One-pot sustainable synthesis of glucosylglycerate from starch and glycerol through artificial in vitro enzymatic cascade.

Glucosylglycerate (R-2-O-α-D-glucopyranosyl-glycerate, GG) is a negatively charged compatible solution with versatile functions. Here, an artificial in vitro enzymatic cascade was designed to feasibly and sustainably produce GG from affordable starch and glycerol. First, Spirochaeta thermophila glucosylglycerate phosphorylase (GGP) was carefully selected because of its excellent heterologous expression, specific activity, and thermostability. The optimized two-enzyme cascade, consisting of alpha-glucan phosphorylase (αGP) and GGP, achieved a remarkable 81 % conversion rate from maltodextrin and D-glycerate. Scaling up this cascade resulted in a practical concentration of 58 g/L GG with a 62 % conversion rate based on the added D-glycerate. Additionally, the production of GG from inexpensive starch and glycerol in one-pot using artificial four-enzyme cascade was successfully implemented, which integrates alditol oxidase and catalase with αGP and GGP. Collectively, this sustainable enzymatic cascade demonstrates the feasibility of the practical synthesis of GG and has the potential to produce other glycosides using the phosphorylase-and-phosphorylase paradigm.

Pushing the boundaries of phosphorylase cascade reaction for cellobiose production I: Kinetic model development.

One-pot cascade reactions of coupled disaccharide phosphorylases enable an efficient transglycosylation via intermediary α-d-glucose 1-phosphate (G1P). Such transformations have promising applications in the production of carbohydrate commodities, including the disaccharide cellobiose for food and feed use. Several studies have shown sucrose and cellobiose phosphorylase for cellobiose synthesis from sucrose, but the boundaries on transformation efficiency that result from kinetic and thermodynamic characteristics of the individual enzyme reactions are not known. Here, we assessed in a step-by-step systematic fashion the practical requirements of a kinetic model to describe cellobiose production at industrially relevant substrate concentrations of up to 600 mM sucrose and glucose each. Mechanistic initial-rate models of the two-substrate reactions of sucrose phosphorylase (sucrose + phosphate → G1P + fructose) and cellobiose phosphorylase (G1P + glucose → cellobiose + phosphate) were needed and additionally required expansion by terms of glucose inhibition, in particular a distinctive two-site glucose substrate inhibition of the cellobiose phosphorylase (from Cellulumonas uda). Combined with mass action terms accounting for the approach to equilibrium, the kinetic model gave an excellent fit and a robust prediction of the full reaction time courses for a wide range of enzyme activities as well as substrate concentrations, including the variable substoichiometric concentration of phosphate. The model thus provides the essential engineering tool to disentangle the highly interrelated factors of conversion efficiency in the coupled enzyme reaction; and it establishes the necessary basis of window of operation calculations for targeted optimizations toward different process tasks.

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.

The Plastidial Glucan Phosphorylase Affects the Maltooligosaccharide Metabolism in Parenchyma Cells of Potato (Solanum tuberosum L.) Tuber Discs.

Maltodextrin metabolism is thought to be involved in both starch initiation and degradation. In this study, potato tuber discs from transgenic lines containing antisense constructs against the plastidial and cytosolic isoforms of α-glucan phosphorylase and phosphoglucomutase were used to evaluate their influences on the conversion of externally supplied glucose-1-phosphate into soluble maltodextrins, as compared to wild-type potato tubers (Solanum tuberosum L. cv. Desiree). Relative maltodextrin amounts analyzed by capillary electrophoresis with laser-induced fluorescence revealed that tuber discs could immediately uptake glucose-1-phosphate and use it to produce maltooligosaccharides with a degree of polymerization of up to 30, as opposed to tubers repressing the plastidial glucan phosphorylase. The results presented here support previous indications that a specific transporter for glucose-1-phosphate may exist in both the plant cells and the plastidial membranes, thereby allowing a glucose-6-phosphate-independent transport. Furthermore, it confirms that the plastidial glucan phosphorylase is responsible for producing longer maltooligosaccharides in the plastids by catalyzing a glucosyl polymerization reaction when glucose-1-phosphate is available. All these findings contribute to a better understanding of the role of the plastidial phosphorylase as a key enzyme directly involved in the synthesis and degradation of glucans and their implication on starch metabolism.

