High-yield synthesis of 2-O-α-D-glucosyl-D-glycerate by a bifunctional glycoside phosphorylase.

Osmolytes are produced by various microorganisms as a defense mechanism to protect cells and macromolecules from damage caused by external stresses in harsh environments. Due to their useful stabilizing properties, these molecules are applied as active ingredients in a wide range of cosmetics and healthcare products. The metabolic pathways and biocatalytic syntheses of glycosidic osmolytes such as 2-O-α-D-glucosyl-D-glycerate often involve the action of a glycoside phosphorylase. Here, we report the discovery of a glucosylglycerate phosphorylase from carbohydrate-active enzyme family GH13 that is also active on sucrose, which contrasts the strict specificity of known glucosylglycerate phosphorylases that can only use α-D-glucose 1-phosphate as glycosyl donor in transglycosylation reactions. The novel enzyme can be distinguished from other phosphorylases from the same family by the presence of an atypical conserved sequence motif at specificity-determining positions in the active site. The promiscuity of the sucrose-active glucosylglycerate phosphorylase can be exploited for the high-yielding and rapid synthesis of 2-O-α-D-glucosyl-D-glycerate from sucrose and D-glycerate. KEY POINTS: • A Xylanimonas protaetiae glycoside phosphorylase can use both d-glycerate and fructose as glucosyl acceptor with high catalytic efficiency • Biocatalytic synthesis of the osmolyte 2-O-α-d-glucosyl-d-glycerate • Positions in the active site of GH13 phosphorylases act as convenient specificity fingerprints.

A large-scale comparative genomics study reveals niche-driven and within-sample intra-species functional diversification in Lacticaseibacillus rhamnosus.

Lacticaseibacillus rhamnosus (L. rhamnosus) is widely recognized as a probiotic species, and it exists in a variety of environments including host gut and dairy products. This work aimed at conducting a large-scale comparative genomics analysis of 384 L. rhamnosus genomes (257 whole-sequence or metagenomic-assembled genomes from gut-associated isolates [122 and 135 retrieved from the UHGG and NCBI databases, respectively] and 127 genomes from dairy isolates [34 from the NCBI database; 93 isolated from a cheese sample and sequenced here]). Our results showed that L. rhamnosus had a large and open pan-genome (15,253 pan-genes identified from all 384 genomes; 15,028 pan-genes if the 93 cheese-originated isolates were excluded). The core-gene phylogenetic tree constructed from the 384 L. rhamnosus genomes comprised five phylogenetic branches, with a random distribution of dairy and gut-associated isolates/genomes across the tree. No significant difference was identified in the overall profile of metabolism-related genes between dairy and gut-associated genomes; however, notably, the gut-associated strains/isolates contained more genes coding for specific metabolic pathways and carbohydrate-active enzymes, e.g., lacto-N-biosidase (EC 3.2.1.140; GT20) and lacto-N-biose phosphorylase/galacto-N-biose phosphorylase (EC 2.4.1.211; GH112). Further, we found that there was obvious intra-species diversification of the 93 cheese-originated L. rhamnosus isolates, forming three clades (Clades A, B, and C) in the reconstructed core-gene phylogenetic tree. There were numerous single nucleotide variations (over 10,000) across the three clades. Moreover, significant differences were observed in the content of metabolism-related genes across clades (p < 0.05, Adonis test), characterized by the enrichment in glycoside hydrolases in Clade C and the possession of unique metabolic pathways in each clade. These results implicated genomics/functional diversification of L. rhamnosus in a single food matrix and niche-driven adaptive evolution of isolates from dairy and host gut-associated origins. Our study shed insights into the selection of candidate strains for food industry applications.

Evaluation of MTAP and p16 immunohistochemical deficiency as surrogate marker for CDKN2A/B homozygous deletion in gliomas.

