蔗糖磷酸化酶及其在糖基化反应中的应用

糖基化反应能有效改善化合物的水溶性、稳定性、生物利用度等性质,利用糖苷水解酶类和糖基转移酶类对生物活性化合物进行糖基化修饰已成为研究热点.相比于糖基转移酶类,糖苷水解酶类在大规模催化中具有来源丰富、成本低的优势.其中,蔗糖磷酸化酶因其卓越的糖基化活性和广泛的底物特异性,在化工领域受到人们的广泛关注.文中综述了蔗糖磷酸化...     

Starch phosphorylase: Role in starch metabolism and biotechnological applications

The α-glucan phosphorylases of the glycosyltransferase family are important enzymes of carbohydrate metabolism in prokaryotes and eukaryotes. The plant α-glucan phosphorylase, commonly called starch phosphorylase (EC 2.4.1.1), is largely known for the phosphorolytic degradation of starch. Starch phosphorylase catalyzes the reversible transfer of glucosyl units from glucose-1-phosphate to the nonreducing end of α-1,4-d-glucan chains with the release of phosphate. Two distinct forms of starch phosphorylase, plastidic phosphorylase and cytosolic phosphorylase, have been consistently observed in higher plants. Starch phosphorylase is industrially useful and a preferred enzyme among all glucan phosphorylases for phosphorolytic reactions for the production of glucose-1-phosphate and for the development of engineered varieties of glucans and starch. Despite several investigations, the precise functional mechanisms of its characteristic multiple forms and the structural details are still eluding us. Recent discoveries have shed some light on their physiological substrates, precise biological functions, and regulatory aspects. In this review, we have highlighted important developments in understanding the role of starch phosphorylases and their emerging applications in industry.     

The structure of a GH149 β-(1 → 3) glucan phosphorylase reveals a new surface oligosaccharide binding site and additional domains that are absent in the disaccharide-specific GH94 glucose-β-(1 → 3)-glucose (laminaribiose) phosphorylase

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.     

Biosynthesis of nucleoside analogues via thermostable nucleoside phosphorylase

Biocatalyzed synthesis of nucleoside analogues was carried out using two thermostable nucleoside phosphorylases from the hyperthermophilic aerobic crenarchaeon Aeropyrum pernix K1. The synthesis of the 2,6-diaminopurine nucleoside and 5-methyluridine was used as a reaction model to test the process. Both the purine nucleoside phosphorylase (apPNP) and uridine phosphorylase (apUP) were functionally expressed in Escherichia coli. The recombinant enzymes were characterized after purification, and both enzymes showed high thermostability and broad substrate specificity. Both enzymes retained 100 % of their activity after 60 min at high temperature, and the optimum temperature for the enzymes was 90–100 °C. The nucleoside phosphorylases obtained from A. pernix are valuable industrial biocatalysts for high-temperature reactions that produce nucleoside drugs in high yields.     

Plastidial phosphorylase is required for normal starch synthesis in Chlamydomonas reinhardtii

Among the three distinct starch phosphorylase activities detected in Chlamydomonas reinhardtii, two distinct plastidial enzymes (PhoA and PhoB) are documented while a single extraplastidial form (PhoC) displays a higher affinity for glycogen as in vascular plants. The two plastidial phosphorylases are shown to function as homodimers containing two 91-kDa (PhoA) subunits and two 110-kDa (PhoB) subunits. Both lack the typical 80-amino-acid insertion found in the higher plant plastidial forms. PhoB is exquisitely sensitive to inhibition by ADP-glucose and has a low affinity for malto-oligosaccharides. PhoA is more similar to the higher plant plastidial phosphorylases: it is moderately sensitive to ADP-glucose inhibition and has a high affinity for unbranched malto-oligosaccharides. Molecular analysis establishes that STA4 encodes PhoB. Chlamydomonas reinhardtii strains carrying mutations at the STA4 locus display a significant decrease in amounts of starch during storage that correlates with the accumulation of abnormally shaped granules containing a modified amylopectin structure and a high amylose content. The wild-type phenotype could be rescued by reintroduction of the cloned wild-type genomic DNA, thereby demonstrating the involvement of phosphorylase in storage starch synthesis.     

