Methicillin-resistant Staphylococcus aureus alters cell wall glycosylation to evade immunity.

Methicillin-resistant Staphylococcus aureus (MRSA) is a frequent cause of difficult-to-treat, often fatal infections in humans1,2. Most humans have antibodies against S. aureus, but these are highly variable and often not protective in immunocompromised patients3. Previous vaccine development programs have not been successful4. A large percentage of human antibodies against S. aureus target wall teichoic acid (WTA), a ribitol-phosphate (RboP) surface polymer modified with N-acetylglucosamine (GlcNAc)5,6. It is currently unknown whether the immune evasion capacities of MRSA are due to variation of dominant surface epitopes such as those associated with WTA. Here we show that a considerable proportion of the prominent healthcare-associated and livestock-associated MRSA clones CC5 and CC398, respectively, contain prophages that encode an alternative WTA glycosyltransferase. This enzyme, TarP, transfers GlcNAc to a different hydroxyl group of the WTA RboP than the standard enzyme TarS7, with important consequences for immune recognition. TarP-glycosylated WTA elicits 7.5-40-fold lower levels of immunoglobulin G in mice than TarS-modified WTA. Consistent with this, human sera contained only low levels of antibodies against TarP-modified WTA. Notably, mice immunized with TarS-modified WTA were not protected against infection with tarP-expressing MRSA, indicating that TarP is crucial for the capacity of S. aureus to evade host defences. High-resolution structural analyses of TarP bound to WTA components and uridine diphosphate GlcNAc (UDP-GlcNAc) explain the mechanism of altered RboP glycosylation and form a template for targeted inhibition of TarP. Our study reveals an immune evasion strategy of S. aureus based on averting the immunogenicity of its dominant glycoantigen WTA. These results will help with the identification of invariant S. aureus vaccine antigens and may enable the development of TarP inhibitors as a new strategy for rendering MRSA susceptible to human host defences.

Crystallographic analysis of TarI and TarJ, a cytidylyltransferase and reductase pair for CDP-ribitol synthesis in Staphylococcus aureus wall teichoic acid biogenesis.

The cell wall of many pathogenic Gram-positive bacteria contains ribitol-phosphate wall teichoic acid (WTA), a polymer that is linked to virulence and regulation of essential physiological processes including cell division. CDP-ribitol, the activated precursor for ribitol-phosphate polymerization, is synthesized by a cytidylyltransferase and reductase pair known as TarI and TarJ, respectively. In this study, we present crystal structures of Staphylococcus aureus TarI and TarJ in their apo forms and in complex with substrates and products. The TarI structures illustrate the mechanism of CDP-ribitol synthesis from CTP and ribitol-phosphate and reveal structural changes required for substrate binding and catalysis. Insights into the upstream step of ribulose-phosphate reduction to ribitol-phosphate is provided by the structures of TarJ. Furthermore, we propose a general topology of the enzymes in a heterotetrameric form built using restraints from crosslinking mass spectrometry analysis. Together, our data present molecular details of CDP-ribitol production that may aid in the design of inhibitors against WTA biosynthesis.

Heat priming improved heat tolerance of photosynthesis, enhanced terpenoid and benzenoid emission and phenolics accumulation in Achillea millefolium.

The mechanism of heat priming, triggering alteration of secondary metabolite pathway fluxes and pools to enhance heat tolerance is not well understood. Achillea millefolium is an important medicinal herbal plant, rich in terpenoids and phenolics. In this study, the potential of heat priming treatment (35°C for 1 hr) to enhance tolerance of Achillea plants upon subsequent heat shock (45°C for 5 min) stress was investigated through recovery (0.5-72 hr). The priming treatment itself had minor impacts on photosynthesis, led to moderate increases in the emission of lipoxygenase (LOX) pathway volatiles and isoprene, and to major elicitation of monoterpene and benzaldehyde emissions in late stages of recovery. Upon subsequent heat shock, in primed plants, the rise in LOX and reduction in photosynthetic rate (A) was much less, stomatal conductance (gs ) was initially enhanced, terpene emissions were greater and recovery of A occurred faster, indicating enhanced heat tolerance. Additionally, primed plants accumulated higher contents of total phenolics and condensed tannins at the end of the recovery. These results collectively indicate that heat priming improved photosynthesis upon subsequent heat shock by enhancing gs and synthesis of volatile and non-volatile secondary compounds with antioxidative characteristics, thereby maintaining the integrity of leaf membranes under stress.

