Structure and Characterization of Phosphoglucomutase 5 from Atlantic and Baltic Herring-An Inactive Enzyme with Intact Substrate Binding.

Phosphoglucomutase 5 (PGM5) in humans is known as a structural muscle protein without enzymatic activity, but detailed understanding of its function is lacking. PGM5 belongs to the alpha-D-phosphohexomutase family and is closely related to the enzymatically active metabolic enzyme PGM1. In the Atlantic herring, Clupea harengus, PGM5 is one of the genes strongly associated with ecological adaptation to the brackish Baltic Sea. We here present the first crystal structures of PGM5, from the Atlantic and Baltic herring, differing by a single substitution Ala330Val. The structure of PGM5 is overall highly similar to structures of PGM1. The structure of the Baltic herring PGM5 in complex with the substrate glucose-1-phosphate shows conserved substrate binding and active site compared to human PGM1, but both PGM5 variants lack phosphoglucomutase activity under the tested conditions. Structure comparison and sequence analysis of PGM5 and PGM1 from fish and mammals suggest that the lacking enzymatic activity of PGM5 is related to differences in active-site loops that are important for flipping of the reaction intermediate. The Ala330Val substitution does not alter structure or biophysical properties of PGM5 but, due to its surface-exposed location, could affect interactions with protein-binding partners.

Allomorphy as a mechanism of post-translational control of enzyme activity.

Enzyme regulation is vital for metabolic adaptability in living systems. Fine control of enzyme activity is often delivered through post-translational mechanisms, such as allostery or allokairy. ß-phosphoglucomutase (ßPGM) from Lactococcus lactis is a phosphoryl transfer enzyme required for complete catabolism of trehalose and maltose, through the isomerisation of ß-glucose 1-phosphate to glucose 6-phosphate via ß-glucose 1,6-bisphosphate. Surprisingly for a gatekeeper of glycolysis, no fine control mechanism of ßPGM has yet been reported. Herein, we describe allomorphy, a post-translational control mechanism of enzyme activity. In ßPGM, isomerisation of the K145-P146 peptide bond results in the population of two conformers that have different activities owing to repositioning of the K145 sidechain. In vivo phosphorylating agents, such as fructose 1,6-bisphosphate, generate phosphorylated forms of both conformers, leading to a lag phase in activity until the more active phosphorylated conformer dominates. In contrast, the reaction intermediate ß-glucose 1,6-bisphosphate, whose concentration depends on the ß-glucose 1-phosphate concentration, couples the conformational switch and the phosphorylation step, resulting in the rapid generation of the more active phosphorylated conformer. In enabling different behaviours for different allomorphic activators, allomorphy allows an organism to maximise its responsiveness to environmental changes while minimising the diversion of valuable metabolites.

Impact of Up- and Downregulation of Metabolites and Mitochondrial Content on pH and Color of the Longissimus Muscle from Normal-pH and Dark-Cutting Beef.

Limited knowledge is currently available on the biochemical basis for the development of dark-cutting beef. The objective of this research was to determine the metabolite profile and mitochondrial content differences between normal-pH and dark-cutting beef. A gas chromatography-mass spectrometer-based nontargeted metabolomic approach indicated downregulation of glycolytic metabolites, including glucose-1- and 6-phosphate and upregulation of tricarboxylic substrates such as malic and fumaric acids occurred in dark-cutting beef when compared to normal-pH beef. Neurotransmitters such as 4-aminobutyric acid and succinate semialdehyde were upregulated in dark-cutting beef than normal-pH beef. Immunohistochemistry indicated a more oxidative fiber type in dark-cutting beef than normal-pH beef. In support, the mitochondrial protein and DNA content were greater in dark-cutting beef. This increased mitochondrial content, in part, could influence oxygen consumption and myoglobin oxygenation/appearance of dark-cutting beef. The current results demonstrate that the more tricarboxylic metabolites and mitochondrial content in dark-cutting beef impact muscle pH and color.

Two Glycosyl 1-Phosphate Polymers and Teichulosonic Acid from Glutamicibacter protophormiae VKM Ac-2104T Cell Wall.