Sucrose phosphorylase as cross-linked enzyme aggregate: Improved thermal stability for industrial applications

Sucrose phosphorylase is an interesting biocatalyst that can glycosylate a variety of small molecules using sucrose as a cheap but efficient donor substrate. The low thermostability of the enzyme, however, limits its industrial applications, as these are preferably performed at 60°C to avoid microbial contamination. Cross-linked enzyme aggregates (CLEAs) of the sucrose phosphorylase from Bifidobacterium adolescentis were found to have a temperature optimum that is 17°C higher than that of the soluble enzyme. Furthermore, the immobilized enzyme displays an exceptional thermostability, retaining all of its activity after 1 week incubation at 60°C. Recycling of the biocatalyst allows its use in at least ten consecutive reactions, which should dramatically increase the commercial potential of its glycosylating activity.     

Pyrimidine Salvage Pathways In Toxoplasma Gondii

ABSTRACT. Pyrimidine salvage enzyme activities in cell-free extracts of Toxoplasma gondii were assayed in order to determine which of these enzyme activities are present in these parasites. Enzyme activities that were detected included phosphoribosyltransferase activity towards uracil (but not cytosine or thymine), nucleoside phosphorylase activity towards uridine, deoxyuridine and thymidine (but not cytidine or deoxycytidine), deaminase activity towards cytidine and deoxycytidine (but not cytosine, cytidine 5'-monophosphate or deoxycytidine 5'-monophosphate), and nucleoside 5'-monophosphate phosphohydrolase activity towards all nucleotides tested. No nucleoside kinase or phosphotransferase activity was detected, indicating that T. gondii lack the ability to directly phosphorylate nucleosides. Toxoplasma gondii appear to have a single non-specific uridine phosphorylase enzyme which can catalyze the reversible phosphorolysis of uridine, deoxyuridine and thymidine, and a single cytidine deaminase activity which can deaminate both cytidine and deoxycytidine. These results indicate that pyrimidine salvage in T. gondii probably occurs via the following reactions: cytidine and deoxycytidine are deaminated by cytidine deaminase to uridine and deoxyuridine, respectively; uridine and deoxyuridine are cleaved to uracil by uridine phosphorylase; and uracil is metabolized to uridine 5'-monophosphate by uracil phosphoribosyltransferase. Thus, uridine 5'-monophosphate is the end-product of both de novo pyrimidine biosynthesis and pyrimidine salvage in T. gondii.     

Two genes in Arabidopsis thaliana encoding GDP-L-galactose phosphorylase are required for ascorbate biosynthesis and seedling viability

Plants synthesize ascorbate from guanosine diphosphate (GDP)-mannose via L-galactose/L-gulose, although uronic acids have also been proposed as precursors. Genes encoding all the enzymes of the GDP-mannose pathway have previously been identified, with the exception of the step that converts GDP-L-galactose to L-galactose 1-P. We show that a GDP-L-galactose phosphorylase, encoded by the Arabidopsis thaliana VTC2 gene, catalyses this step in the ascorbate biosynthetic pathway. Furthermore, a homologue of VTC2, At5g55120, encodes a second GDP-L-galactose phosphorylase with similar properties to VTC2. Two At5g55120 T-DNA insertion mutants (vtc5-1 and vtc5-2) have 80% of the wild-type ascorbate level. Double mutants were produced by crossing the loss-of-function vtc2-1 mutant with each of the two vtc5 alleles. These show growth arrest immediately upon germination and the cotyledons subsequently bleach. Normal growth was restored by supplementation with ascorbate or L-galactose, indicating that both enzymes are necessary for ascorbate generation. vtc2-1 leaves contain more mannose 6-P than wild-type. We conclude that the GDP-mannose pathway is the only significant source of ascorbate in A. thaliana seedlings, and that ascorbate is essential for seedling growth. A. thaliana leaves accumulate more ascorbate after acclimatization to high light intensity. VTC2 expression and GDP-L-galactose phosphorylase activity rapidly increase on transfer to high light, but the activity of other enzymes in the GDP-mannose pathway is little affected. VTC2 and At5g55120 (VTC5) expression also peak in at the beginning of the light cycle and are controlled by the circadian clock. The GDP-L-galactose phosphorylase step may therefore play an important role in controlling ascorbate biosynthesis.     