A direct spectropolarimetric assay of arabinose 5-phosphate isomerase.

Arabinose 5-phosphate isomerase (API) catalyzes the reversible isomerization of Ribulose 5-phosphate (Ru5P) to Arabinose 5-Phosphate (Ar5P) for the production of 3-deoxy-2-octulosonic acid 8-phosphate (KDO), a component of bacterial lipopolysaccharide (LPS) of gram-negative bacteria. API is an attractive target for therapeutic development against gram-negative bacterial pathogens. The current assay method of API activity utilizes a general reaction for keto sugar determination in a secondary, 3-h color development reaction with 25 N sulfuric acid which poses hazard to both personnel and instrumentation. We therefore aimed to develop a more user friendly assay of the enzyme. Since Ru5P absorbs in the UV region and contains at least 2 chiral centers, it can be expected to display circular dichroism (CD). A wavelength scan revealed indeed Ru5P displays a pronounced negative ellipticity of 30,560 mDeg M-1cm-1 at 279 nm in Tris buffer pH 9.1 but Ar5P does not have any CD. API enzymatic reactions were monitored directly and continuously in real time by following the disappearance of CD from the Ru5P substrate, or by the appearance of CD from Ar5P substrate. The CD signal at this wavelength was not affected by absorption of the enzyme protein or of small molecules, or turbidity of the solution. Common additives in protein and enzyme reaction mixtures such as detergents, metals, and 5% dimethylsulfoxide did not interfere with the CD signal. Assay reactions of 1-3 min consistently yielded reproducible results. Introduction of accessories in a spectropolarimeter will easily adapt this assay to high throughput format for screening thousands of small molecules as inhibitor candidates of API.

Characterization and Function of the 1-Deoxy-D-xylose-5-Phosphate Synthase (DXS) Gene Related to Terpenoid Synthesis in Pinus massoniana.

In the methyl-D-erythritol-4-phosphate (MEP) pathway, 1-deoxy-D-xylose-5-phosphate synthase (DXS) is considered the key enzyme for the biosynthesis of terpenoids. In this study, PmDXS (MK970590) was isolated from Pinus massoniana. Bioinformatics analysis revealed homology of MK970590 with DXS proteins from other species. Relative expression analysis suggested that PmDXS expression was higher in roots than in other plant parts, and the treatment of P. massoniana seedlings with mechanical injury via 15% polyethylene glycol 6000, 10 mM H2O2, 50 µM ethephon (ETH), 10 mM methyl jasmonate (MeJA), and 1 mM salicylic acid (SA) resulted in an increased expression of PmDXS. pET28a-PmDXS was expressed in Escherichia coli TransB (DE3) cells, and stress analysis showed that the recombinant protein was involved in resistance to NaCl and drought stresses. The subcellular localization of PmDXS was in the chloroplast. We also cloned a full-length 1024 bp PmDXS promoter. GUS expression was observed in Nicotiana benthamiana roots, stems, and leaves. PmDXS overexpression significantly increased carotenoid, chlorophyll a, and chlorophyll b contents and DXS enzyme activity, suggesting that DXS is important in isoprenoid biosynthesis. This study provides a theoretical basis for molecular breeding for terpene synthesis regulation and resistance.

X-ray crystallography-based structural elucidation of enzyme-bound intermediates along the 1-deoxy-d-xylulose 5-phosphate synthase reaction coordinate.