Homozygous deletion (HD) of the CDKN2A/B locus has emerged as an unfavourable prognostic marker in diffuse gliomas, both IDH-mutant and IDH-wild-type. Testing for CDKN2A/B deletions can be performed by a variety of approaches, including copy number variation (CNV) analysis based on gene array analysis, next generation sequencing (NGS) or fluorescence in situ hybridisation (FISH), but questions remain regarding the accuracy of testing modalities. In this study, we assessed: (1) the utility of S-methyl-5'-thioadenosine phosphorylase (MTAP) and cellular tumour suppressor protein pl61NK4a (p16) immunostainings as surrogate markers for CDKN2A/B HD in gliomas, and (2) the prognostic value of MTAP, across different histological tumour grades and IDH mutation status. One hundred consecutive cases of diffuse and circumscribed gliomas (Cohort 1) were collected, in order to correlate MTAP and p16 expression with the CDKN2A/B status in the CNV plot of each tumour. IDH1 R132H, ATRX and MTAP immunohistochemistry was performed on next generation tissue microarrays (ngTMAs) of 251 diffuse gliomas (Cohort 2) for implementing survival analysis. Complete loss of MTAP and p16 by immunohistochemistry was 100% and 90% sensitive as well as 97% and 89% specific for CDKN2A/B HD, respectively, as identified on CNV plot. Only two cases (2/100) with MTAP and p16 loss of expression did not demonstrate CDKN2A/B HD in CNV plot; however, FISH analysis confirmed the HD for CDKN2A/B. Moreover, MTAP deficiency was associated with shortened survival in IDH-mutant astrocytomas (n=75; median survival 61 vs 137 months; p<0.0001), IDH-mutant oligodendrogliomas (n=59; median survival 41 vs 147 months; p<0.0001) and IDH-wild-type gliomas (n=117; median survival 13 vs 16 months; p=0.011). In conclusion, MTAP immunostaining is an important complement for diagnostic work-up of gliomas, because of its excellent correlation with CDKN2A/B status, robustness, rapid turnaround time and low costs, and provides significant prognostic value in IDH-mutant astrocytomas and oligodendrogliomas, while p16 should be used cautiously.

Blue light promotes ascorbate synthesis by deactivating the PAS/LOV photoreceptor that inhibits GDP-L-galactose phosphorylase.

Ascorbate (vitamin C) is an essential antioxidant in fresh fruits and vegetables. To gain insight into the regulation of ascorbate metabolism in plants, we studied mutant tomato plants (Solanum lycopersicum) that produce ascorbate-enriched fruits. The causal mutation, identified by a mapping-by-sequencing strategy, corresponded to a knock-out recessive mutation in a class of photoreceptor named PAS/LOV protein (PLP), which acts as a negative regulator of ascorbate biosynthesis. This trait was confirmed by CRISPR/Cas9 gene editing and further found in all plant organs, including fruit that accumulated 2 to 3 times more ascorbate than in the WT. The functional characterization revealed that PLP interacted with the 2 isoforms of GDP-L-galactose phosphorylase (GGP), known as the controlling step of the L-galactose pathway of ascorbate synthesis. The interaction with GGP occurred in the cytoplasm and the nucleus, but was abolished when PLP was truncated. These results were confirmed by a synthetic approach using an animal cell system, which additionally demonstrated that blue light modulated the PLP-GGP interaction. Assays performed in vitro with heterologously expressed GGP and PLP showed that PLP is a noncompetitive inhibitor of GGP that is inactivated after blue light exposure. This discovery provides a greater understanding of the light-dependent regulation of ascorbate metabolism in plants.

Pho1 cooperates with DPE1 to control short maltooligosaccharide mobilization during starch synthesis initiation in rice endosperm.