Two glycosyl 1-phosphate polymers containing monoglycosyl 1-phosphate, -6)-α-D-Glcp-(1-P-, and diglycosyl 1-phosphate, -6)-α-D-GalpNAc-(1→6)-α-D-GlcpNAc-(1-P-, in the repeating unit were identified in the cell wall of Glutamicibacter protophormiae VKM Ac-2104T (formerly, Arthrobacter protophormiae). The structures of these polymers were described for the first time in prokaryotes. Teichulosonic acid, the third identified polymer, with 3-deoxy-D-glycero-α-D-galacto-non-2-ulopyranosonic acid (Kdn) and ß-D-glucopyranose residues in the main chain, →6)-ß-D-Glcp-(1→8)-α-Kdn-(2→, has been previously detected in a number of actinobacteria. The structures of these glycopolymers were established based on the results of chemical analysis and one-dimensional 1H, 13C, and 31P NMR spectroscopy using two-dimensional homonuclear (1H,1H COZY, TOCSY, ROESY) and heteronuclear (1H,13C HSQC, HSQC-TOCSY, HMBC, and 1H,31P HMBC) techniques.

Esters of Glucose-2-Phosphate: Occurrence and Chemistry.

Phosphodiesters of glucose-2-phosphate (G2P) are found only in few natural compounds such as agrocinopine D and agrocin 84. Agrocinopine D is a G2P phosphodiester produced by plants infected by Agrobacterium fabrum C58 and recognized by the bacterial periplasmic binding protein AccA for being transported into the bacteria before cleavage by the phosphodiesterase AccF, releasing G2P, which promotes virulence by binding the repressor protein AccR. The G2P amide agrocin 84 is a natural antibiotic produced by the non-pathogenic Agrobacterium radiobacter K84 strain used as a biocontrol agent by competing with Agrobacterium fabrum C58. G2P esters are also found in irregular glycogen structures. The rare glucopyranosyl-2-phophoryl moiety found in agrocin 84 is the key structural signature enabling its action as a natural antibiotic. Likewise, G2P and G2P esters can also dupe the Agrobacterium agrocinopine catabolism cascade. Such observations illustrate the importance of G2P esters on which we have recently focused our interest. After a brief review of the reported phosphorylation coupling methods and the choice of carbohydrate building blocks used in G2P chemistry, a flexible access to glucose-2-phosphate esters using the phosphoramidite route is proposed.

Enhanced Glycogen Metabolism Supports the Survival and Proliferation of HPV-Infected Keratinocytes in Condylomata Acuminata.

Condylomata acuminata (CA) is caused by human papillomavirus (HPV) infections of keratinocytes and is a common sexually transmitted disease. The main clinical feature and risk of CA is the high recurrence of genital warts formed by infected keratinocytes. Metabolic reprogramming of most types of mammalian cells including keratinocytes can provide energy and intermediates essential for their survival. Here, we report that HPV infection develops a hypoxic microenvironment in CA warts by inducing the accumulation of glycogen and increased glycogen metabolism in the infected keratinocytes in a hypoxia-inducible factor 1α (HIF-1α) -dependent pathway. Our in vitro studies show that the increased glycogen metabolism is essential for the survival and proliferation of keratinocytes. Regarding its mechanism of action, glycogenolysis generates glucose-1-phosphate that fluxes into the pentose phosphate pathway and, then, generates abundant nicotinamide adenine dinucleotide phosphate, thereby ensuring high levels of glutathione in keratinocytes under hypoxia. The abrogation of glycogen synthesis and glycogenolysis decreases the ratio of glutathione and glutathione disulfide and increases the level of ROS, further resulting in the impairment of keratinocyte survival. Collectively, our work offers an insight into the metabolic reprogramming in the development of CA and implies that the intervention of glycogen metabolism would be a promising therapeutic target for CA.

Efficient one-pot enzymatic synthesis of trehalose 6-phosphate using GH65 α-glucoside phosphorylases.

