Carbohydrate synthesis by disaccharide phosphorylases: reactions, catalytic mechanisms and application in the glycosciences

Disaccharide phosphorylases are glycosyltransferases (EC 2.4.1.α) of specialized carbohydrate metabolism in microorganisms. They catalyze glycosyl transfer to phosphate using a disaccharide as donor substrate. Phosphorylases for the conversion of naturally abundant disaccharides including sucrose, maltose, α,α-trehalose, cellobiose, chitobiose, and laminaribiose have been described. Structurally, these disaccharide phosphorylases are often closely related to glycoside hydrolases and transglycosidases. Mechanistically, they are categorized according the stereochemical course of the reaction catalyzed, whereby the anomeric configuration of the disaccharide donor substrate may be retained or inverted in the sugar 1-phosphate product. Glycosyl transfer with inversion is thought to occur through a single displacement-like catalytic mechanism, exemplified by the reaction coordinate of cellobiose/chitobiose phosphorylase. Reaction via configurational retention takes place through the double displacement-like mechanism employed by sucrose phosphorylase. Retaining α,α-trehalose phosphorylase (from fungi) utilizes a different catalytic strategy, perhaps best described by a direct displacement mechanism, to achieve stereochemical control in an overall retentive transformation. Disaccharide phosphorylases have recently attracted renewed interest as catalysts for synthesis of glycosides to be applied as food additives and cosmetic ingredients. Relevant examples are lacto-N-biose and glucosylglycerol whose enzymatic production was achieved on multikilogram scale. Protein engineering of phosphorylases is currently pursued in different laboratories with the aim of broadening the donor and acceptor substrate specificities of naturally existing enzyme forms, to eventually generate a toolbox of new catalysts for glycoside synthesis.     

Potato and rabbit muscle phosphorylases: comparative studies on the structure,function and regulation of regulatory and nonregulatory enzymes

Summary Phosphorylases (EC 2.4.1.1) from potato and rabbit muscle are similar in many of their structural and kinetic properties, despite differences in regulation of their enzyme activity. Rabbit muscle phosphorylase is subject to both allosteric and covalent controls, while potato phosphorylase is an active species without any regulatory mechanism. Both phosphorylases are composed of subunits of approximately 100 000 molecular weight, and contain a firmly bound pyridoxal 5-phosphate. Their actions follow a rapid equilibrium random Bi Bi mechanism. From the sequence comparison between the two phosphorylases, high homologies of widely distributed regions have been found, suggesting that they may have evolved from the same ancestral protein. By contrast, the sequences of the N-terminal region are remarkably different from each other. Since this region of the muscle enzyme forms the phosphorylatable and AMP-binding sites as well as the subunit-subunit contact region, these results provide the structural basis for the difference in the regulatory properties between potato and rabbit muscle phosphorylases. Judged from CD spectra, the surface structures of the potato enzyme might be significantly different from that of the muscle enzyme. Indeed, the subunit-subunit interaction in the potato enzyme is tighter than that in the muscle enzyme, and the susceptibility of the two enzymes toward modification reagents and proteolytic enzymes are different. Despite these differences, the structural and functional features of the cofactor, pyridoxal phosphate, site are surprisingly well conserved in these phosphorylases. X-ray crystallographic studies on rabbit muscle phosphorylase have shown that glucose-1-phosphate and orthophosphate bind to a common region close to the 5-phosphate of the cofactor. The muscle enzyme has a glycogen storage site for binding of the enzyme to saccharide substrate, which is located away from the cofactor site. We have obtained, in our reconstitution studies, evidence for binding of saccharide directly to the cofactor site of potato phosphorylase. This difference in the topography of the functional sites explains the previously known different specificities for saccharide substrates in the two phosphorylases. Based on a combination of these and other studies, it is now clear that the 5-phosphate group of pyridoxal phosphate plays a direct role in the catalysis of this enzyme. Information now available on the reaction mechanism of phosphorylase is briefly described.     