1-Deoxy-d-xylulose 5-phosphate synthase (DXPS) uses thiamine diphosphate (ThDP) to convert pyruvate and d-glyceraldehyde 3-phosphate (d-GAP) into 1-deoxy-d-xylulose 5-phosphate (DXP), an essential bacterial metabolite. DXP is not utilized by humans; hence, DXPS has been an attractive antibacterial target. Here, we investigate DXPS from Deinococcus radiodurans (DrDXPS), showing that it has similar kinetic parameters Kmd-GAP and Kmpyruvate (54 ± 3 and 11 ± 1 µm, respectively) and comparable catalytic activity (kcat = 45 ± 2 min-1) with previously studied bacterial DXPS enzymes and employing it to obtain missing structural data on this enzyme family. In particular, we have determined crystallographic snapshots of DrDXPS in two states along the reaction coordinate: a structure of DrDXPS bound to C2α-phosphonolactylThDP (PLThDP), mimicking the native pre-decarboxylation intermediate C2α-lactylThDP (LThDP), and a native post-decarboxylation state with a bound enamine intermediate. The 1.94-Å-resolution structure of PLThDP-bound DrDXPS delineates how two active-site histidine residues stabilize the LThDP intermediate. Meanwhile, the 2.40-Å-resolution structure of an enamine intermediate-bound DrDXPS reveals how a previously unknown 17-Å conformational change removes one of the two histidine residues from the active site, likely triggering LThDP decarboxylation to form the enamine intermediate. These results provide insight into how the bi-substrate enzyme DXPS limits side reactions by arresting the reaction on the less reactive LThDP intermediate when its cosubstrate is absent. They also offer a molecular basis for previous low-resolution experimental observations that correlate decarboxylation of LThDP with protein conformational changes.

Conformational dynamics of 1-deoxy-d-xylulose 5-phosphate synthase on ligand binding revealed by H/D exchange MS.

The enzyme 1-deoxy-d-xylulose 5-phosphate synthase (DXPS) is a key enzyme in the methylerythritol 4-phosphate pathway and is a target for the development of antibiotics, herbicides, and antimalarial drugs. DXPS catalyzes the formation of 1-deoxy-d-xylulose 5-phosphate (DXP), a branch point metabolite in isoprenoid biosynthesis, and is also used in the biosynthesis of thiamin (vitamin B1) and pyridoxal (vitamin B6). Previously, we found that DXPS is unique among the superfamily of thiamin diphosphate (ThDP)-dependent enzymes in stabilizing the predecarboxylation intermediate, C2-alpha-lactyl-thiamin diphosphate (LThDP), which has subsequent decarboxylation that is triggered by d-glyceraldehyde 3-phosphate (GAP). Herein, we applied hydrogen-deuterium (H/D) exchange MS (HDX-MS) of full-length Escherichia coli DXPS to provide a snapshot of the conformational dynamics of this enzyme, leading to the following conclusions. (i) The high sequence coverage of DXPS allowed us to monitor structural changes throughout the entire enzyme, including two segments (spanning residues 183-238 and 292-317) not observed by X-ray crystallography. (ii) Three regions of DXPS (spanning residues 42-58, 183-199, and 278-298) near the active center displayed both EX1 (monomolecular) and EX2 (bimolecuar) H/D exchange (HDX) kinetic behavior in both ligand-free and ligand-bound states. All other peptides behaved according to the common EX2 kinetic mechanism. (iii) The observation of conformational changes on DXPS provides support for the role of conformational dynamics in the DXPS mechanism: The closed conformation of DXPS is critical for stabilization of LThDP, whereas addition of GAP converts DXPS to the open conformation that coincides with decarboxylation of LThDP and DXP release.

Active Site Histidines Link Conformational Dynamics with Catalysis on Anti-Infective Target 1-Deoxy-d-xylulose 5-Phosphate Synthase.

The product of 1-deoxy-d-xyluose 5-phosphate (DXP) synthase, DXP, feeds into the bacterial biosynthesis of isoprenoids, thiamin diphosphate (ThDP), and pyridoxal phosphate. DXP is essential for human pathogens but not utilized by humans; thus, DXP synthase is an attractive anti-infective target. The unique ThDP-dependent mechanism and structure of DXP synthase offer ideal opportunities for selective targeting. Upon reaction with pyruvate, DXP synthase uniquely stabilizes the predecarboxylation intermediate, C2α-lactylThDP (LThDP), in a closed conformation. Subsequent binding of d-glyceraldehyde 3-phosphate induces an open conformation that is proposed to destabilize LThDP, triggering decarboxylation. Evidence for the closed and open conformations has been revealed by hydrogen-deuterium exchange mass spectrometry and X-ray crystallography, which indicate that H49 and H299 are involved in conformational dynamics and movement of the fork and spoon motifs away from the active site is important for the closed-to-open transition. Interestingly, H49 and H299 are critical for DXP formation and interact with the predecarboxylation intermediate in the closed conformation. H299 is removed from the active site in the open conformation of the postdecarboxylation state. In this study, we show that substitution at H49 and H299 negatively impacts LThDP formation by shifting the conformational equilibrium of DXP synthase toward an open conformation. We also present a method for monitoring the dynamics of the spoon motif that uncovered a previously undetected role for H49 in coordinating the closed conformation. Overall, our results suggest that H49 and H299 are critical for the closed, predecarboxylation state providing the first direct link between catalysis and conformational dynamics.