Sucrose phosphorylase: a powerful transglucosylation catalyst for synthesis of α-D-glucosides as industrial fine chemicals

Abstract

Sucrose phosphorylase is a bacterial transglucosidase that catalyzes conversion of sucrose and phosphate into α-D-glucose-1-phosphate and D-fructose. The enzyme utilizes a glycoside hydrolase-like double displacement mechanism that involves a catalytically competent β-glucosyl enzyme intermediate. In addition to reaction with phosphate, glucosylated sucrose phosphorylase can undergo hydrolysis to yield α-D-glucose or it can decompose via glucosyl transfer to a hydroxy group in suitable acceptor molecules, giving new α-D-glucosidic products. The glucosyl acceptor specificity of sucrose phosphorylase is reviewed, focusing on applications of the enzyme in glucoside synthesis. Polyhydroxylated compounds such as sugars and sugar alcohols are often glucosylated efficiently. Aryl alcohols and different carboxylic acids also serve as acceptors for enzymatic transglucosylation. The natural osmolyte 2-O-(α-D-glucopyranosyl)-sn-glycerol (GG) was prepared by regioselective glucosylation of glycerol from sucrose using the phosphorylase from Leuconostoc mesenteroides. An industrial process for production of GG as active ingredient of cosmetic formulations has been recently developed. General advantages of sucrose phosphorylase as a transglucosylation catalyst lie in the use of sucrose as a high-energy glucosyl donor and the usually weak hydrolase activity of the enzyme towards substrate and product.     

Effect of molecular crowding on the enzymes of glycogenolysis

Cell cytoplasm contains high concentrations of macromolecules occupying a significant part of the cell volume (crowding conditions). According to modern concepts, crowding has a pronounced effect on the rate and equilibrium of biochemical reactions and stimulates the formation of more compact structures. This review considers different aspects of the crowding effect in vivo and in vitro, its role in regulation of cell volume, the effect of crowding on various interactions, such as protein-ligand and protein-protein interactions, as well as on protein denaturation, conformation transitions of macromolecules, and supramolecular structure formation. The influence of crowding arising from the presence of high concentrations of osmolytes on the interactions of the enzymes of glycogenolysis has been demonstrated. It has been established that, in accordance with predictions of crowding theory, trimethylamine N-oxide (TMAO) and betaine highly stimulate the association of phosphorylase kinase (PhK) and its interaction with glycogen. However, high concentrations of proline, betaine, and TMAO completely suppress the formation of PhK complex with phosphorylase b (Phb). The protective effect of osmolyte-induced molecular crowding on Phb denaturation by guanidine hydrochloride is shown. The influence of crowding on the interaction of Phb with allosteric inhibitor FAD has been revealed. The results show that, under crowding conditions, the equilibrium of the isomerization of Phb shifts towards a more compact dimeric state with decreased affinity for FAD.     

Enzymatic activities of uridine and thymidine phosphorylase in normal and cancerous uterine cervical tissues

In this study, the preliminary analyses were conducted of enzymatic activities of uridine phosphorylase (UP) and thymidine phosphorylase (TP) in normal tissues and cancer tissues of the uterine cervix. The study was performed on 27 patients of cervical cancer, treated first in our hospital. Normal cervical tissues obtained from 15 patients undergoing hysterectomy for benign diseases were used as controls. The supernatant of the homogenated cervical tissues and the stroma (5-FU and ribose-1-P or deoxyribose-1-P) were analyzed by high performance liquid chromatography, and then the UP and TP activities calculated. TP activity was significantly greater than UP activity (P < 0.0001). Both UP and TP showed significantly greater activity in cancer tissues than in normal tissues (P < 0.0001). In the TP activity of the cancer tissues, there was no significant difference among the histological types, while the TP activity tended to be significantly higher in the cases with lymph node metastasis. These results showed that the TP-mediated route seemed important as the 5FU metabolic pathway in the uterine cervical tissues, and TP enzymatic activity might be associated with lymph node metastasis.     

Further studies of primer-independent phosphorylase isozymes in the algae

Both Oscillatoria princeps and Cyanidium caldarium contain phosphorylase isozymes that can cause the synthesis of polyglucan from glucose-1-phosphate in the absence of added maltodextrin ‘primer’. In addition, O. princeps contains a primer-dependent phosphorylase isozyme. When the phosphorylase fractions isolated from extracts of the algae were treated with α-amylase, the primer-independent isozyme became primer-dependent and shifted from the position it was normally found at after polyacrylamide gel electrophoresis. This primer-independent isozyme became less mobile towards the anode, and was found at the locus usually occupied by the primer-dependent isozyme. It was not possible to restore its mobility towards the anode and its primer-independent properties by preincubation with maltoheptaose. The indication is that this isozyme is a glucoprotein and that the glucan component is chemically bonded to the protein.     

A bioluminescent assay for glycogen phosphorylase in cultured cells

A new method for the determination of glycogen phosphorylase (1,4 alpha-D-glucose:orthophosphate alpha-glucosyltransferase, EC 2.4.1.1) in cultured cells is described. The assay utilizes bacterial luciferase (EC 2.7) in a liquid scintillation spectrometer to measure NAD(P)H formed in a coupled enzyme reaction comprising glucose-6-phosphate dehydrogenase (EC 1.1.1.49) and phosphoglucomutase (EC 2.7.5.1). This assay is highly sensitive, easily detecting as little as 10 microU phosphorylase, fast and simple to perform. With modifications this procedure can be extended to measure other glycogenolytic enzymes and intermediates.     