Soft-Shell Clam, Mya arenaria, a Convenient Laboratory Animal for Screening Pathogens of Bivalve Mollusks

Attempts to introduce infectious or foreign material into oysters and other bivalve mollusks usually involve force or trauma because of immediate, prolonged adduction of the tightly closing valves. The soft-shell clam, Mya arenaria, is unable to seal its valves completely and relaxes readily, exposing soft tissue and a large siphon. This species is free from fouling organisms and is readily available at all seasons in the New England and mid-Atlantic areas. Suspensions of five strains of Vibrio sp. that cause bacillary necrosis in larval and juvenile bivalve mollusks were injected into the heart, siphon tissue, and the incurrent and excurrent siphon lumina of soft-shell clams. All vibrio strains caused significant mortality, usually within 2 days. Heaviest losses resulted from heart and excurrent siphon injections. No mortality occurred in control clams injected with seawater, broth, Serratia sp., and Escherichia coli. The soft-shell clam appears to be a useful animal for testing the pathogenicity of marine microorganisms for bivalve mollusks.     

Alpha-glucan phosphorylase from Escherichia coli. Cloning of the gene, and purification and characterization of the protein

By using a synthetic oligonucleotide probe identical to a part of the gene for the Escherichia coli major outer membrane lipoprotein, we have cloned a gene from E. coli chromosomal DNA. However, the cloned gene was not one of the lipoprotein genes. The amino acid sequence deduced from its nucleotide sequence shows extensive similarities instead to alpha-glucan phosphorylase (EC 2.4.1.1). The gene, glgP, is located immediately downstream from glgA, the gene for glycogen synthase. The glgP gene was inserted into pUC9 vector and expressed in the presence of the lac inducer. The gene product was purified to apparent homogeneity as shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In all chromatographies, the protein was eluted accompanied by a low phosphorylase activity. The final preparation showed phosphorolytic activity to various alpha-glucans, although the specific activity was extremely low compared to other alpha-glucan phosphorylases under the standard assay conditions. Its enzymatic activity, however, increased almost linearly as the concentration of glucan increased, reaching a value comparable with those of other phosphorylases. The amino acid sequence deduced was compared with those of alpha-glucan phosphorylases from other sources.     

The evolution of catalytic residues and enzyme mechanism within the bacterial nucleoside phosphorylase superfamily 1

Nucleoside phosphorylases are essential for the salvage and catabolism of nucleotides in bacteria and other organisms, and members of this enzyme superfamily have been of interest for the development of antimicrobial and cancer therapies. The nucleotide phosphorylase superfamily 1 encompasses a number of different enzymes which share a general superfold and catalytic mechanism, while they differ in the nature of the nucleophiles used and in the nature of characteristic active site residues. Recently, one subfamily, the uridine phosphorylases, has been subdivided into two types which differ with respect to the mechanism of transition state stabilization, as dictated by differences in critical amino acid residues. Little is known about the phylogenetic distribution and relationship of the two different types, as well as the relationship to other NP-1 superfamily members. Here comparative genomic analysis illustrates that UP-1s and UP-2s fall into monophyletic groups and are biased with respect to species representation. UP-1 evolved in Gram negative bacteria, while Gram positive species tend to predominantly contain UP-2. PNP (a sister clade to all UPs) contains both Gram positive and Gram negative species. The findings imply that the nucleoside phosphorylase superfamily 1 evolved through a series of three important duplications, leading to the separate, monophyletic enzyme families, coupled to individual lateral transfer events. Extensive horizontal transfer explains the occurrence of unexpected uridine phosphorylases in some genomes. This study provides a basis for understanding the evolution of uridine and purine nucleoside phosphorylases with respect to DNA/RNA metabolism and with potential utility in the design of antimicrobial and anti-tumor drugs.     

The food and feeding ecology of Gerreidae, Bleeker 1859, in the estuaries of Natal

The food and feeding ecology of five species of Genes in the estuaries of Natal, were investigated from 1978 to 1980. The Kosi system, consisting of an estuary and three main lakes was selected as the main study area due to an abundance of Genes . Four other estuarine systems were sampled.
At Kosi polychaetes were important at the estuary, siphon tips (distal ±5 mm) of the bivalve Hiatula lunulata were most commonly taken in Lakes Makhawulani and Mpungwini, while chironomid larvae were important in Lake Nhlange. Ivlev's electivity test showed that Genes positively selected bivalve siphon tips when searching for food. The food taken by Genes from other estuaries was similar to that at Kosi, although the proportions of the different prey varied. At Kosi between three and five species occurred sympatri-cally. Resource segregation was through differences in diet and feeding periodicity and a superabundance of food ( H. lunulata siphon tips) during summer and autumn. During winter and spring when food may be limiting, most Genes leave shelf areas of the Kosi system, only G. acinaces remains in large numbers. Little is known of the diet of other fish which feed on benthic invertebrates in Kosi but there is probably little direct competition with Gerreidae, although Acanthopagrus berda and Pomadasys commersonni have been recorded feeding on the siphons of the bivalve Solen corneus , the former in Durban Bay and the latter at Kosi. In areas where the bivalve H. lunulata occurs it is suggested that Gerreidae have developed optimal foraging techniques which enhance resource partitioning.     