Crystal structure of a xylulose 5-phosphate phosphoketolase. Insights into the substrate specificity for xylulose 5-phosphate.

Phosphoketolases (PK) are TPP-dependent enzymes which play essential roles in carbohydrate metabolism of numerous bacteria. Depending on the substrate specificity PKs can be subdivided into xylulose 5-phosphate (X5P) specific PKs (XPKs) and PKs which accept both X5P and fructose 6-phosphate (F6P) (XFPKs). Despite their key metabolic importance, so far only the crystal structures of two XFPKs have been reported. There are no reported structures for any XPKs and for any complexes between PK and substrate. One of the major unknowns concerning PKs mechanism of action is related to the structural determinants of PKs substrate specificity for X5P or F6P. We report here the crystal structure of XPK from Lactococcus lactis (XPK-Ll) at 2.1 Šresolution. Using small angle X-ray scattering (SAXS) we proved that XPK-Ll is a dimer in solution. Towards better understanding of PKs substrate specificity, we performed flexible docking of TPP-X5P and TPP-F6P on crystal structures of XPK-Ll, two XFPKs and transketolase (TK). Calculated structure-based binding energies consistently support XPK-Ll preference for X5P. Analysis of structural models thus obtained show that substrates adopt moderately different conformation in PKs active sites following distinct networks of polar interactions. Based on the here reported structure of XPK-Ll we propose the most probable amino acid residues involved in the catalytic steps of reaction mechanism. Altogether our results suggest that PKs substrate preference for X5P or F6P is the outcome of a fine balance between specific binding network and dissimilar catalytic residues depending on the enzyme (XPK or XFPK) - substrate (X5P or F6P) couples.

CDP-glycerol inhibits the synthesis of the functional O-mannosyl glycan of α-dystroglycan.

α-Dystroglycan (α-DG) is a highly glycosylated cell-surface laminin receptor. Defects in the O-mannosyl glycan of an α-DG with laminin-binding activity can cause α-dystroglycanopathy, a group of congenital muscular dystrophies. In the biosynthetic pathway of functional O-mannosyl glycan, fukutin (FKTN) and fukutin-related protein (FKRP), whose mutated genes underlie α-dystroglycanopathy, sequentially transfer ribitol phosphate (RboP) from CDP-Rbo to form a tandem RboP unit (RboP-RboP) required for the synthesis of the laminin-binding epitope on O-mannosyl glycan. Both RboP- and glycerol phosphate (GroP)-substituted glycoforms have recently been detected in recombinant α-DG. However, it is unclear how GroP is transferred to the O-mannosyl glycan or whether GroP substitution affects the synthesis of the O-mannosyl glycan. Here, we report that, in addition to having RboP transfer activity, FKTN and FKRP can transfer GroP to O-mannosyl glycans by using CDP-glycerol (CDP-Gro) as a donor substrate. Kinetic experiments indicated that CDP-Gro is a less efficient donor substrate for FKTN than is CDP-Rbo. We also show that the GroP-substituted glycoform synthesized by FKTN does not serve as an acceptor substrate for FKRP and that therefore further elongation of the outer glycan chain cannot occur with this glycoform. Finally, CDP-Gro inhibited the RboP transfer activities of both FKTN and FKRP. These results suggest that CDP-Gro inhibits the synthesis of the functional O-mannosyl glycan of α-DG by preventing further elongation of the glycan chain. This is the first report of GroP transferases in mammals.

AAV-mediated transfer of FKRP shows therapeutic efficacy in a murine model but requires control of gene expression.