Screening of Induced Mutants Led to the Identification of Starch Biosynthetic Genes Associated with Improved Resistant Starch in Wheat.

Several health benefits are obtained from resistant starch, also known as healthy starch. Enhancing resistant starch with genetic modification has huge commercial importance. The variation of resistant starch content is narrow in wheat, in relation to which limited improvement has been attained. Hence, there is a need to produce a wheat population that has a wide range of variations in resistant starch content. In the present study, stable mutants were screened that showed significant variation in the resistant starch content. A megazyme kit was used for measuring the resistant starch content, digestible starch, and total starch. The analysis of variance showed a significant difference in the mutant population for resistant starch. Furthermore, four diverse mutant lines for resistant starch content were used to study the quantitative expression patterns of 21 starch metabolic pathway genes; and to evaluate the candidate genes for resistant starch biosynthesis. The expression pattern of 21 starch metabolic pathway genes in two diverse mutant lines showed a higher expression of key genes regulating resistant starch biosynthesis (GBSSI and their isoforms) in the high resistant starch mutant lines, in comparison to the parent variety (J411). The expression of SBEs genes was higher in the low resistant starch mutants. The other three candidate genes showed overexpression (BMY, Pho1, Pho2) and four had reduced (SSIII, SBEI, SBEIII, ISA3) expression in high resistant starch mutants. The overexpression of AMY and ISA1 in the high resistant starch mutant line JE0146 may be due to missense mutations in these genes. Similarly, there was a stop_gained mutation for PHO2; it also showed overexpression. In addition, the gene expression analysis of 21 starch metabolizing genes in four different mutants (low and high resistant starch mutants) shows that in addition to the important genes, several other genes (phosphorylase, isoamylases) may be involved and contribute to the biosynthesis of resistant starch. There is a need to do further study about these new genes, which are responsible for the fluctuation of resistant starch in the mutants.

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.

Comparative Study of Starch Phosphorylase Genes and Encoded Proteins in Various Monocots and Dicots with Emphasis on Maize.

Starch phosphorylase (PHO) is a multimeric enzyme with two distinct isoforms: plastidial starch phosphorylase (PHO1) and cytosolic starch phosphorylase (PHO2). PHO1 specifically resides in the plastid, while PHO2 is found in the cytosol. Both play a critical role in the synthesis and degradation of starch. This study aimed to report the detailed structure, function, and evolution of genes encoding PHO1 and PHO2 and their protein ligand-binding sites in eight monocots and four dicots. "True" orthologs of PHO1 and PHO2 of Oryza sativa were identified, and the structure of the enzyme at the protein level was studied. The genes controlling PHO2 were found to be more conserved than those controlling PHO1; the variations were mainly due to the variable sequence and length of introns. Cis-regulatory elements in the promoter region of both genes were identified, and the expression pattern was analyzed. The real-time quantitative polymerase chain reaction indicated that PHO2 was expressed in all tissues with a uniform pattern of transcripts, and the expression pattern of PHO1 indicates that it probably contributes to the starch biosynthesis during seed development in Zea mays. Under abscisic acid (ABA) treatment, PHO1 was found to be downregulated in Arabidopsis and Hordeum vulgare. However, we found that ABA could up-regulate the expression of both PHO1 and PHO2 within 12 h in Zea mays. In all monocots and dicots, the 3D structures were highly similar, and the ligand-binding sites were common yet fluctuating in the position of aa residues.

Whole-cell biosynthesis of cytarabine by an unnecessary protein-reduced Escherichia coli that coexpresses purine and uracil phosphorylase.

Currently, whole-cell catalysts face challenges due to the complexity of reaction systems, although they have a cost advantage over pure enzymes. In this study, cytarabine was synthesized by purified purine phosphorylase 1 (PNP1) and uracil phosphorylase (UP), and the conversion of cytarabine from adenine arabinoside reached 72.3 ± 4.3%. However, the synthesis was unsuccessful by whole-cell catalysis due to interference from unnecessary proteins (UNPs) in cells. Thus, we carried out a large-scale gene editing involving 377 genes in the genome of Escherichia coli to reduce the negative effect of UNPs on substrate conversion and cytarabine production. Finally, the PNP1 and UP activities of the obtained mutant were increased significantly compared with the parental strain, and more importantly, the conversion rate of cytarabine by whole-cell catalysis reached 67.4 ± 2.5%. The lack of 148 proteins and downregulation of 783 proteins caused by gene editing were equivalent to partial purification of the enzymes within cells, and thus, we provided inspiration to solve the problem caused by UNP interference, which is ubiquitous in the field of whole-cell catalysis.