KEY MESSAGE: Plastidial α-glucan phosphorylase is a key factor that cooperates with plastidial disproportionating enzyme to control short maltooligosaccharide mobilization during the initiation process of starch molecule synthesis in developing rice endosperm. Storage starch synthesis is essential for grain filling. However, little is known about how cereal endosperm controls starch synthesis initiation. One of core events for starch synthesis initiation is short maltooligosaccharide (MOS) mobilization consisting of long MOS primer production and excess MOS breakdown. By mutant analyses and biochemical investigations, we present here functional identifications of plastidial α-glucan phosphorylase (Pho1) and disproportionating enzyme (DPE1) during starch synthesis initiation in rice (Oryza sativa) endosperm. Pho1 deficiency impaired MOS mobilization, triggering short MOS accumulation and starch synthesis reduction during early seed development. The mutant seeds differed significantly in MOS level and starch content at 15 days after flowering and exhibited diverse endosperm phenotypes during mid-late seed development: ranging from pseudonormal to shrunken (Shr), severely or excessively Shr. The level of DPE1 was almost normal in the PN seeds but significantly reduced in the Shr seeds. Overexpression of DPE1 in pho1 resulted in plump seeds only. DPE1 deficiency had no obvious effects on MOS mobilization. Knockout of DPE1 in pho1 completely blocked MOS mobilization, resulting in severely and excessively Shr seeds only. These findings show that Pho1 cooperates with DPE1 to control short MOS mobilization during starch synthesis initiation in rice endosperm.

Overexpression of banana GDP-L-galactose phosphorylase (GGP) modulates the biosynthesis of ascorbic acid in Arabidopsis thaliana.

l-Ascorbic acid (AsA) is a potent antioxidant and essential micronutrient for the growth and development of plants and animals. AsA is predominantly synthesized by the Smirnoff-Wheeler (SW) pathway in plants where the GDP-L-galactose phosphorylase (GGP) gene encodes the rate-limiting step. In the present study, AsA was estimated in twelve banana cultivars, where Nendran carried the highest (17.2 mg/100 g) amount of AsA in ripe fruit pulp. Five GGP genes were identified from the banana genome database, and they were located at chromosome 6 (4 MaGGPs) and chromosome 10 (1 MaGGP). Based on in-silico analysis, three potential MaGGP genes were isolated from the cultivar Nendran and subsequently overexpressed in Arabidopsis thaliana. Significant enhancement in AsA (1.52 to 2.20 fold) level was noted in the leaves of all three MaGGPs overexpressing lines as compared to non-transformed control plants. Among all, MaGGP2 emerged as a potential candidate for AsA biofortification in plants. Further, the complementation assay of Arabidopsis thaliana vtc-5-1 and vtc-5-2 mutants with MaGGP genes overcome the AsA deficiency that showed improved plant growth as compared to non-transformed control plants. This study lends strong affirmation towards development of AsA biofortified plants, particularly the staples that sustain the personages in developing countries.

Phosphorylase phosphatase and flash activation of skeletal muscle glycogen phosphorylase-A tribute to Edmond H. Fischer.

Glycogen is a polymerized form of glucose that serves as an energy reserve in all types of organisms. In animals glycogen synthesis and degradation, especially in liver and skeletal muscle, are regulated by hormonal and physiological signals that reciprocally control the opposing activities of glycogen synthase and glycogen phosphorylase. These enzymes are under allosteric control by binding of metabolites (e.g., ATP, AMP, G6P) and covalent control by reversible phosphorylation by kinase and phosphatase all assembled together on glycogen. More than 50 years ago Edmond Fischer and colleagues showed "flash activation" of phosphorylase in glycogen particles. This involved transient and extensive inhibition of protein phosphatase but even today the phenomenon is not understood. Phosphatase regulation is known to rely on regulatory subunits including glycogen binding subunits that serve as scaffolds, binding catalytic subunit, glycogen, and substrates. This tribute article to Edmond Fischer highlights his thoughts and ideas about the transient inhibition of phosphorylase phosphatase during flash activation of phosphorylase and speculates that phosphatase regulation in glycogen particles might involve a/b hybrids of phosphorylase.

Functional characterization of a novel GH94 glycoside phosphorylase, 3-O-ß-d-glucopyranosyl ß-d-glucuronide phosphorylase, and implication of the metabolic pathway of acidic carbohydrates in Paenibacillus borealis.