Limb Girdle Muscular Dystrophies type 2I (LGMD2I), a recessive autosomal muscular dystrophy, is caused by mutations in the Fukutin Related Protein (FKRP) gene. It has been proposed that FKRP, a ribitol-5-phosphate transferase, is a participant in α-dystroglycan (αDG) glycosylation, which is important to ensure the cell/matrix anchor of muscle fibers. A LGMD2I knock-in mouse model was generated to express the most frequent mutation (L276I) encountered in patients. The expression of FKRP was not altered neither at transcriptional nor at translational levels, but its function was impacted since abnormal glycosylation of αDG was observed. Skeletal muscles were functionally impaired from 2 months of age and a moderate dystrophic pattern was evident starting from 6 months of age. Gene transfer with a rAAV2/9 vector expressing Fkrp restored biochemical defects, corrected the histological abnormalities and improved the resistance to eccentric stress in the mouse model. However, injection of high doses of the vector induced a decrease of αDG glycosylation and laminin binding, even in WT animals. Finally, intravenous injection of the rAAV-Fkrp vector into a dystroglycanopathy mouse model due to Fukutin (Fktn) knock-out indicated a dose-dependent toxicity. These data suggest requirement for a control of FKRP expression in muscles.

A computational study of the molecular basis of antibiotic resistance in a DXR mutant.

Proteins of the independent mevalonate pathway for isoprenoid biosynthesis are important targets for the development of new antibacterial compounds as this pathway is present in most pathogenic organisms such as Mycobacterium tuberculosis, DPlasmodium falciparum and Escherichia coli, but is not present in mammalian species, including humans. Deoxy-D-xylulose 5-phosphate reductoisomerase (DXR) is an important target in this pathway and the most effective DXR inhibitor to date is fosmidomycin, which is used to treat malaria and, more recently, tuberculosis. Recently, Armstrong C. M. et al. showed that a mutant of DXR, S222T, induces a loss of the fosmidomycin inhibition efficiency, even though the bacteria culture is still viable and able to produce isoprenoids. As this represents a potential fosmidomycin-resistant mutation, it is important to understand the mechanism of this apparent mutation-induced resistance to fosmidomycin. Here, we used molecular dynamics simulations and Molecular Mechanics/Poisson Boltzmann Surface Area analysis to understand the structural and energetic basis of the resistance. Our results suggest that the point mutation results in changes to the structural dynamics of an active site loop that probably protects the active site and facilitates enzymatic reaction. From the simulation analysis, we also showed that the mutation results in changes in the interaction energy profiles in a way that can explain the observed activity of the mutant protein toward the natural inhibitor deoxy-D-xylulose 5-phosphate. These results should be taken into consideration in future efforts to develop new therapeutic antibiotic compounds that target DXR.

The catalytic mechanism for aerobic formation of methane by bacteria.

Methane is a potent greenhouse gas that is produced in significant quantities by aerobic marine organisms. These bacteria apparently catalyse the formation of methane through the cleavage of the highly unreactive carbon-phosphorus bond in methyl phosphonate (MPn), but the biological or terrestrial source of this compound is unclear. However, the ocean-dwelling bacterium Nitrosopumilus maritimus catalyses the biosynthesis of MPn from 2-hydroxyethyl phosphonate and the bacterial C-P lyase complex is known to convert MPn to methane. In addition to MPn, the bacterial C-P lyase complex catalyses C-P bond cleavage of many alkyl phosphonates when the environmental concentration of phosphate is low. PhnJ from the C-P lyase complex catalyses an unprecedented C-P bond cleavage reaction of ribose-1-phosphonate-5-phosphate to methane and ribose-1,2-cyclic-phosphate-5-phosphate. This reaction requires a redox-active [4Fe-4S]-cluster and S-adenosyl-L-methionine, which is reductively cleaved to L-methionine and 5'-deoxyadenosine. Here we show that PhnJ is a novel radical S-adenosyl-L-methionine enzyme that catalyses C-P bond cleavage through the initial formation of a 5'-deoxyadenosyl radical and two protein-based radicals localized at Gly 32 and Cys 272. During this transformation, the pro-R hydrogen from Gly 32 is transferred to the 5'-deoxyadenosyl radical to form 5'-deoxyadenosine and the pro-S hydrogen is transferred to the radical intermediate that ultimately generates methane. A comprehensive reaction mechanism is proposed for cleavage of the C-P bond by the C-P lyase complex that uses a covalent thiophosphate intermediate for methane and phosphate formation.

Targeting the Unique Mechanism of Bacterial 1-Deoxy-d-xylulose-5-phosphate Synthase.