A century of exercise physiology: key concepts in regulation of glycogen metabolism in skeletal muscle.

Glycogen is a branched, glucose polymer and the storage form of glucose in cells. Glycogen has traditionally been viewed as a key substrate for muscle ATP production during conditions of high energy demand and considered to be limiting for work capacity and force generation under defined conditions. Glycogenolysis is catalyzed by phosphorylase, while glycogenesis is catalyzed by glycogen synthase. For many years, it was believed that a primer was required for de novo glycogen synthesis and the protein considered responsible for this process was ultimately discovered and named glycogenin. However, the subsequent observation of glycogen storage in the absence of functional glycogenin raises questions about the true role of the protein. In resting muscle, phosphorylase is generally considered to be present in two forms: non-phosphorylated and inactive (phosphorylase b) and phosphorylated and constitutively active (phosphorylase a). Initially, it was believed that activation of phosphorylase during intense muscle contraction was primarily accounted for by phosphorylation of phosphorylase b (activated by increases in AMP) to a, and that glycogen synthesis during recovery from exercise occurred solely through mechanisms controlled by glucose transport and glycogen synthase. However, it now appears that these views require modifications. Moreover, the traditional roles of glycogen in muscle function have been extended in recent years and in some instances, the original concepts have undergone revision. Thus, despite the extensive amount of knowledge accrued during the past 100 years, several critical questions remain regarding the regulation of glycogen metabolism and its role in living muscle.

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.

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.

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.

Discovery of a Kojibiose Hydrolase by Analysis of Specificity-Determining Correlated Positions in Glycoside Hydrolase Family 65.

The Glycoside Hydrolase Family 65 (GH65) is an enzyme family of inverting α-glucoside phosphorylases and hydrolases that currently contains 10 characterized enzyme specificities. However, its sequence diversity has never been studied in detail. Here, an in-silico analysis of correlated mutations was performed, revealing specificity-determining positions that facilitate annotation of the family's phylogenetic tree. By searching these positions for amino acid motifs that do not match those found in previously characterized enzymes from GH65, several clades that may harbor new functions could be identified. Three enzymes from across these regions were expressed in E. coli and their substrate profile was mapped. One of those enzymes, originating from the bacterium Mucilaginibacter mallensis, was found to hydrolyze kojibiose and α-1,2-oligoglucans with high specificity. We propose kojibiose glucohydrolase as the systematic name and kojibiose hydrolase or kojibiase as the short name for this new enzyme. This work illustrates a convenient strategy for mapping the natural diversity of enzyme families and smartly mining the ever-growing number of available sequences in the quest for novel specificities.

Thermostable adenosine 5'-monophosphate phosphorylase from Thermococcus kodakarensis forms catalytically active inclusion bodies.

Catalytically active inclusion bodies (CatIBs) produced in Escherichia coli are an interesting but currently underexplored strategy for enzyme immobilization. They can be purified easily and used directly as stable and reusable heterogenous catalysts. However, very few examples of CatIBs that are naturally formed during heterologous expression have been reported so far. Previous studies have revealed that the adenosine 5'-monophosphate phosphorylase of Thermococcus kodakarensis (TkAMPpase) forms large soluble multimers with high thermal stability. Herein, we show that heat treatment of soluble protein from crude extract induces aggregation of active protein which phosphorolyse all natural 5'-mononucleotides. Additionally, inclusion bodies formed during the expression in E. coli were found to be similarly active with 2-6 folds higher specific activity compared to these heat-induced aggregates. Interestingly, differences in the substrate preference were observed. These results show that the recombinant thermostable TkAMPpase is one of rare examples of naturally formed CatIBs.