Localization of the muscle, liver, and brain glycogen phosphorylase genes on linkage maps of mouse chromosomes 19, 12, and 2, respectively

Mammalian glycogen phosphorylases comprise a family of three isozymes, muscle, liver, and brain, which are expressed selectively and to varying extents in a wide variety of cell types. To better understand the regulation of phosphorylase gene expression, we isolated partial cDNAs for all three isozymes from the rat and used these to map the corresponding genes in the mouse. Chromosome mapping was accomplished by comparing the segregation of phosphorylase restriction fragment length polymorphisms (RFLPs) with 16 reference loci in a multipoint interspecies backcross between Mus musculus domesticus and Mus spretus. The genes encoding muscle, liver, and brain phosphorylases (Pygm, Pygl, and Pygb) are assigned to mouse chromosomes 19, 12, and 2, respectively. Their location on separate chromosomes indicates that distinct cis-acting elements govern the differential expression of phosphorylase isozymes in various tissues. Our findings significantly extend the genetic maps of mouse chromosomes 2, 12, and 19 and can be used to define the location of phosphorylase genes in man more precisely. Finally, this analysis suggests that the previously mapped "muscle-deficient" mutation in mouse, mdf, is closely linked to the muscle phosphorylase gene. However, muscle phosphorylase gene structure and expression appear to be unaltered in mdf/mdf mice, indicating that this mutation is not an animal model for the human genetic disorder McArdle's disease.     

Bacterial alpha-glucan phosphorylases

Although glycogen and other alpha-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 non-mammalian and mammalian alpha-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.     

The family of glycogen phosphorylases: structure and function

Glycogen phosphorylase plays a central role in the mobilization of carbohydrate reserves in a wide variety of organisms and tissues. While rabbit muscle phosphorylase remains the most studied and best characterized of phosphorylases, recombinant DNA techniques have led to the recent appearance of primary sequence data for a wide variety of phosphorylase enzymes. The functional properties of rabbit muscle phosphorylases are reviewed and then compared to properties of phosphorylases from other tissues and organisms. Tissue expression patterns and the chromosomal localization of mammalian phosphorylases are described. Differences in functional properties among phosphorylases are related to new structural information. Evolutionary relationships among phosphorylases as afforded by comparative analysis of proteins and gene sequences are discussed.     

Mechanistic differences among retaining disaccharide phosphorylases: insights from kinetic analysis of active site mutants of sucrose phosphorylase and alpha,alpha-trehalose phosphorylase

Sucrose phosphorylase utilizes a glycoside hydrolase-like double displacement mechanism to convert its disaccharide substrate and phosphate into alpha-d-glucose 1-phosphate and fructose. Site-directed mutagenesis was employed to characterize the proposed roles of Asp(196) and Glu(237) as catalytic nucleophile and acid-base, respectively, in the reaction of sucrose phosphorylase from Leuconostoc mesenteroides. The side chain of Asp(295) is suggested to facilitate the catalytic steps of glucosylation and deglucosylation of Asp(196) through a strong hydrogen bond (23 kJ/mol) with the 2-hydroxyl of the glucosyl oxocarbenium ion-like species believed to be formed in the transition states flanking the beta-glucosyl enzyme intermediate. An assortment of biochemical techniques used to examine the mechanism of alpha-retaining glucosyl transfer by Schizophyllum commune alpha,alpha-trehalose phosphorylase failed to provide evidence in support of a similar two-step catalytic reaction via a covalent intermediate. Mutagenesis studies suggested a putative active-site structure for this trehalose phosphorylase that is typical of retaining glycosyltransferases of fold family GT-B and markedly different from that of sucrose phosphorylase. While ambiguity remains regarding the chemical mechanism by which the trehalose phosphorylase functions, the two disaccharide phosphorylases have evolved strikingly different reaction coordinates to achieve catalytic efficiency and stereochemical control in their highly analogous substrate transformations.     

