Enzymatic Phosphorolysis Research Articles

Effect of Free Cysteine Residues to Serine Mutation on Cellodextrin Phosphorylase.

Cellodextrin phosphorylase (CDP) plays a key role in energy-efficient cellulose metabolism of anaerobic bacteria by catalyzing phosphorolysis of cellodextrin to produce cellobiose and glucose 1-phosphate, which can be utilized for glycolysis without consumption of additional ATP. As the enzymatic phosphorolysis reaction is reversible, CDP is also employed to produce cellulosic materials in vitro. However, the enzyme is rapidly inactivated by oxidation, which hinders in vitro utilization in aerobic environments. It has been suggested that the cysteine residues of CDP, which do not form disulfide bonds, are responsible for the loss of activity, and the aim of the present work was to test this idea. For this purpose, we replaced all 11 free cysteine residues of CDP from Acetivibrio thermocellus (formerly known as Clostridium thermocellum) with serine, which structurally resembles cysteine in our previous work. Herein, we show that the resulting CDP variant, named CDP-CS, has comparable activity to the wild-type enzyme, but shows increased stability to oxidation during long-term storage. X-Ray crystallography indicated that the mutations did not markedly alter the overall structure of the enzyme. Ensemble refinement of the crystal structures of CDP and CDP-CS indicated that the C372S and C625S mutations reduce structural fluctuations in the protein main chain, which may contribute to the increased stability of CDP-CS to oxidation.

Efficient Synthesis of β-Glucose 1-Phosphate through Enzymatic Phosphorolysis and Baker's Yeast Fermentation

Efficient Synthesis of β-Glucose 1-Phosphate through Enzymatic Phosphorolysis and Baker's Yeast Fermentation

Synthesis of α-D-Ribose 1-Phosphate and 2-Deoxy-α-D-Ribose 1-Phosphate Via Enzymatic Phosphorolysis of 7-Methylguanosine and 7-Methyldeoxyguanosine.

A simple and efficient method for the preparation of α-D-ribose 1-phosphate and 2-deoxy-α-D-ribose 1-phosphate, key intermediates in nucleoside metabolism and important starting compounds for the enzymatic synthesis of various modified nucleosides, has been proposed. It consists in near-irreversible enzymatic phosphorolysis of readily prepared hydroiodide salts of 7-methylguanosine and 7-methyl-2'-deoxyguanosine, respectively, in the presence of purine nucleoside phosphorylase. α-D-Ribose 1-phosphate and 2-deoxy-α-D-ribose 1-phosphate are obtained in near quantitative yields (by HPLC analysis) and 74%-94% yields after their isolation and purification. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Preparation of α-D-ribose 1-phosphate barium salt (4a) Alternate Protocol 1: Preparation of 2-deoxy-α-D-ribose 1-phosphate barium salt (4b) Basic Protocol 2: Preparation of α-D-ribose 1-phosphate bis(cyclohexylammonium) salt (5a) Alternate Protocol 2: Preparation of 2-deoxy-α-D-ribose 1-phosphate bis(cyclohexylammonium) salt (5b).

Acceleration of cellodextrin phosphorolysis for bioelectricity generation from cellulosic biomass by integrating a synthetic two-enzyme complex into an in vitro synthetic enzymatic biosystem

BackgroundCellulosic biomass, the earth’s most abundant renewable resource, can be used as substrates for biomanufacturing biofuels or biochemicals via in vitro synthetic enzymatic biosystems in which the first step is the enzymatic phosphorolysis of cellodextrin to glucose 1-phosphate (G1P) by cellodextrin phosphorylase (CDP). However, almost all the CDPs prefer cellodextrin synthesis to phosphorolysis, resulting in the low reaction rate of cellodextrin phosphorolysis for biomanufacturing.ResultsTo increase the reaction rate of cellodextrin phosphorolysis, synthetic enzyme complexes containing CDP and phosphoglucomutase (PGM) were constructed to convert G1P to glucose 6-phosphate (G6P) rapidly, which is an important intermediate for biomanufacturing. Four self-assembled synthetic enzyme complexes were constructed with different spatial organizations based on the high-affinity and high-specific interaction between cohesins and dockerins from natural cellulosomes. Thus, the CDP–PGM enzyme complex with the highest enhancement of initial reaction rate was integrated into an in vitro synthetic enzymatic biosystem for generating bioelectricity from cellodextrin. The in vitro biosystem containing the best CDP–PGM enzyme complex exhibited a much higher current density (3.35-fold) and power density (2.14-fold) than its counterpart biosystem containing free CDP and PGM mixture.ConclusionsHereby, we first reported bioelectricity generation from cellulosic biomass via in vitro synthetic enzymatic biosystems. This work provided a strategy of how to link non-energetically favorable reaction (cellodextrin phosphorolysis) and energetically favorable reaction (G1P to G6P) together to circumvent unfavorable reaction equilibrium and shed light on improving the reaction efficiency of in vitro synthetic enzymatic biosystems through the construction of synthetic enzyme complexes.