Effect of Short-Term Cold Treatment on Carbohydrate Metabolism in Potato Leaves.

Plants are often challenged by an array of unfavorable environmental conditions. During cold exposure, many changes occur that include, for example, the stabilization of cell membranes, alterations in gene expression and enzyme activities, as well as the accumulation of metabolites. In the presented study, the carbohydrate metabolism was analyzed in the very early response of plants to a low temperature (2 °C) in the leaves of 5-week-old potato plants of the Russet Burbank cultivar during the first 12 h of cold treatment (2 h dark and 10 h light). First, some plant stress indicators were examined and it was shown that short-term cold exposure did not significantly affect the relative water content and chlorophyll content (only after 12 h), but caused an increase in malondialdehyde concentration and a decrease in the expression of NDA1, a homolog of the NADH dehydrogenase gene. In addition, it was shown that the content of transitory starch increased transiently in the very early phase of the plant response (3-6 h) to cold treatment, and then its decrease was observed after 12 h. In contrast, soluble sugars such as glucose and fructose were significantly increased only at the end of the light period, where a decrease in sucrose content was observed. The availability of the monosaccharides at constitutively high levels, regardless of the temperature, may delay the response to cold, involving amylolytic starch degradation in chloroplasts. The decrease in starch content, observed in leaves after 12 h of cold exposure, was preceded by a dramatic increase in the transcript levels of the key enzymes of starch degradation initiation, the α-glucan, water dikinase (GWD-EC 2.7.9.4) and the phosphoglucan, water dikinase (PWD-EC 2.7.9.5). The gene expression of both dikinases peaked at 9 h of cold exposure, as analyzed by real-time PCR. Moreover, enhanced activities of the acid invertase as well as of both glucan phosphorylases during exposure to a chilling temperature were observed. However, it was also noticed that during the light phase, there was a general increase in glucan phosphorylase activities for both control and cold-stressed plants irrespective of the temperature. In conclusion, a short-term cold treatment alters the carbohydrate metabolism in the leaves of potato, which leads to an increase in the content of soluble sugars.

pH-Independent Heat Capacity Changes during Phosphorolysis Catalyzed by the Pyrimidine Nucleoside Phosphorylase from Geobacillus thermoglucosidasius.

Enzyme-catalyzed reactions sometimes display curvature in their Eyring plots in the absence of denaturation, indicative of a change in activation heat capacity. However, the effects of pH and (de)protonation on this phenomenon have remained unexplored. Herein, we report a kinetic characterization of the thermophilic pyrimidine nucleoside phosphorylase from Geobacillus thermoglucosidasius across a two-dimensional working space covering 35 °C and 3 pH units with two substrates displaying different pKa values. Our analysis revealed the presence of a measurable activation heat capacity change ΔCp⧧ in this reaction system, which showed no significant dependence on medium pH or substrate charge. Our results further describe the remarkable effects of a single halide substitution that has a minor influence on ΔCp⧧ but conveys a significant kinetic effect by decreasing the activation enthalpy, causing a >10-fold rate increase. Collectively, our results present an important piece in the understanding of enzymatic systems across multidimensional working spaces where the choice of reaction conditions can affect the rate, affinity, and thermodynamic phenomena independently of one another.

ß-Glucan phosphorylases in carbohydrate synthesis.

ß-Glucan phosphorylases are carbohydrate-active enzymes that catalyze the reversible degradation of ß-linked glucose polymers, with outstanding potential for the biocatalytic bottom-up synthesis of ß-glucans as major bioactive compounds. Their preference for sugar phosphates (rather than nucleotide sugars) as donor substrates further underlines their significance for the carbohydrate industry. Presently, they are classified in the glycoside hydrolase families 94, 149, and 161 ( www.cazy.org ). Since the discovery of ß-1,3-oligoglucan phosphorylase in 1963, several other specificities have been reported that differ in linkage type and/or degree of polymerization. Here, we present an overview of the progress that has been made in our understanding of ß-glucan and associated ß-glucobiose phosphorylases, with a special focus on their application in the synthesis of carbohydrates and related molecules. KEY POINTS: • Discovery, characteristics, and applications of ß-glucan phosphorylases. • ß-Glucan phosphorylases in the production of functional carbohydrates.