Validating the Incorporation of 13C and 15N in a Shorebird That Consumes an Isotopically Distinct Chemosymbiotic Bivalve

The wealth of field studies using stable isotopes to make inferences about animal diets require controlled validation experiments to make proper interpretations. Despite several pleas in the literature for such experiments, validation studies are still lagging behind, notably in consumers dwelling in chemosynthesis-based ecosystems. In this paper we present such a validation experiment for the incorporation of ¹³C and ¹⁵N in the blood plasma of a medium-sized shorebird, the red knot (Calidris canutus canutus), consuming a chemosymbiotic lucinid bivalve (Loripes lucinalis). Because this bivalve forms a symbiosis with chemoautotrophic sulphide-oxidizing bacteria living inside its gill, the bivalve is isotopically distinct from ‘normal’ bivalves whose food has a photosynthetic basis. Here we experimentally tested the hypothesis that isotope discrimination and incorporation dynamics are different when consuming such chemosynthesis-based prey. The experiment showed that neither the isotopic discrimination factor, nor isotopic turnover time, differed between birds consuming the chemosymbiotic lucinid and a control group consuming a photosynthesis-based bivalve. This was true for ¹³C as well as for ¹⁵N. However, in both groups the ¹⁵N discrimination factor was much higher than expected, which probably had to do with the birds losing body mass over the course of the experiment.     

Thermophilic nucleoside phosphorylases: Their properties,characteristics and applications

Nucleoside phosphorylases catalyze the reversible phosphorolysis of pyrimidine and purine nucleosides in the presence of phosphate. They are valuable catalysts in the synthesis of nucleosides and their analogues, which are often used as pharmaceuticals or their precursors. Thermostable nucleoside phosphorylases are promising biocatalysts, as they withstand harsh reaction conditions such as high pH or the addition of organic solvents. In this review, the characteristics and properties of thermostable nucleoside phosphorylases are described. Differences in amino acid content and protein structure were compared to their mesophilic homologues to identify features involved in thermostability. Substrate spectra of thermostable nucleoside phosphorylases were analyzed, and it is shown that thermostable nucleoside phosphorylases have a wider substrate spectrum than their mesophilic counterparts. Thus, thermostable nucleoside phosphorylases are interesting biocatalysts for industrial applications.     

侏罗纪双壳类动物群的分布与古气候和古地理因素

生物群的地理分布受各种生物和环境因素的影响,对于不同地史晚期以及不同的生物类型,控制生物群分布的主导因素也不同的。就侏罗纪古大西洋双壳类动物群而言古地理和古气候条件是影响其分布的主要环境因素。     

用流式细胞仪测定扇贝血细胞吞噬活性

采用流式细胞仪技术对栉孔扇贝(Chlamysfarreri)和海湾扇贝(Argopectenirradians)血细胞吞噬活性进行了测定。通过对缓冲体系的筛选和比较,建立了扇贝血细胞吞噬活性的流式细胞仪检测方法,并应用该技术对两种扇贝血细胞的吞噬活性进行了比较研究。结果发现在TBS(0.05mol.L-1Tris-HCl,pH7.4;2%glucose;2%NaCl;20mmol.L-1EDTA)和PBS体系中,扇贝血细胞的吞噬活性几乎完全受到抑制,在CMPBS(0.1mol.L-1PBS,pH7.4;2%NaCl;2%glucose;1.0mmol.L-1CaCl2;0.5mmol.L-1MgCl2)体系中细胞吞噬活性略有升高,而在海水缓冲液中细胞吞噬活性最高,其中海湾扇贝和栉孔扇贝血细胞的吞噬率分别达到了26.73%和19.89%,且海湾扇贝血细胞的吞噬率显著高于栉孔扇贝(P<0.05)。研究结果为进一步探讨扇贝血细胞吞噬功能提供了方法依据。     

Bacillary necrosis, a disease of larval and juvenile bivalve mollusks. I. Etiology and epizootiology

Tubiash, Haskell S. (U.S. Bureau of Commercial Fisheries, Milford, Conn.), Paul E. Chanley, and Einar Leifson. Bacillary necrosis, a disease of larval and juvenile bivalve mollusks. I. Etiology and epizootiology. J. Bacteriol. 90:1036-1044. 1965.-Lethal bacterial infections of a variety of hatchery-spawned bivalve mollusk larvae and juveniles have been studied. The symptoms of the disease and the course of the infection are described. Four biotypes and five antigenic types of bacteria, pathogenic for the larvae of five species of bivalve mollusks, were isolated and described in some detail. All are gram-negative motile rods. Comparative studies were made of a fairly large number of similar bacteria isolated from presumably normal marine fauna. None of these was pathogenic for the bivalve larvae nor did they have antigens in common with the pathogenic group. The four biotypes had a number of characteristics in common that rarely were present in other cultures from marine fauna. Several antibiotic preparations proved to be of value in the treatment and control of the infection.     