Stoichiometric Conversion of Cellulosic Biomass by in Vitro Synthetic Enzymatic Biosystems for Biomanufacturing

Cellulosic biomass is the earth’s most abundant renewable resource, which is considered to be a promising feedstock for manufacturing biofuels and biochemicals. In this study, stoichiometric enzymatic phosphorolysis of cellulosic biomass for manufacturing biochemicals or biofuels by in vitro synthetic enzymatic biosystems was designed. Three cascade phosphorolytic enzymes, cellodextrin phosphorylase, cellobiose phosphorylase, and polyphosphate-dependent glucokinase, were used for the biotransformation of cellodextrins to high-energy phosphorylated sugars (that is, glucose 1-phosphate and glucose 6-phosphate). A series of downstream exergonic reactions then converted these high-energy phosphorylated sugars into myo-inositol, resulting in a near-stoichiometric conversion of cellodextrins with a high product yield of 98% (w/w). Moreover, this enzymatic biosystem can even work for the acid-treated biomass hydrolysate containing microorganism-toxic compounds. The construction of this in vitro synthetic enzymat...

Advanced water splitting for green hydrogen gas production through complete oxidation of starch by in vitro metabolic engineering

Starch is a natural energy storage compound and is hypothesized to be a high-energy density chemical compound or solar fuel. In contrast to industrial hydrolysis of starch to glucose, an alternative ATP-free phosphorylation of starch was designed to generate cost-effective glucose 6-phosphate by using five thermophilic enzymes (i.e., isoamylase, alpha-glucan phosphorylase, 4-α-glucanotransferase, phosphoglucomutase, and polyphosphate glucokinase). This enzymatic phosphorolysis is energetically advantageous because the energy of α-1,4-glycosidic bonds among anhydroglucose units is conserved in the form of phosphorylated glucose. Furthermore, we demonstrated an in vitro 17-thermophilic enzyme pathway that can convert all glucose units of starch, regardless of branched and linear contents, with water to hydrogen at a theoretic yield (i.e., 12 H2 per glucose), three times of the theoretical yield from dark microbial fermentation. The use of a biomimetic electron transport chain enabled to achieve a maximum volumetric productivity of 90.2mmol of H2/L/h at 20g/L starch. The complete oxidation of starch to hydrogen by this in vitro synthetic (enzymatic) biosystem suggests that starch as a natural solar fuel becomes a high-density hydrogen storage compound with a gravimetric density of more than 14% H2-based mass and an electricity density of more than 3000Wh/kg of starch.

An Investigation of Enzymatic Phosphorolysis of α(1→4)-Linked Oligo-d-glucosaminides by Thermostable α-Glucan Phosphorylase Catalysis

This paper reports an investigation of enzymatic phosphorolysis of α(1→4)- linked oligo-d-glucosaminide substrates by thermo stable α-glucan phosphorylase (from Aquifex aeolicus VF5) catalysis. α-Glucan phosphorylase catalyzes both phosphorolysis and glucosylation/polymerization at the nonreducing end of α(1→4)-linked glucose substrates depending on conditions. The authors also found that α(1→4)-linked d-glucosaminide polymer (chitosan stereoisomer) was obtained by the thermostable α-glucan phosphorylase-catalyzed enzymatic polymerization of α-d-glucosamine 1-phosphate from a maltooligosaccharide primer. In the present study, we intend to reveal whether the enzyme catalyzes phosphorolysis of substrates containing such d-glucosaminide chains. The α(1→4)-linked oligo-d-glucosaminide substrates elongated from maltotriose were first prepared by the thermostable α-glucan phosphorylase-catalyzed enzymatic polymerization of α-d-glucosamine 1-phosphate from the maltotriose primer and subsequent purification by preparative HPLC. The phosphorolysis of the resulting substrates are conducted in the presence of thermostable α-glucan phosphorylase in phosphate buffer. The analytical results of the products fully supported the occurrence of phosphorolysis at the nonreducing end of both the chains of d-glucosamine-α(1→4)-d-glucosamine and d-glucosamine-α(1→4)-d-glucose sequences.