Trehalose 6-phosphate (Tre6P) is an important intermediate for trehalose biosynthesis. Recent researches have revealed that Tre6P is an endogenous signaling molecule that regulates plant development and stress responses. The necessity of Tre6P in physiological studies is expected to be increasing. To achieve the cost-effective production of Tre6P, a novel approach is required. In this study, we utilized trehalose 6-phosphate phosphorylase (TrePP) from Lactococcus lactis to produce Tre6P. In the reverse phosphorolysis by the TrePP, 91.9 mM Tre6P was produced from 100 mM ß-glucose 1-phosphate (ß-Glc1P) and 100 mM glucose 6-phosphate (Glc6P). The one-pot reaction of TrePP and maltose phosphorylase (MP) enabled production of 65 mM Tre6P from 100 mM maltose, 100 mM Glc6P, and 20 mM inorganic phosphate. Addition of ß-phosphoglucomutase to this reaction produced Glc6P from ß-Glc1P and thus reduced requirement of Glc6P as a starting material. Within the range of 20-469 mM inorganic phosphate tested, the 54 mM concentration yielded the highest amount of Tre6P (33 mM). Addition of yeast increased the yield because of its glucose consumption. Finally, from 100 mmol maltose and 60 mmol inorganic phosphate, we successfully achieved production of 37.5 mmol Tre6P in a one-pot reaction (100 mL), and 9.4 g Tre6P dipotassium salt was obtained.

Identification of Two Arabidopsis thaliana Plasma Membrane Transporters Able to Transport Glucose 1-Phosphate.

Primary carbohydrate metabolism in plants includes several sugar and sugar-derivative transport processes. Over recent years, evidences have shown that in starch-related transport processes, in addition to glucose 6-phosphate, maltose, glucose and triose-phosphates, glucose 1-phosphate also plays a role and thereby increases the possible fluxes of sugar metabolites in planta. In this study, we report the characterization of two highly similar transporters, At1g34020 and At4g09810, in Arabidopsis thaliana, which allow the import of glucose 1-phosphate through the plasma membrane. Both transporters were expressed in yeast and were biochemically analyzed to reveal an antiport of glucose 1-phosphate/phosphate. Furthermore, we showed that the apoplast of Arabidopsis leaves contained glucose 1-phosphate and that the corresponding mutant of these transporters had higher glucose 1-phosphate amounts in the apoplast and alterations in starch and starch-related metabolism.

Three-Enzyme Phosphorylase Cascade for Integrated Production of Short-Chain Cellodextrins.

Cellodextrins are linear ß-1,4-gluco-oligosaccharides that are soluble in water up to a degree of polymerization (DP) of ≈6. Soluble cellodextrins have promising applications as nutritional ingredients. A DP-controlled, bottom-up synthesis from expedient substrates is desired for their bulk production. Here, a three-enzyme glycoside phosphorylase cascade is developed for the conversion of sucrose and glucose into short-chain (soluble) cellodextrins (DP range 3-6). The cascade reaction involves iterative ß-1,4-glucosylation of glucose from α-glucose 1-phosphate (αGlc1-P) donor that is formed in situ from sucrose and phosphate. With final concentration and yield of the soluble cellodextrins set as targets for biocatalytic synthesis, three major factors of reaction efficiency are identified and partly optimized: the ratio of enzyme activity, the ratio of sucrose and glucose, and the phosphate concentration used. The efficient use of the phosphate/αGlc1-P shuttle for cellodextrin production is demonstrated and the soluble product at 40 g L-1 is obtained under near-complete utilization of the donor substrate offered (88 mol% from 200 mm sucrose). The productivity is 16 g (L h)-1 . Through a simple two-step route, the soluble cellodextrins are recovered from the reaction mixture in ≥95% purity and ≈92% yield. Overall, this study provides the basis for their integrated production.

Two Homologous Enzymes of the GalU Family in Rhodococcus opacus 1CP-RoGalU1 and RoGalU2.