Glycoside hydrolase family 94 (GH94) contains enzymes that reversibly catalyze the phosphorolysis of ß-glycosides. We conducted this study to investigate a GH94 protein (PBOR_13355) encoded in the genome of Paenibacillus borealis DSM 13188 with low sequence identity to known phosphorylases. Screening of acceptor substrates for reverse phosphorolysis in the presence of α-d-glucose 1-phosphate as a donor substrate showed that PBOR_13355 utilized d-glucuronic acid and p-nitrophenyl ß-d-glucuronide as acceptors. In the reaction with d-glucuronic acid, 3-O-ß-d-glucopyranosyl-d-glucuronic acid was synthesized. PBOR_13355 showed a higher apparent catalytic efficiency to p-nitrophenyl ß-d-glucuronide than to d-glucuronic acid, and thus, PBOR_13355 was concluded to be a novel glycoside phosphorylase, 3-O-ß-d-glucopyranosyl ß-d-glucuronide phosphorylase. PBOR_13360, encoded by the gene immediately downstream of the PBOR_13355 gene, was shown to be ß-glucuronidase. Collectively, PBOR_13355 and PBOR_13360 are predicted to work together in the cytosol to metabolize oligosaccharides containing the 3-O-ß-d-glucopyranosyl ß-d-glucuronide structure released from bacterial and plant acidic carbohydrates.

Kiwifruit MYBS1-like and GBF3 transcription factors influence l-ascorbic acid biosynthesis by activating transcription of GDP-L-galactose phosphorylase 3.

Plant-derived Vitamin C (l-ascorbic acid (AsA)) is crucial for human health and wellbeing and thus increasing AsA content is of interest to plant breeders. In plants GDP-l-galactose phosphorylase (GGP) is a key biosynthetic control step and here evidence is presented for two new transcriptional activators of GGP. AsA measurement, transcriptomics, transient expression, hormone application, gene editing, yeast 1/2-hybrid, and electromobility shift assay (EMSA) methods were used to identify two positively regulating transcription factors. AceGGP3 was identified as the most highly expressed GGP in Actinidia eriantha fruit, which has high fruit AsA. A gene encoding a 1R-subtype myeloblastosis (MYB) protein, AceMYBS1, was found to bind the AceGGP3 promoter and activate its expression. Overexpression and gene-editing show AceMYBS1 effectively increases AsA accumulation. The bZIP transcription factor AceGBF3 (a G-box binding factor), also was shown to increase AsA content, and was confirmed to interact with AceMYBS1. Co-expression experiments showed that AceMYBS1 and AceGBF3 additively promoted AceGGP3 expression. Furthermore, AceMYBS1, but not GBF3, was repressed by abscisic acid, resulting in reduced AceGGP3 expression and accumulation of AsA. This study sheds new light on the roles of MYBS1 homologues and ABA in modulating AsA synthesis, and adds to the understanding of mechanisms underlying AsA accumulation.

Inorganic phosphate self-sufficient whole-cell biocatalysts containing two co-expressed phosphorylases facilitate cellobiose production.

Cellobiose, a natural disaccharide, attracts extensive attention as a potential functional food/feed additive. In this study, we present an inorganic phosphate (Pi) self-sufficient biotransformation system to produce cellobiose by co-expressing sucrose phosphorylase (SP) and cellobiose phosphorylase (CBP). The Bifidobacterium adolescentis SP (BASP) and Cellvibrio gilvus CBP (CGCBP) were co-expressed in Escherichia coli. Escherichia coli cells containing BASP and CGCBP were used as whole-cell catalysts to convert sucrose and glucose to cellobiose. The effects of reaction pH, temperature, Pi concentration, and substrate concentration were investigated. In the optimum biotransformation conditions, 800 mM cellobiose was produced from 1.0 M sucrose, 1.0 M glucose, and 50 mM Pi, within 12 hr. The by-product fructose and residual substrate (sucrose and glucose) were efficiently removed by treatment with yeast, to help purify the product cellobiose. The wider applicability of this Pi self-sufficiency strategy was demonstrated in the production of laminaribiose by co-expressing SP and laminaribiose phosphorylase. This study suggests that the Pi self-sufficiency strategy through co-expressing two phosphorylases has the advantage of great flexibility for enhanced production of cellobiose (or laminaribiose).

Discovery of solabiose phosphorylase and its application for enzymatic synthesis of solabiose from sucrose and lactose.