Examining the role of phosphate in glycosyl transfer reactions of Cellulomonas uda cellobiose phosphorylase using d-glucal as donor substrate

Cellobiose phosphorylase from Cellulomonas uda (CuCPase) is shown to utilize d-glucal as slow alternative donor substrate for stereospecific glycosyl transfer to inorganic phosphate, giving 2-deoxy-α-d-glucose 1-phosphate as the product. When performed in D2O, enzymatic phosphorolysis of d-glucal proceeds with incorporation of deuterium in equatorial position at C-2, implying a stereochemical course of reaction where substrate becomes protonated from below its six-membered ring through stereoselective re side attack at C-2. The proposed catalytic mechanism, which is supported by results of docking studies, involves direct protonation of d-glucal by the enzyme-bound phosphate, which then performs nucleophilic attack on the reactive C-1 of donor substrate. When offered d-glucose next to d-glucal and phosphate, CuCPase produces 2-deoxy-β-d-glucosyl-(1→4)-d-glucose and 2-deoxy-α-d-glucose 1-phosphate in a ratio governed by mass action of the two acceptor substrates present. Enzymatic synthesis of 2-deoxy-β-d-glucosyl-(1→4)-d-glucose is effectively promoted by catalytic concentrations of phosphate, suggesting that catalytic reaction proceeds through a quaternary complex of CuCPase, d-glucal, phosphate, and d-glucose. Conversion of d-glucal and phosphate presents a convenient single-step synthesis of 2-deoxy-α-d-glucose 1-phosphate that is difficult to prepare chemically.

Production, Characterization and Synthetic Application of a Purine Nucleoside Phosphorylase from Aeromonas hydrophila

AbstractPurine nucleoside phosphorylase (PNP) from Aeromonas hydrophila encoded by the deoD gene has been over‐expressed in Escherichia coli, purified, characterized about its substrate specificity and used for the preparative synthesis of some 6‐substituted purine‐9‐ribosides. Substrate specificity towards natural nucleosides showed that this PNP catalyzes the phosphorolysis of both 6‐oxo‐ and 6‐aminopurine (deoxy)ribonucleosides. A library of nucleoside analogues was synthesized and then submitted to enzymatic phosphorolysis as well. This assay revealed that 1‐, 2‐, 6‐ and 7‐modified nucleosides are accepted as substrates, whereas 8‐substituted nucleosides are not. A few transglycosylation reactions were carried out using 7‐methylguanosine iodide (4) as a D‐ribose donor and 6‐substituted purines as acceptor. In particular, following this approach, 2‐amino‐6‐chloropurine‐9‐riboside (2c), 6‐methoxypurine‐9‐riboside (2d) and 2‐amino‐6‐(methylthio)purine‐9‐riboside (2g) were synthesized in very high yield and purity.

Properties of Purine Nucleoside Phosphorylase (PNP) of Mammalian and Bacterial Origin

Purine nucleoside phosphorylase (PNP), from calf spleen, human erythrocytes and E. coli have been examined with regard to structural requirements of substrates and inhibitors. Kinetic parameters (Km, Vmax/Km) for a variety of N(1) and/or N(7)-methylated analogues of guanosine, inosine and adenosine have been evaluated for all three enzymes. The substrate and/or inhibitor properties of purine riboside, 1,6-dihydropurine riboside, some deazapurine nucleosides: 3-deaza- and 7-deazainosine, 1,3-dideazapurine riboside (ribobenzimidazole), and a variety of acyclonucleosides, have been determined with mammalian and bacterial enzymes. Overall results indicate distinct similarities of kinetic properties and structural requirements of the two mammalian enzymes, although there are some differences as well. The N(1) and O6 of the purine ring are necessary for substrate-inhibitor activity and constitute a binding site for the mammalian (but not the bacterial) enzymes. Moreover, nucleosides lacking the N(3) undergo phosphorolysis and those lacking N(7) are inhibitors (but not substrates). Methylation of the ring N(7) leads to two overlapping effects: labilization of the glycosidic bond, and impediment to protonation at this site by the enzyme, a postulated prerequisite for enzymatic phosphorolysis. It is proposed that a histidine interacts with N(1) as a donor and O6 as an acceptor. Alternatively N(1)-H and C(2)-NH2 may serve as donors for hydrogen bonds with a glutamate residue. The less specific E. coli enzyme phosphorolyses all purine ring modified nucleosides but 7-deazainosine which is only an inhibitor. On the other hand, the bacterial enzyme exhibits decreased activity towards N(7)-methylated nucleosides and lack of affinity for a majority of the tested acyclonucleoside inhibitors of the mammalian enzymes. The foregoing results underline the fundamental differences between mammalian and bacterial enzymes, including variations in the binding sites for the purine ring.