Uridine-5'-diphosphate (UDP)-glucose is reported as one of the most versatile building blocks within the metabolism of pro- and eukaryotes. The activated sugar moiety is formed by the enzyme UDP-glucose pyrophosphorylase (GalU). Two homologous enzymes (designated as RoGalU1 and RoGalU2) are encoded by most Rhodococcus strains, known for their capability to degrade numerous compounds, but also to synthesize natural products such as trehalose comprising biosurfactants. To evaluate their functionality respective genes of a trehalose biosurfactant producing model organism-Rhodococcus opacus 1CP-were cloned and expressed, proteins produced (yield up to 47 mg per L broth) and initially biochemically characterized. In the case of RoGalU2, the Vmax was determined to be 177 U mg-1 (uridine-5'-triphosphate (UTP)) and Km to be 0.51 mM (UTP), respectively. Like other GalUs this enzyme seems to be rather specific for the substrates UTP and glucose 1-phosphate, as it accepts only dTTP and galactose 1-phoshate in addition, but both with solely 2% residual activity. In comparison to other bacterial GalU enzymes the RoGalU2 was found to be somewhat higher in activity (factor 1.8) even at elevated temperatures. However, RoGalU1 was not obtained in an active form thus it remains enigmatic if this enzyme participates in metabolism.

Enzymatic synthesis of functional amylosic materials and amylose analog polysaccharides.

Because polysaccharides have very complicated chemical structures constructed by a great diversity of monosaccharide residues and glycosidic linkages, enzymatic approaches have been identified as powerful tools to precisely synthesize polysaccharides as the reactions progress in highly controlled regio- and stereoarrangements. α-Glucan phosphorylase (GP) is one of the enzymes that have acted as catalysts for the practical production of well-defined polysaccharides. GP can catalyze enzymatic polymerization of α-d-glucose 1-phosphate (Glc-1-P) as a monomer from a maltooligosaccharide primer to produce a pure amylose with well-defined structure via the formation of α(1→4)-glycosidic linkages. Here, the author presents methods which achieve the enzymatic synthesis of functional amylosic materials and amylose analog polysaccharides by GP-catalyzed enzymatic polymerization approaches. As the polymerization progresses at the non-reducing end of the primer, it can be conducted using polymeric primers that are modified at the reducing end and covalently attached on suitable polymeric chains. By using such polymeric primers, various amylose-grafted functional materials can be enzymatically synthesized. For example, the detailed protocol for the synthesis of amylose-grafted poly(γ-glutamic acid) is described. GP shows loose specificity for the recognition of substrates, which allows to recognize some monosaccharide 1-phosphates as analog substrates of Glc-1-P. Representatively, the experimental procedure of the GP-catalyzed enzymatic polymerization of α-d-glucosamine 1-phosphate as the analog substrate is presented to synthesize an α(1→4)-linked glucosamine polymer, that is called amylosamine. By means of a similar approach catalyzed by GP, several amylose analog polysaccharides have been obtained.

Synthesis of a non-natural glucose-2-phosphate ester able to dupe the acc system of Agrobacterium fabrum.

The first non-natural derivative of the rare d-glucose-2-phosphate (G2P), namely glucose-2-(O-lactic acid phosphate) (G2LP), has been synthesized. When used as sole carbon source, G2LP enables bacterial growth of the plant pathogenic strain Agrobacterium fabrum C58 (formerly referred to as Agrobacterium tumefaciens). X-ray crystallography and affinity measurements investigations reveal that G2LP binds the periplasmic binding protein (PBP) AccA similarly to the natural compounds and with the same affinity. Moreover, enzymatic assays show that it is able to serve as substrate of the phosphodiesterase AccF. The properties found for G2LP demonstrate that the very unusual glucose-2-phosphoryl residue, present in G2LP, can be used as structural feature for designing non-natural systems fully compatible with the Acc cascade of A. fabrum.

Characterization of the Transglycosylation Reaction of 4-α-Glucanotransferase (MalQ) and Its Role in Glycogen Breakdown in Escherichia coli.