Glycoside phosphorylases (GPs), which catalyze the reversible phosphorolysis of glycosides, are promising enzymes for the efficient production of glycosides. Various GPs with new catalytic activities are discovered from uncharacterized proteins phylogenetically distant from known enzymes in the past decade. In this study, we characterized Paenibacillus borealis PBOR_28850 protein, belonging to glycoside hydrolase family 94. Screening of acceptor substrates for reverse phosphorolysis, in which α-D-glucose 1-phosphate was used as the donor substrate, revealed that the recombinant PBOR_28850 produced in Escherichia coli specifically utilized D-galactose as an acceptor and produced solabiose (ß-D-Glcp-(1 → 3)-D-Gal). This indicates that PBOR_28850 is a new GP, solabiose phosphorylase. PBOR_28850 catalyzed the phosphorolysis and synthesis of solabiose through a sequential bi-bi mechanism involving the formation of a ternary complex. The production of solabiose from lactose and sucrose has been established. Lactose was hydrolyzed to D-galactose and D-glucose by ß-galactosidase. Phosphorolysis of sucrose and synthesis of solabiose were then coupled by adding sucrose, sucrose phosphorylase, and PBOR_28850 to the reaction mixture. Using 210 mmol lactose and 280 mmol sucrose, 207 mmol of solabiose was produced. Yeast treatment degraded the remaining monosaccharides and sucrose without reducing solabiose. Solabiose with a purity of 93.7% was obtained without any chromatographic procedures.

Fusion of cellobiose phosphorylase and potato alpha-glucan phosphorylase facilitates substrate channeling for enzymatic conversion of cellobiose to starch.

We previously reported an in vitro enzymatic pathway for conversion of nonfood cellulose to starch (PNAS,110 (18): 7182-7187, 2013), in which the two sequential enzymes cellobiose phosphorylase (CBP) from Clostridium thermocellum and potato alpha-glucan phosphorylase (PGP) from Solanum tuberosum were the two key enzymes responsible for the whole conversion rate. In this work CBP and PGP were fused to form a large enzyme and it turned out that the fusion protein could exhibit a good bifunctionality when PGP moiety was put at the N-terminus and CBP moiety at the C-terminus (designated as PGP-CBP). Although the coupled reaction rate of PGP-CBP was decreased by 23.0% compared with the free enzymes, substrate channeling between the two active sites in PGP-CBP was formed, demonstrated by the introduction of the competing enzyme of PGP to the reaction system. The potential of PGP-CBP fusion enzyme being applied to the conversion of cellulose to amylose was discussed.

Ethylene response factor AcERF91 affects ascorbate metabolism via regulation of GDP-galactose phosphorylase encoding gene (AcGGP3) in kiwifruit.

Kiwifruit is known as 'the king of vitamin C' because of the high content of ascorbic acid (AsA) in the fruit. Deciphering the regulatory network and identification of the key regulators mediating AsA biosynthesis is vital for fruit nutrition and quality improvement. To date, however, the key transcription factors regulating AsA metabolism during kiwifruit developmental and ripening processes remains largely unknown. Here, we generated a putative transcriptional regulatory network mediating ascorbate metabolism by transcriptome co-expression analysis. Further studies identified an ethylene response factor AcERF91 from this regulatory network, which is highly co-expressed with a GDP-galactose phosphorylase encoding gene (AcGGP3) during fruit developmental and ripening processes. Through dual-luciferase reporter and yeast one-hybrid assays, it was shown that AcERF91 is able to bind and directly activate the activity of the AcGGP3 promoter. Furthermore, transient expression of AcERF91 in kiwifruit fruits resulted in a significant increase in AsA content and AcGGP3 transcript level, indicating a positive role of AcERF91 in controlling AsA accumulation via regulation of the expression of AcGGP3. Overall, our results provide a new insight into the regulation of AsA metabolism in kiwifruit.

The role of GDP-l-galactose phosphorylase in the control of ascorbate biosynthesis.