Determination of glycogen and enzymes of glycogen metabolism in human hair follicles

The skin epithelium and its organelles use glycogen as well as glucose as source of energy. Therefore the characterisation of glycogen metabolism and the enzymes involved is important in the study of mechanisms regulating the normal or abnormal differentiation of skin organelles such as sebaceous glands and hair follicles.The present paper describes fluorimetric methods for the determination of glycogen and for the measurements of phosphorylase and phosphorylase kinase activity in one and the same lysate of minute tissue samples. The methods were tested for their suitability on freshly isolated human hair follicles and cultured hair follicle cells. The possible use of these techniques for studies on the pathophysiology of acne and hirsutism is discussed.     

The crystal structure of 5'-deoxy-5'-methylthioadenosine phosphorylase II from Sulfolobus solfataricus, a thermophilic enzyme stabilized by intramolecular disulfide bonds

The crystal structure of Sulfolobus solfataricus 5'-deoxy-5'-methylthioadenosine phosphorylase II (SsMTAPII) in complex with 5'-deoxy-5'-methylthioadenosine (MTA) and sulfate was determined to 1.45A resolution. The hexameric structure of SsMTAPII is a dimer-of-trimers with one active site per monomer. The oligomeric assembly of the trimer and the monomer topology of SsMTAPII are almost identical with trimeric human 5'-deoxy-5'-methylthioadenosine phosphorylase (hMTAP). SsMTAPII is the first reported hexameric member in the trimeric class of purine nucleoside phosphorylase (PNP) from Archaea. Unlike hMTAP, which is highly specific for MTA, SsMTAPII also accepts adenosine as a substrate. The residues at the active sites of SsMTAPII and hMTAP are almost identical. The broad substrate specificity of SsMTAPII may be due to the flexibility of the C-terminal loop. SsMTAPII is extremely thermoactive and thermostable. The three-dimensional structure of SsMTAPII suggests that the unique dimer-of-trimers quaternary structure, a CXC motif at the C terminus, and two pairs of intrasubunit disulfide bridges may play an important role in its thermal stability.     

Production of RNA by a polymerase protein encapsulated within phospholipid vesicles

Catalyzed polymerization reactions represent a primary anabolic activity of all cells. It can be assumed that early cells carried out such reactions, in which macromolecular catalysts were encapsulated within some type of boundary membrane. In the experiments described here, we show that a template-independent RNA polymerase (polynucleotide phosphorylase) can be encapsulated in dimyristoyl phosphatidylcholine vesicles without substrate. When the substrate adenosine diphosphate (ADP) was provided externally, long-chain RNA polymers were synthesized within the vesicles. Substrate flux was maximized by maintaining the vesicles at the phase transition temperature of the component lipid. A protease was introduced externally as an additional control. Free enzyme was inactivated under identical conditions. RNA products were visualized in situ by ethidium bromide fluorescence. The products were harvested from the liposomes, radiolabeled, and analyzed by polyacrylamide gel electrophoresis. Encapsulated catalysts represent a model for primitive cellular systems in which an RNA polymerase was entrapped within a protected microenvironment.Abbreviations ADP adenosine diphosphate - DMPC dimyristoyl phosphatidylcholine - EDTA ethylenediaminetetraacetic acid - LUV large unilamellar vesicle - MLV multilamellar vesicle - PAGE polyacrylamide gel electrophoresis - PNPase or PNP polynucleotide phosphorylase - SUV small unilamellar vesicle Correspondence to.: A.C. Chakrabarti     

α-1,4-Glucan phosphorylase forms from leaves of spinach (Spinacia oleracea L.) I. In situ localization by indirect immunofluorescence

Antisera were raised against two forms of -1,4-glucan phosphorylase (EC 2.4.1.1) which had been purified from leaves of Spinacia oleracea L. Immunoglobulin G preparations were isolated from the antisera, and their specificity was ensured by immunoplobulin G preparations were used for in situ localization of the two phosphorylase forms in spinach leaf thin sections by indirect immuno-fluorescence. Both enzyme forms were present in the palisade and spongy parenchyma and in the guard cells, but their intracellular distribution was complementary. One phosphorylase form (designated as the chloroplastic form) was restricted to the stromal space of chloroplasts whereas the other (the non-chloroplastic form) was present only in the cytoplasm of chloroplast-containing cells. Thus, the phosphorylases represent two distinct compartment-specific enzyme forms which reside within the same photosynthetically active mesophyll cell.Abbreviations DBM diazobenzyloxymethyl - FITC fluorescein isothiocyanate - IgG immunoglobulin G - PMSF phenylmethylsulfonyl fluoride     