We first confirmed the involvement of MalQ (4-α-glucanotransferase) in Escherichia coli glycogen breakdown by both in vitro and in vivo assays. In vivo tests of the knock-out mutant, ΔmalQ, showed that glycogen slowly decreased after the stationary phase compared to the wild-type strain, indicating the involvement of MalQ in glycogen degradation. In vitro assays incubated glycogen-mimic substrate, branched cyclodextrin (maltotetraosyl-ß-CD: G4- ß-CD) and glycogen phosphorylase (GlgP)-limit dextrin with a set of variable combinations of E. coli enzymes, including GlgX (debranching enzyme), MalP (maltodextrin phosphorylase), GlgP and MalQ. In the absence of GlgP, the reaction of MalP, GlgX and MalQ on substrates produced glucose-1-P (glc-1-P) 3-fold faster than without MalQ. The results revealed that MalQ led to disproportionate G4 released from GlgP-limit dextrin to another acceptor, G4, which is phosphorylated by MalP. In contrast, in the absence of MalP, the reaction of GlgX, GlgP and MalQ resulted in a 1.6-fold increased production of glc-1-P than without MalQ. The result indicated that the G4-branch chains of GlgP-limit dextrin are released by GlgX hydrolysis, and then MalQ transfers the resultant G4 either to another branch chain or another G4 that can immediately be phosphorylated into glc-1-P by GlgP. Thus, we propose a model of two possible MalQ-involved pathways in glycogen degradation. The operon structure of MalP-defecting enterobacteria strongly supports the involvement of MalQ and GlgP as alternative pathways in glycogen degradation.

Expression of manB Gene from Escherichia coli in Lactococcus lactis and Characterization of Its Bifunctional Enzyme, Phosphomannomutase.

Phosphomannomutase (ManB) converts mannose-6-phosphate (M-6-P) to mannose-1-phosphate (M-1-P), which is a key metabolic precursor for the production of GDP-D-mannose used for production of glycoconjugates and post-translational modification of proteins. The aim of this study was to express the manB gene from Escherichia coli in Lactococcus lactis subsp. cremoris NZ9000 and to characterize the encoded enzyme. The manB gene from E. coli K12, of 1,371 bp and encoding 457 amino acids (52 kDa), was cloned and overexpressed in L. lactis NZ9000 using the nisin-controlled expression system. The enzyme was purified by Ni-NTA column chromatography and exhibited a specific activity of 5.34 units/mg, significantly higher than that of other previously reported ManB enzymes. The pH and temperature optima were 8.0 and 50°C, respectively. Interestingly, the ManB used in this study had two substrate specificity for both mannose-1-phosphate and glucose-1-phosphate, and the specific activity for glucose-1-phosphate was 3.76 units/mg showing 70% relative activity to that of mannose-1-phosphate. This is the first study on heterologous expression and characterization of ManB in lactic acid bacteria. The ManB expression system constructed in this study canbe used to synthesize rare sugars or glycoconjugates.

Mechanism of Substrate Recognition and Catalysis of the Haloalkanoic Acid Dehalogenase Family Member α-Phosphoglucomutase.

α-Phosphoglucomutase (αPGM), in its phosphorylated state, catalyzes the interconversion of α-d-glucose 1-phosphate and α-d-glucose 6-phosphate. The αPGM of Lactococcus lactis is a type C2B member of the haloalkanoic acid dehalogenase (HAD) enzyme family and is comprised of a Rossmann-fold catalytic domain and inserted α/ß-fold cap domain. The active site is formed at the domain-domain interface. Herein, we report the results from a kinetic-based study of L. lactis αPGM catalysis, which demonstrate enzyme activation by autocatalyzed phosphorylation of Asp8 with αG1P, the intermediacy of αG1,6bisP in the phospho Ll-αPGM-catalyzed conversion of αG1P to G6P, and the reorientation of the αG1,6bisP intermediate via dissociation to solvent and rebinding. In order to provide insight into the structural determinants of L. lactis αPGM substrate recognition and catalysis, metal cofactor and substrate specificities were determined as were the contributions made by active-site residues toward catalytic efficiency. Lastly, the structure and catalytic mechanism of L. lactis αPGM are compared with those of HAD family phosphomutases L. lactis ß-phosphoglucomutase and eukayotic α-phosphomannomutase to provide insight into the evolution of phosphohexomutases from HAD family phosphatases.

Identification of a Direct Biosynthetic Pathway for UDP-N-Acetylgalactosamine from Glucosamine-6-Phosphate in Thermophilic Crenarchaeon Sulfolobus tokodaii.