The enzymes involved in l-ascorbate biosynthesis in photosynthetic organisms (the Smirnoff-Wheeler [SW] pathway) are well established. Here, we analyzed their subcellular localizations and potential physical interactions and assessed their role in the control of ascorbate synthesis. Transient expression of C terminal-tagged fusions of SW genes in Nicotiana benthamiana and Arabidopsis thaliana mutants complemented with genomic constructs showed that while GDP-d-mannose epimerase is cytosolic, all the enzymes from GDP-d-mannose pyrophosphorylase (GMP) to l-galactose dehydrogenase (l-GalDH) show a dual cytosolic/nuclear localization. All transgenic lines expressing functional SW protein green fluorescent protein fusions driven by their endogenous promoters showed a high accumulation of the fusion proteins, with the exception of those lines expressing GDP-l-galactose phosphorylase (GGP) protein, which had very low abundance. Transient expression of individual or combinations of SW pathway enzymes in N. benthamiana only increased ascorbate concentration if GGP was included. Although we did not detect direct interaction between the different enzymes of the pathway using yeast-two hybrid analysis, consecutive SW enzymes, as well as the first and last enzymes (GMP and l-GalDH) associated in coimmunoprecipitation studies. This association was supported by gel filtration chromatography, showing the presence of SW proteins in high-molecular weight fractions. Finally, metabolic control analysis incorporating known kinetic characteristics showed that previously reported feedback repression at the GGP step, combined with its relatively low abundance, confers a high-flux control coefficient and rationalizes why manipulation of other enzymes has little effect on ascorbate concentration.

Analysis of the diversity of the glycoside hydrolase family 130 in mammal gut microbiomes reveals a novel mannoside-phosphorylase function.

Mannoside phosphorylases are involved in the intracellular metabolization of mannooligosaccharides, and are also useful enzymes for the in vitro synthesis of oligosaccharides. They are found in glycoside hydrolase family GH130. Here we report on an analysis of 6308 GH130 sequences, including 4714 from the human, bovine, porcine and murine microbiomes. Using sequence similarity networks, we divided the diversity of sequences into 15 mostly isofunctional meta-nodes; of these, 9 contained no experimentally characterized member. By examining the multiple sequence alignments in each meta-node, we predicted the determinants of the phosphorolytic mechanism and linkage specificity. We thus hypothesized that eight uncharacterized meta-nodes would be phosphorylases. These sequences are characterized by the absence of signal peptides and of the catalytic base. Those sequences with the conserved E/K, E/R and Y/R pairs of residues involved in substrate binding would target ß-1,2-, ß-1,3- and ß-1,4-linked mannosyl residues, respectively. These predictions were tested by characterizing members of three of the uncharacterized meta-nodes from gut bacteria. We discovered the first known ß-1,4-mannosyl-glucuronic acid phosphorylase, which targets a motif of the Shigella lipopolysaccharide O-antigen. This work uncovers a reliable strategy for the discovery of novel mannoside-phosphorylases, reveals possible interactions between gut bacteria, and identifies a biotechnological tool for the synthesis of antigenic oligosaccharides.

A bifunctional salvage pathway for two distinct S-adenosylmethionine by-products that is widespread in bacteria, including pathogenic Escherichia coli.

S-adenosyl-l-methionine (SAM) is a necessary cosubstrate for numerous essential enzymatic reactions including protein and nucleotide methylations, secondary metabolite synthesis and radical-mediated processes. Radical SAM enzymes produce 5'-deoxyadenosine, and SAM-dependent enzymes for polyamine, neurotransmitter and quorum sensing compound synthesis produce 5'-methylthioadenosine as by-products. Both are inhibitory and must be addressed by all cells. This work establishes a bifunctional oxygen-independent salvage pathway for 5'-deoxyadenosine and 5'-methylthioadenosine in both Rhodospirillum rubrum and Extraintestinal Pathogenic Escherichia coli. Homologous genes for this pathway are widespread in bacteria, notably pathogenic strains within several families. A phosphorylase (Rhodospirillum rubrum) or separate nucleoside and kinase (Escherichia coli) followed by an isomerase and aldolase sequentially function to salvage these two wasteful and inhibitory compounds into adenine, dihydroxyacetone phosphate and acetaldehyde or (2-methylthio)acetaldehyde during both aerobic and anaerobic growth. Both SAM by-products are metabolized with equal affinity during aerobic and anaerobic growth conditions, suggesting that the dual-purpose salvage pathway plays a central role in numerous environments, notably the human body during infection. Our newly discovered bifunctional oxygen-independent pathway, widespread in bacteria, salvages at least two by-products of SAM-dependent enzymes for carbon and sulfur salvage, contributing to cell growth.