Two pathways from glycogen to glucose-6-phosphate in sea urchin eggs with special reference to the difference between the species in the contribution ratio of each pathway

In the unfertilized eggs of the sea urchins, Anthocidaris crassispina, Pseudocentrotus depressus, Hemicentrotus pulcherrimus, Mespilia globulus, Temnopleurus toreumaticus, Toxopeneustes pileolus, and Clypeaster japonicus, the activities of phosphorylase [EC 2.4.1.1], phosphoglucomutase [EC 2.7.5.1], exo-l,4-α-glucosidase [EC 3.2.1.3], and hexokinase [EC 2.7.1.1] are very similar. In all species, only phosphorylase activity is higher in fertilized eggs than in unfertilized eggs. The concentrations of glycogen, glucose, GIP, G6P, ATP, ADP, and Pi; the products and substrates in reactions catalyzed by these enzymes, were measured in these eggs. Based on the concentrations of these compounds in the eggs, it is assumed that G6P is produced by the combined action of glucosidase and hexokinase in all species examined, and that it is also produced in the reaction catalyzed by phosphorylase and phosphoglucomutase in all species except A crassispina and P depressus. Glycogen was found both in supernatant and in precipitate fractions, which were obtained by adding perchloric acid. Glycogen in the precipitate seems to be protein-bound. Whole glycogen level in the eggs is almost the same in all species examined, but the level of acid-soluble glycogen, as well as GIP, is markedly lower in the eggs of A crassispina and P depressus than in the eggs of other species examined. Protein-bound glycogen is utilized by glucosidase activity but not by phosphorylase activity, in contrast to acid-soluble glycogen, which is utilized by both enzyme activities. Hence, it is assumed that the failure of G6P production by phosphorylase and phosphoglucomutase-in A crassispina and P depressus eggs is due to a low level of acid-soluble glycogen in these eggs.     

Glycogen phosphorylase from flight muscle of the hawk moth,Manduca sexta: purification and properties of three interconvertible forms and the effect of flight on their interconversion

Glycogen phosphorylase (EC 2.4.1.1) of Manduca sexta flight muscle was separated into three distinct peaks of activity on diethylaminoethyl-Sephacel. The three fractions of phosphorylase activity were further purified by affinity chromatography on AMP-Sepharose and shown to have the same relative molecular mass (=178000) on polyacrylamide gradient gel electrophoresis under non-denaturating conditions and to produce subunits of molecular mass =92000 on SDS gelelectrophoresis. On the basis of their kinetic properties with respect to the activator AMP and the inhibitor caffeine, the three fractions of phosphorylase activity were assigned as follows: peak 1=phosphorylase b (unphosphorylated form), peak 3=phosphorylase a (phosphorylated form); peak 2 represented a phospho-dephospho hybrid in which only one subunit of the dimeric enzyme was phosphorylated. This hypothesis was corroborated as the various forms could be interconverted in vitro by either dephosphorylation by an endogenous protein phosphatase producing the b form, or by phosphorylation catalyzed by purified phosphorylase kinase from rabbit muscle producing phosphorylase ab and a. From muscle of resting moths more phosphorylase was isolated in the b form (41%) than in the forms ab (28%) and a (31%), respectively. This proportion was changed in favour of the fully phosphorylated a form after a brief interval of flight when 68% of the phosphorylase activity was represented by the a form and only 13% by the b form. Unlike the phosphorylated forms a and ab of phosphorylase, the b form had low affinities for the substrates and for the activator AMP, and was virtually inactive if near-physiological concentrations of substrates and effectors were employed in the assays. The results demonstrate that in Manduca flight muscle three forms of phosphorylase coexist and that their interconversion is a mechanism for regulating phosphorylase activity in vivo.Abbreviations DEAE diethylaminoethyl - EDTA ethylenediamine tetraacetate - EGTA ethyleneglycol-bis(-aminoethylether)N,N-tetra-acetic acid - M _r relative molecular mass - NMR nuclear magnetic resonance - PAGGE polyacrylamide gradient gel electrophoresis - P_i morganic phosphate - SDS sodium dodecylsulphate - TRIS tris(hydroxymethyl)-aminomethane - V _max maximum activity