α-D-glucopyranosyl-4-thiomaltotetraose (GSG4), both at 2.9 Å resolution; and the 2.1 Å resolution ternary complex of MalP with thio-oligosaccharide and phosphate (GSG4-P). The results show a pentasaccharide bound across the catalytic site of MalP with sugars occupying sub-sites-1 to ⍣4. Binding of GSG4 is identical to the natural pentasaccharide, indicating that the inactive thio compound is a close mimic of the natural substrate. The ternary MalP-GSG4-P complex shows the phosphate group poised to attack the glycosidic bond and promote phosphorolysis. In all three complexes the pentasaccharide exhibits an altered conformation across sub-sites-1 and ⍣1, the site of catalysis, from the preferred conformation for α(1-4)-linked glucosyl polymers.
Journal of Biotechnology, 2007
Sucrose phosphorylase catalyzes the reversible conversion of sucrose (␣-d-glucopyranosyl-1,2--d-fructofuranoside) and phosphate into d-fructose and ␣-d-glucose 1-phosphate. We report on the molecular cloning and expression of the structural gene encoding sucrose phosphorylase from Leuconostoc mesenteroides (LmSPase) in Escherichia coli DH10B. The recombinant enzyme, containing an 11 amino acid-long N-terminal metal affinity fusion peptide, was overproduced 60-fold in comparison with the natural enzyme. It was purified to apparent homogeneity using copper-loaded Chelating Sepharose and obtained in 20% yield with a specific activity of 190 U mg −1 . LmSPase was covalently attached onto Eupergit C with a binding efficiency of 50% and used for the continuous production of ␣-d-glucose 1-phosphate from sucrose and phosphate (600 mM each) in a packed-bed immobilised enzyme reactor (30 • C, pH 7.0). The reactor was operated at a stable conversion of 91% (550 mM product) and productivity of approximately 11 g l −1 h −1 for up to 600 h. A kinetic study of transglucosylation by soluble LmSPase was performed using ␣-d-glucose 1-phosphate as the donor substrate and various alcohols as acceptors. d-and l-arabitol were found to be good glucosyl acceptors.
FEBS Letters, 2006
Mutagenesis of Asp-196 into Ala yielded an inactive variant of Leuconostoc mesenteroides sucrose phosphorylase (D196A). External azide partly complemented the catalytic defect in D196A with a second-order rate constant of 0.031 M À1 s À1 (pH 5, 30°C) while formate, acetate and halides could not restore activity. The mutant utilized azide to convert a-D D-glucose 1-phosphate into b-D D-glucose 1-azide, reflecting a change in stereochemical course of glucosyl transfer from a-retaining in wild-type to inverting in D196A. Phosphorolysis of b-D D-glucose 1-azide by D196A occurred through a ternary complex kinetic mechanism, in marked contrast to the wild-type whose reactions feature a common glucosyl enzyme intermediate and Ping-Pong kinetics. Therefore, Asp-196 is identified unambiguously as the catalytic nucleophile of sucrose phosphorylase, and its substitution by Ala forces the reaction to proceed via single nucleophilic displacement. D196A is not detectably active as a-glucosynthase.
Enzymatic synthesis using glycoside phosphorylases
Carbohydrate Research, 2015
Carbohydrate phosphorylases are readily accessible but under-explored catalysts for glycoside synthesis. Their use of accessible and relatively stable sugar phosphates as donor substrates underlies their potential. A wide range of these enzymes has been reported of late, displaying a range of preferences for sugar donors, acceptors and glycosidic linkages. This has allowed this class of enzymes to be used in the synthesis of diverse carbohydrate structures, including at the industrial scale. As more phosphorylase enzymes are discovered, access to further difficult to synthesise glycosides will be enabled. Herein we review reported phosphorylase enzymes and the glycoside products that they have been used to synthesise.
Proteins: Structure, Function, and Bioinformatics, 2019
Glycoside phosphorylases (GPs) with specificity for β-(1 ! 3)-gluco-oligosaccharides are potential candidate biocatalysts for oligosaccharide synthesis. GPs with this linkage specificity are found in two families thus far-glycoside hydrolase family 94 (GH94) and the recently discovered glycoside hydrolase family 149 (GH149). Previously, we reported a crystallographic study of a GH94 laminaribiose phosphorylase with specificity for disaccharides, providing insight into the enzyme's ability to recognize its' sugar substrate/product. In contrast to GH94, characterized GH149 enzymes were shown to have more flexible chain length specificity, with preference for substrate/product with higher degree of polymerization. In order to advance understanding of the specificity of GH149 enzymes, we herein solved X-ray crystallographic structures of GH149 enzyme Pro_7066 in the absence of substrate and in complex with laminarihexaose (G6). The overall domain organization of Pro_7066 is very similar to that of GH94 family enzymes. However, two additional domains flanking its catalytic domain were found only in the GH149 enzyme. Unexpectedly, the G6 complex structure revealed an oligosaccharide surface binding site remote from the catalytic site, which, we suggest, may be associated with substrate targeting. As such, this study reports the first structure of a GH149 phosphorylase enzyme acting on β-(1 ! 3)-gluco-oligosaccharides and identifies structural elements that may be involved in defining the specificity of the GH149 enzymes.
Bacterial α-glucan phosphorylases
FEMS Microbiology Letters, 1999
Although glycogen and other K-1,4-D-glucan storage polysaccharides are present in many bacteria, only few glucan phosphorylases from bacteria have been identified and characterised on the protein or gene level. All bacterial phosphorylases follow the same catalytic mechanisms as their plant and vertebrate counterparts, but differ considerably in terms of their substrate specificity and regulation. The catalytic domains are highly conserved while the regulatory sites are only poorly conserved. The degree of conservation between bacterial and mammalian phosphorylases is comparable to that of other nonmammalian and mammalian K-glucan phosphorylases. Only for maltodextrin phosphorylase from E. coli the physiological role of the enzyme in the utilisation of maltodextrins is known in detail; that of all other phosphorylases remains still unclear. Roles in regulation of endogenous glycogen metabolism in periods of starvation, and sporulation, stress response or quick adaptation to changing environments are imaginable. z : S 0 3 7 8 -1 0 9 7 ( 9 8 ) 0 0 5 8 0 -1