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.

A Family of Dual-Activity Glycosyltransferase-Phosphorylases Mediates Mannogen Turnover and Virulence in Leishmania Parasites.

Parasitic protists belonging to the genus Leishmania synthesize the non-canonical carbohydrate reserve, mannogen, which is composed of ß-1,2-mannan oligosaccharides. Here, we identify a class of dual-activity mannosyltransferase/phosphorylases (MTPs) that catalyze both the sugar nucleotide-dependent biosynthesis and phosphorolytic turnover of mannogen. Structural and phylogenic analysis shows that while the MTPs are structurally related to bacterial mannan phosphorylases, they constitute a distinct family of glycosyltransferases (GT108) that have likely been acquired by horizontal gene transfer from gram-positive bacteria. The seven MTPs catalyze the constitutive synthesis and turnover of mannogen. This metabolic rheostat protects obligate intracellular parasite stages from nutrient excess, and is essential for thermotolerance and parasite infectivity in the mammalian host. Our results suggest that the acquisition and expansion of the MTP family in Leishmania increased the metabolic flexibility of these protists and contributed to their capacity to colonize new host niches.

An NAD+ Phosphorylase Toxin Triggers Mycobacterium tuberculosis Cell Death.

Toxin-antitoxin (TA) systems regulate fundamental cellular processes in bacteria and represent potential therapeutic targets. We report a new RES-Xre TA system in multiple human pathogens, including Mycobacterium tuberculosis. The toxin, MbcT, is bactericidal unless neutralized by its antitoxin MbcA. To investigate the mechanism, we solved the 1.8 Å-resolution crystal structure of the MbcTA complex. We found that MbcT resembles secreted NAD+-dependent bacterial exotoxins, such as diphtheria toxin. Indeed, MbcT catalyzes NAD+ degradation in vitro and in vivo. Unexpectedly, the reaction is stimulated by inorganic phosphate, and our data reveal that MbcT is a NAD+ phosphorylase. In the absence of MbcA, MbcT triggers rapid M. tuberculosis cell death, which reduces mycobacterial survival in macrophages and prolongs the survival of infected mice. Our study expands the molecular activities employed by bacterial TA modules and uncovers a new class of enzymes that could be exploited to treat tuberculosis and other infectious diseases.

The Construction of an In Vitro Synthetic Enzymatic Biosystem that Facilitates Laminaribiose Biosynthesis from Maltodextrin and Glucose.

Laminaribiose is a reducing disaccharide linked by a ß-1,3 glycosidic bond; it is also a precursor for building blocks in the pharmaceutical industry, a powerful germinating agent and antiseptic, as well as a potential prebiotic. In this study, an in vitro enzymatic biosystem composed of α-glucan phosphorylase, laminaribiose phosphorylase, isoamylase, and 4-glucanotransferase is designed for the one-pot synthesis of laminaribiose from low-cost maltodextrin and glucose. Through condition optimization, 51 mM laminaribiose is produced from 10 g L-1 maltodextrin (55.5 mM glucose equivalent) and 90 mM glucose. The product yield based on maltodextrin is 91.9%. To investigate the industrial potential of this in vitro enzymatic biosystem, the production of laminaribiose from high concentrations of substrates is also examined, and 179 mM laminaribiose is produced from 50 g L-1 of maltodextrin and 450 mM glucose. This in vitro enzymatic biosystem comprised of thermophilic enzymes can drastically decrease the manufacturing cost of laminaribiose and provide a green method for the production of other disaccharides using phosphorylases.