Monomeric purine nucleoside phosphorylase from rabbit liver. Purification and characterization.

Rabbit liver purine nucleoside phosphorylase (purine nucleoside: orthophosphate ribosyltransferase EC 2.4.2.1.) was purified to homogeneity by column chromatography and ammonium sulfate fractionation. Homogeneity was established by disc gel electrophoresis in presence and absence of sodium dodecyl sulfate, and isoelectric focusing. Molecular weights of 46,000 and 39,000 were determined, respectively, by gel filtration and by sodium dodecyl sulfate-polyacrylamide disc gel electrophoresis. Product inhibition was observed with guanine and hypoxanthine as strong competitive inhibitors for the enzymatic phosphorolysis of guanosine. Respective Kis calculated were 1.25 x 10(-5) M for guanine and 2.5 x 10(-5) M for hypoxanthine. Ribose 1-phosphate, another product of the reaction, gave noncompetitive inhibition with guanosine as variable substrate, and an inhibition constant of 3.61 x 10(-4) M was calculated. The protection of essential --SH groups on the enzyme, by 2-mercaptoethanol or dithiothreitol, was necessary for the maintenance of enzyme activity. Noncompetitive inhibition was observed for p-chloromercuribenzoate with an inhibition constant of 5.68 x 10(-6)M. Complete reversal of this inhibition by an excess of 2-mercaptoethanol or dithiothreitol was demonstrated. In the presence of methylene blue, the enzyme showed a high sensitivity to photooxidation and a dependence of photoinactivation on pH, strongly implicating histidine as the susceptible group at the active site of the enzyme. The pKa values determined for ionizable groups of the active site of the enzyme were near pH 5.5 and pH 8.5 The chemical and kinetic evidences suggest that histidine and cysteine may be essential for catalysis. Inorganic orthophosphate (Km 1.54 x 10(-2) M) was an obligatory anion requirement, and arsenate substituted for phosphate with comparable results. Guanosine (Km 5.00 x 10(-5) M), deoxyguanosine (Km 1.00 x 10(-4)M) and inosine (Km 1.33 x 10(-4)M), were substrates for enzymatic phosphorolysis. Xanthosine was an extremely poor substrate, and adenosine was not phosphorylyzed at 20-fold excess of the homogeneous enzyme. Guanine (Km 1.82 x 10(-5)M),ribose 1-phosphate (Km 1.34 x 10(-4) M) and hypoxanthine were substrates for the reverse reaction, namely, the enzymatic synthesis of nucleosides. The initial velocity studies of the saturation of the enzyme with guanosine, at various fixed concentrations of inorganic orthophosphate, suggest a sequential bireactant catalytic mechanism for the enzyme.

Inhibitory action of thymine ribonucleoside and other pyrimidine derivatives on the cleavage of 5-iodo-deoxy-uridine by mouse-liver extract

(1) The action of various pyrimidine derivatives on the enzymatic phosphorolysis of [ 125I]-iodo-2'-deoxyuridine ([ 125I]IUdR) by a particulate-free fraction (100 000 × g) derived from mouse liver has been examined. (2) The most efficient inhibitor of phosphorolysis investigated was thymine ribofuranoside (TR), the ribose analogue of thymidine (TdR), which is not itself cleaved by the enzyme extract. (3) Although the apparent affinities of IUdR and TdR for the enzyme system were comparable, the maximum velocity of cleavage of the IUdR was approximately 5 times greater. This difference could explain the observed shorter duration of the homologue in the cell pool in vivo compared with TdR. (4) The competitive inhibitory action of thymine ribonucleoside is approximately 20 times less effective for IUdR cleavage than for TdR cleavage by the enzyme extract. This difference offers a probable explanation for the 100-fold molar excess of thymine ribonucleoside necessary to inhibit loss of [ 125I]IUdR from the cell pool in vivo. (5) An extract from rat liver contained 5 times greater IUdR cleaving capacity than an equivalent extract of mouse liver.