Most organisms, from Bacteria to Eukarya, synthesize UDP-N-acetylglucosamine (UDP-GlcNAc) from fructose-6-phosphate via a four-step reaction, and UDP-N-acetylgalactosamine (UDP-GalNAc) can only be synthesized from UDP-GlcNAc by UDP-GlcNAc 4-epimerase. In Archaea, the bacterial-type UDP-GlcNAc biosynthetic pathway was reported for Methanococcales. However, the complete biosynthetic pathways for UDP-GlcNAc and UDP-GalNAc present in one archaeal species are unidentified. Previous experimental analyses on enzymatic activities of the ST0452 protein, identified from the thermophilic crenarchaeon Sulfolobus tokodaii, predicted the presence of both a bacterial-type UDP-GlcNAc and an independent UDP-GalNAc biosynthetic pathway in this archaeon. In the present work, functional analyses revealed that the recombinant ST2186 protein possessed an glutamine:fructose-6-phosphate amidotransferase activity and that the recombinant ST0242 protein possessed a phosphoglucosamine-mutase activity. Along with the acetyltransferase and uridyltransferase activities of the ST0452 protein, the activities of the ST2186 and ST0242 proteins confirmed the presence of a bacterial-type UDP-GlcNAc biosynthetic pathway in S. tokodaii In contrast, the UDP-GlcNAc 4-epimerase homologue gene was not detected within the genomic data. Thus, it was expected that galactosamine-1-phosphate or galactosamine-6-phosphate (GalN-6-P) was provided by conversion of glucosamine-1-phosphate or glucosamine-6-phosphate (GlcN-6-P). A novel epimerase converting GlcN-6-P to GalN-6-P was detected in a cell extract of S. tokodaii, and the N-terminal sequence of the purified protein indicated that the novel epimerase was encoded by the ST2245 gene. Along with the ST0242 phosphogalactosamine-mutase activity, this observation confirmed the presence of a novel UDP-GalNAc biosynthetic pathway from GlcN-6-P in S. tokodaii Discovery of the novel pathway provides a new insight into the evolution of nucleotide sugar metabolic pathways.IMPORTANCE In this work, a novel protein capable of directly converting glucosamine-6-phosphate to galactosamine-6-phosphate was successfully purified from a cell extract of the thermophilic crenarchaeon Sulfolobus tokodaii Confirmation of this novel activity using the recombinant protein indicates that S. tokodaii possesses a novel UDP-GalNAc biosynthetic pathway derived from glucosamine-6-phosphate. The distributions of this and related genes indicate the presence of three different types of UDP-GalNAc biosynthetic pathways: a direct pathway using a novel enzyme and two conversion pathways from UDP-GlcNAc using known enzymes. Additionally, Crenarchaeota species lacking all three pathways were found, predicting the presence of one more unknown pathway. Identification of these novel proteins and pathways provides important insights into the evolution of nucleotide sugar biosynthesis, as well as being potentially important industrially.

Metabolomics reveals critical adrenergic regulatory checkpoints in glycolysis and pentose-phosphate pathways in embryonic heart.

Cardiac energy demands during early embryonic periods are sufficiently met through glycolysis, but as development proceeds, the oxidative phosphorylation in mitochondria becomes increasingly vital. Adrenergic hormones are known to stimulate metabolism in adult mammals and are essential for embryonic development, but relatively little is known about their effects on metabolism in the embryonic heart. Here, we show that embryos lacking adrenergic stimulation have ∼10-fold less cardiac ATP compared with littermate controls. Despite this deficit in steady-state ATP, neither the rates of ATP formation nor degradation was affected in adrenergic hormone-deficient hearts, suggesting that ATP synthesis and hydrolysis mechanisms were fully operational. We thus hypothesized that adrenergic hormones stimulate metabolism of glucose to provide chemical substrates for oxidation in mitochondria. To test this hypothesis, we employed a metabolomics-based approach using LC/MS. Our results showed glucose 1-phosphate and glucose 6-phosphate concentrations were not significantly altered, but several downstream metabolites in both glycolytic and pentose-phosphate pathways were significantly lower compared with controls. Furthermore, we identified glyceraldehyde-3-phosphate dehydrogenase and glucose-6-phosphate dehydrogenase as key enzymes in those respective metabolic pathways whose activity was significantly (p < 0.05) and substantially (80 and 40%, respectively) lower in adrenergic hormone-deficient hearts. Addition of pyruvate and to a lesser extent ribose led to significant recovery of steady-state ATP concentrations. These results demonstrate that without adrenergic stimulation, glucose metabolism in the embryonic heart is severely impaired in multiple pathways, ultimately leading to insufficient metabolic substrate availability for successful transition to aerobic respiration needed for survival.