The bacterial metabolite 1-deoxy-d-xyulose 5-phosphate (DXP) is essential in bacterial central metabolism feeding into isoprenoid, thiamin diphosphate (ThDP), and pyridoxal phosphate de novo biosynthesis. Halting its production through the inhibition of DXP synthase is an attractive strategy for the development of novel antibiotics. Recent work has revealed that DXP synthase utilizes a unique random sequential mechanism that requires formation of a ternary complex among pyruvate-derived C2α-lactylthiamin diphosphate (LThDP), d-glyceraldehyde 3-phosphate (d-GAP), and enzyme, setting it apart from all other known ThDP-dependent enzymes. Herein, we describe the development of bisubstrate inhibitors bearing an acetylphosphonate (AP) pyruvate mimic and a distal negative charge mimicking the phosphoryl group of d-GAP, designed to target the unique form of DXP synthase that binds LThDP and d-GAP in a ternary complex. A d-phenylalanine-derived triazole acetylphosphonate (d-PheTrAP) emerged as the most potent inhibitor in this series, displaying slow, tight-binding inhibition with a Ki* of 90 ± 10 nM, forward ( k1) and reverse ( k2) isomerization rates of 1.1 and 0.14 min-1, respectively, and exquisite selectivity (>15000-fold) for DXP synthase over mammalian pyruvate dehydrogenase. d-PheTrAP is the most potent, selective DXP synthase inhibitor described to date and represents the first inhibitor class designed specifically to exploit the unique E-LThDP-GAP ternary complex in ThDP enzymology.

A two-step evolutionary process establishes a non-native vitamin B6 pathway in Bacillus subtilis.

Pyridoxal 5'-phosphate (PLP), the most important form of vitamin B6 serves as a cofactor for many proteins. Two alternative pathways for de novo PLP biosynthesis are known: the short deoxy-xylulose-5-phosphate (DXP)-independent pathway, which is present in the Gram-positive model bacterium Bacillus subtilis and the longer DXP-dependent pathway, which has been intensively studied in the Gram-negative model bacterium Escherichia coli. Previous studies revealed that bacteria contain many promiscuous enzymes causing a so-called 'underground metabolism', which can be important for the evolution of novel pathways. Here, we evaluated the potential of B. subtilis to use a truncated non-native DXP-dependent PLP pathway from E. coli for PLP synthesis. Adaptive laboratory evolution experiments revealed that two non-native enzymes catalysing the last steps of the DXP-dependent PLP pathway and two genomic alterations are sufficient to allow growth of vitamin B6 auxotrophic bacteria as rapid as the wild type. Thus, the existence of an underground metabolism in B. subtilis facilitates the generation of a pathway for synthesis of PLP using parts of a non-native vitamin B6 pathway. The introduction of non-native enzymes into a metabolic network and rewiring of native metabolism could be helpful to generate pathways that might be optimized for producing valuable substances.

Identification of a d-Arabinose-5-Phosphate Isomerase in the Gram-Positive Clostridium tetani.

d-Arabinose-5-phosphate (A5P) isomerases (APIs) catalyze the interconversion of d-ribulose-5-phosphate and d-arabinose-5-phosphate. Various Gram-negative bacteria, such as the uropathogenic Escherichia coli strain CFT073, contain multiple API paralogs (KdsD, GutQ, KpsF, and c3406) that have been assigned various cellular functions. The d-arabinose-5-phosphate formed by these enzymes seems to play important roles in the biosynthesis of lipopolysaccharide (LPS) and group 2 K-antigen capsules, as well as in the regulation of the cellular d-glucitol uptake and uropathogenic infectivity/virulence. The genome of a Gram-positive pathogenic bacterium, Clostridium tetani, contains a gene encoding a putative API, C. tetani API (CtAPI), even though C. tetani lacks both LPS and capsid biosynthetic genes. To better understand the physiological role of d-arabinose-5-phosphate in this Gram-positive organism, recombinant CtAPI was purified and characterized. CtAPI displays biochemical characteristics similar to those of APIs from Gram-negative organisms and complements the API deficiency of an E. coli API knockout strain. Thus, CtAPI represents the first d-arabinose-5-phosphate isomerase to be identified and characterized from a Gram-positive bacterium.IMPORTANCE The genome of Clostridium tetani, a pathogenic Gram-positive bacterium and the causative agent of tetanus, contains a gene (the CtAPI gene) that shares high sequence similarity with those of genes encoding d-arabinose-5-phosphate isomerases. APIs play an important role within Gram-negative bacteria in d-arabinose-5-phosphate production for lipopolysaccharide biosynthesis, capsule formation, and regulation of cellular d-glucitol uptake. The significance of our research is in identifying and characterizing CtAPI, the first Gram-positive API. Our findings show that CtAPI is specific to the interconversion of arabinose-5-phosphate and ribulose-5-phosphate while having no activity with the other sugars and sugar phosphates tested. We have speculated a regulatory role for this API in C. tetani, an organism that does not produce lipopolysaccharide.