Influence of low-temperature processing of the brackish-water bivalve, Corbicula japonica, on the ornithine content of its extract

The ornithine content of an extract of the brackish-water bivalve, Corbicula japonica, increased when the bivalve was frozen. It was not influenced by the period of freezing. This phenomenon was not apparent in the scallop, little-neck clam, or hard clam. We applied various low-temperature conditions for processing the bivalve from 4 degrees C to -10 degrees C and measured the ornithine content of each extract. The ornithine content was maximized by processing at - 4 degrees C. The increase in this ornithine content was reduced when the bivalve was stored at 5 degrees C or 15 degrees C after processing at - 4 degrees C, this decrease being reversed when the bivalve was again processed at - 4 degrees C after warming. Low-temperature processing of the brackish-water bivalve therefore increased the ornithine content of the extract.     

An i-type lysozyme from the Asiatic hard clam Meretrix meretrix potentially functioning in host immunity

Lysozymes function in animal immunity. Three types of lysozyme have been identified in animal kingdom and most lysozymes identified from bivalve molluscs belong to the invertebrate (i) type. In this research, we cloned and sequenced a new i-type lysozyme, named MmeLys, from the Asiatic hard clam Meretrix meretrix. MmeLys cDNA was constituted of 552 bp, with a 441 bp open reading frame encoding a 146 amino acid polypeptide. The encoded polypeptide was predicted to have a 15 amino acid signal peptide, and a 131 amino acid mature protein with a theoretical mass of 14601.44 Da and an isoelectric point (pI) of 7.14. MmeLys amino acid sequence bore 64% identity with the Manila clam (Venerupis philippinarum) i-type lysozyme and was grouped with other veneroid i-type lysozymes in a bivalve lysozyme phylogenetic tree predicted using Neighbor-Jointing method. Recombinantly expressed MmeLys showed lysozyme activity and strong antibacterial activity against Gram positive and Gram negative bacteria. MmeLys mRNA and protein were detected to be mainly produced in hepatopancreas and gill by the methods of semi-quantitative RT-PCR and western blotting. In addition, MmeLys gene expression increased following Vibrio parahaemolyticus challenge. Results of this research indicated that MmeLys represents a new i-type lysozyme that likely functions in M. meretrix immunity.     

The dynamics of the sodium, potassium, calcium, magnesium contents in the fresh water mollusc zebra musselDreissenia polymorpha during stress

The sodium and magnesium concentration in the hemolymph of the zebra musselDreissenia polymorpha decreased by 25.1 and 25.6%, respectively, in 7.5 h after catching and transportation. One day later, the content of all four cations studied in the molluscan body was significantly reduced. After 7 days, the decrease in the tissue concentration, as compared to initial levels, was 2.7, 2.5, 4, and 4.4 times for sodium, potassium, calcium, and magnesium, respectively. After 18 days of acclimation, the concentrations of cations in the hemolymph and molluscan body did not differ from the initial values. Comparative analysis shows that bivalve molluscs and fish have a common negative mechanism connected with a loss of salt from the organism during the initial period of stress. Possible causes are discussed of the decrease in the salt content in the body of the marine bivalve molluscs to minimal values during their migration to the fresh water in the course of evolution.     

The dynamics of the sodium, potassium, calcium, magnesium contents in the fresh water mollusc zebra musselDreissenia polymorpha during stress

The sodium and magnesium concentration in the hemolymph of the zebra musselDreissenia polymorpha decreased by 25.1 and 25.6%, respectively, in 7.5 h after catching and transportation. One day later, the content of all four cations studied in the molluscan body was significantly reduced. After 7 days, the decrease in the tissue concentration, as compared to initial levels, was 2.7, 2.5, 4, and 4.4 times for sodium, potassium, calcium, and magnesium, respectively. After 18 days of acclimation, the concentrations of cations in the hemolymph and molluscan body did not differ from the initial values. Comparative analysis shows that bivalve molluscs and fish have a common negative mechanism connected with a loss of salt from the organism during the initial period of stress. Possible causes are discussed of the decrease in the salt content in the body of the marine bivalve molluscs to minimal values during their migration to the fresh water in the course of evolution.