Human NUDT22 Is a UDP-Glucose/Galactose Hydrolase Exhibiting a Unique Structural Fold.

Human NUDT22 belongs to the diverse NUDIX family of proteins, but has, until now, remained uncharacterized. Here we show that human NUDT22 is a Mg2+-dependent UDP-glucose and UDP-galactose hydrolase, producing UMP and glucose 1-phosphate or galactose 1-phosphate. We present the structure of human NUDT22 alone and in a complex with the substrate UDP-glucose. These structures reveal a partially conserved NUDIX fold domain preceded by a unique N-terminal domain responsible for UDP moiety binding and recognition. The NUDIX domain of NUDT22 contains a modified NUDIX box identified using structural analysis and confirmed through functional analysis of mutants. Human NUDT22's distinct structure and function as a UDP-carbohydrate hydrolase establish a unique NUDIX protein subfamily.

Glucose-1-phosphate uridylyltransferase from Erwinia amylovora: Activity, structure and substrate specificity.

Erwinia amylovora, a Gram-negative plant pathogen, is the causal agent of Fire Blight, a contagious necrotic disease affecting plants belonging to the Rosaceae family, including apple and pear. E. amylovora is highly virulent and capable of rapid dissemination in orchards; effective control methods are still lacking. One of its most important pathogenicity factors is the exopolysaccharide amylovoran. Amylovoran is a branched polymer made by the repetition of units mainly composed of galactose, with some residues of glucose, glucuronic acid and pyruvate. E. amylovora glucose-1-phosphate uridylyltransferase (UDP-glucose pyrophosphorylase, EC 2.7.7.9) has a key role in amylovoran biosynthesis. This enzyme catalyses the production of UDP-glucose from glucose-1-phosphate and UTP, which the epimerase GalE converts into UDP-galactose, the main building block of amylovoran. We determined EaGalU kinetic parameters and substrate specificity with a range of sugar 1-phosphates. At time point 120min the enzyme catalysed conversion of the sugar 1-phosphate into the corresponding UDP-sugar reached 74% for N-acetyl-α-d-glucosamine 1-phosphate, 28% for α-d-galactose 1-phosphate, 0% for α-d-galactosamine 1-phosphate, 100% for α-d-xylose 1-phosphate, 100% for α-d-glucosamine 1-phosphate, 70% for α-d-mannose 1-phosphate, and 0% for α-d-galacturonic acid 1-phosphate. To explain our results we obtained the crystal structure of EaGalU and augmented our study by docking the different sugar 1-phosphates into EaGalU active site, providing both reliable models for substrate binding and enzyme specificity, and a rationale that explains the different activity of EaGalU on the sugar 1-phosphates used. These data demonstrate EaGalU potential as a biocatalyst for biotechnological purposes, as an alternative to the enzyme from Escherichia coli, besides playing an important role in E. amylovora pathogenicity.

Cellodextrin phosphorylase from Ruminiclostridium thermocellum: X-ray crystal structure and substrate specificity analysis.

The GH94 glycoside hydrolase cellodextrin phosphorylase (CDP, EC 2.4.1.49) produces cellodextrin oligomers from short ß-1→4-glucans and α-D-glucose 1-phosphate. Compared to cellobiose phosphorylase (CBP), which produces cellobiose from glucose and α-D-glucose 1-phosphate, CDP is biochemically less well characterised. Herein, we investigate the donor and acceptor substrate specificity of recombinant CDP from Ruminiclostridium thermocellum and we isolate and characterise a glucosamine addition product to the cellobiose acceptor with the non-natural donor α-D-glucosamine 1-phosphate. In addition, we report the first X-ray crystal structure of CDP, along with comparison to the available structures from CBPs and other closely related enzymes, which contributes to understanding of the key structural features necessary to discriminate between monosaccharide (CBP) and oligosaccharide (CDP) acceptor substrates.