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.

A pentose bisphosphate pathway for nucleoside degradation in Archaea.

Owing to the absence of the pentose phosphate pathway, the degradation pathway for the ribose moieties of nucleosides is unknown in Archaea. Here, in the archaeon Thermococcus kodakarensis, we identified a metabolic network that links the pentose moieties of nucleosides or nucleotides to central carbon metabolism. The network consists of three nucleoside phosphorylases, an ADP-dependent ribose-1-phosphate kinase and two enzymes of a previously identified NMP degradation pathway, ribose-1,5-bisphosphate isomerase and type III ribulose-1,5-bisphosphate carboxylase/oxygenase. Ribose 1,5-bisphosphate and ribulose 1,5-bisphosphate are intermediates of this pathway, which is thus designated the pentose bisphosphate pathway.

Glycosylation with ribitol-phosphate in mammals: New insights into the O-mannosyl glycan.

BACKGROUND: O-mannosyl glycans have been found in a limited number of glycoproteins of the brain, nerves, and skeletal muscles, particularly in α-dystroglycan (α-DG). Defects in O-mannosyl glycan on α-DG are the primary cause of a group of congenital muscular dystrophies, which are collectively termed α-dystroglycanopathy. Recent studies have revealed various O-mannosyl glycan structures, which can be classified as core M1, core M2, and core M3 glycans. Although many dystroglycanopathy genes are involved in core M3 processing, the structure and biosynthesis of core M3 glycan remains only partially understood. SCOPE OF REVIEW: This review presents recent findings about the structure, biosynthesis, and pathology of O-mannosyl glycans. MAJOR CONCLUSIONS: Recent studies have revealed that the entire structure of core M3 glycan, including ribitol-5-phosphate, is a novel structure in mammals; its unique biosynthetic pathway has been elucidated by the identification of new causative genes for α-dystroglycanopathies and their functions. GENERAL SIGNIFICANCE: O-mannosyl glycan has a novel, unique structure that is important for the maintenance of brain and muscle functions. These findings have opened up a new field in glycoscience. These studies will further contribute to the understanding of the pathomechanism of α-dystroglycanopathy and the development of glycotherapeutics. This article is part of a Special Issue entitled Neuro-glycoscience, edited by Kenji Kadomatsu and Hiroshi Kitagawa.

Engineering a functional 1-deoxy-D-xylulose 5-phosphate (DXP) pathway in Saccharomyces cerevisiae.

Isoprenoids are used in many commercial applications and much work has gone into engineering microbial hosts for their production. Isoprenoids are produced either from acetyl-CoA via the mevalonate pathway or from pyruvate and glyceraldehyde 3-phosphate via the 1-deoxy-D-xylulose 5-phosphate (DXP) pathway. Saccharomyces cerevisiae exclusively utilizes the mevalonate pathway to synthesize native isoprenoids and in fact the alternative DXP pathway has never been found or successfully reconstructed in the eukaryotic cytosol. There are, however, several advantages to isoprenoid synthesis via the DXP pathway, such as a higher theoretical yield, and it has long been a goal to transplant the pathway into yeast. In this work, we investigate and address barriers to DXP pathway functionality in S. cerevisiae using a combination of synthetic biology, biochemistry and metabolomics. We report, for the first time, functional expression of the DXP pathway in S. cerevisiae. Under low aeration conditions, an engineered strain relying solely on the DXP pathway for isoprenoid biosynthesis achieved an endpoint biomass 80% of that of the same strain using the mevalonate pathway.