Phosphorolytic Cleavage Research Articles

Ambient temperature structure of phosphoketolase from Bifidobacterium longum determined by serial femtosecond X-ray crystallography.

Phosphoketolase and transketolase are thiamine diphosphate-dependent enzymes and play a central role in the primary metabolism of bifidobacteria: the bifid shunt. The enzymes both catalyze phosphorolytic cleavage of xylulose 5-phosphate or fructose 6-phosphate in the first reaction step, but possess different substrate specificity in the second reaction step, where phosphoketolase and transketolase utilize inorganic phosphate (Pi) and D-ribose 5-phosphate, respectively, as the acceptor substrate. Structures of Bifidobacterium longum phosphoketolase holoenzyme and its complex with a putative inhibitor, phosphoenolpyruvate, were determined at 2.5 Å resolution by serial femtosecond crystallography using an X-ray free-electron laser. In the complex structure, phosphoenolpyruvate was present at the entrance to the active-site pocket and plugged the channel to thiamine diphosphate. The phosphate-group position of phosphoenolpyruvate coincided well with those of xylulose 5-phosphate and fructose 6-phosphate in the structures of their complexes with transketolase. The most striking structural change was observed in a loop consisting of Gln546-Asp547-His548-Asn549 (the QN-loop) at the entrance to the active-site pocket. Contrary to the conformation of the QN-loop that partially covers the entrance to the active-site pocket (`closed form') in the known crystal structures, including the phosphoketolase holoenzyme and its complexes with reaction intermediates, the QN-loop in the current ambient structures showed a more compact conformation with a widened entrance to the active-site pocket (`open form'). In the phosphoketolase reaction, the `open form' QN-loop may play a role in providing the binding site for xylulose 5-phosphate or fructose 6-phosphate in the first step, and the `closed form' QN-loop may help confer specificity for Pi in the second step.

Crystal structures of a new class of pyrimidine/purine nucleoside phosphorylase revealed a Cupin fold.

Nucleotides metabolism is a fundamental process in all organisms. Two families of nucleoside phosphorylases (NP) that catalyze the phosphorolytic cleavage of the glycosidic bond in nucleosides have been found, including the trimeric or hexameric NP-I and dimeric NP-II family enzymes. Recent studies revealed another class of NP protein in Escherichia coli named Pyrimidine/purine nucleoside phosphorylase (ppnP), which can catalyze the phosphorolysis of diverse nucleosides and yield d-ribose 1-phosphate and the respective free bases. Here, we solved the crystal structures of ppnP from E. coli and the other three species. Our studies revealed that the structure of ppnP belongs to the RlmC-like Cupin fold and showed as a rigid dimeric conformation. Detail analysis revealed a potential nucleoside binding pocket full of hydrophobic residues, and the residues involved in the dimer and pocket formation are all well conserved in bacteria. Since the Cupin fold is a large superfamily in the biosynthesis of natural products, our studies provide the structural basis for understanding, and the directed evolution of NP proteins.

Properties and functions of TP/PD-ECGF — enzyme and angiogenic factor in norm and in neoplastic pathology

Thymidine phosphorylase is a protein which may has a dual action: it is a rate-limiting enzyme in thymidine metabolism and it is similar to the platelet – derived endothelial cell growth factor (PD/ECGF). The enzyme catalyzes the reversible reaction of phosphorolytic cleavage of thymidine to thymine and deoxyribose-1-phosphate. It has been found that TP has higher activity in tumor tissues. Also it is involved in a proliferative process in a wide variety of chronic inflammatory diseases. Increased expression of PD/ECGF in many tumors is associated with aggressive disease and/or poor prognosis. Its known that high TP activity is related to malignant angiogenesis and invasion.
 On the other hand, TP inhibits a hypoxia induced apoptotic pathway and enhances expression of various inflammatory cytokines and interferons. This apparent role of enzyme in tumor progression has prompted investigation a large number of TP inhibitors for applicability in chemotherapy backbone regimens. The enzymatic activity of PD/ECGF is being able to generate 5-fluorouracile from capecitabine and other precursors. Thus TP is identified as a prime target for developing novel anticancer therapies. The serum TP level in cancer patients provides useful prognostic information regarding both responses to chemotherapy and length of survival and should be used in planning appropriate therapy. TP could be suggested that control of individual enzyme activity in blood serum may be used as informative tool for monitoring of patients and treatment optimization.

Creation of strain – producer of bacterial purine nucleoside phosphorylase fused with human annexin A5

It is known that bacterial purine nucleoside phosphorylase (PNPase), unlike mammalian PNPase, is capable of phosphorolytic cleavage of adenosine and its derivatives to form free nitrogen bases. This makes it possible to use bacterial PNPase (provided the problem of delivering this enzyme or its gene to target cells is solved) as a prodrug therapy for cancer. In addition, PNPase in a tumor bed can destroy extracellular adenosine, which is known to protect cancer cells from antitumor immunity.As a result of the study, a new strain of Escherichia coli was constructed, producing a chimeric protein whose molecule consists of a homologous PNPase fused to human annexin A5, a protein showing affinity for cancer cells.The production capacity of the producer strain of the chimeric protein “Annexin-PNPase” with respect to PNPase calculated from the results of the inosine phosphorolysis reaction, was 10,200 units/ml of culture liquid. The obtained strain is intended for creation of a technology for obtaining new antitumor preparations.

Dissecting cellobiose metabolic pathway and its application in biorefinery through consolidated bioprocessing in Myceliophthora thermophila

BackgroundLignocellulosic biomass has long been recognized as a potential sustainable source for industrial applications. The costs associated with conversion of plant biomass to fermentable sugar represent a significant barrier to the production of cost-competitive biochemicals. Consolidated bioprocessing (CBP) is considered a potential breakthrough for achieving cost-efficient production of biomass-based fuels and commodity chemicals. During the degradation of cellulose, cellobiose (major end-product of cellulase activity) is catabolized by hydrolytic and phosphorolytic pathways in cellulolytic organisms. However, the details of the two intracellular cellobiose metabolism pathways in cellulolytic fungi remain to be uncovered.ResultsUsing the engineered malic acid production fungal strain JG207, we demonstrated that the hydrolytic pathway by β-glucosidase and the phosphorolytic pathway by phosphorylase are both used for intracellular cellobiose metabolism in Myceliophthora thermophila, and the yield of malic acid can benefit from the energy advantages of phosphorolytic cleavage. There were obvious differences in regulation of the two cellobiose catabolic pathways depending on whether M. thermophila JG207 was grown on cellobiose or Avicel. Disruption of Mtcpp in strain JG207 led to decreased production of malic acid under cellobiose conditions, while expression levels of all three intracellular β-glucosidase genes were significantly up-regulated to rescue the impairment of the phosphorolytic pathway under Avicel conditions. When the flux of the hydrolytic pathway was reduced, we found that β-glucosidase encoded by bgl1 was the dominant enzyme in the hydrolytic pathway and deletion of bgl1 resulted in significant enhancement of protein secretion but reduction of malate production. Combining comprehensive manipulation of both cellobiose utilization pathways and enhancement of cellobiose uptake by overexpression of a cellobiose transporter, the final strain JG412Δbgl2Δbgl3 produced up to 101.2 g/L and 77.4 g/L malic acid from cellobiose and Avicel, respectively, which corresponded to respective yields of 1.35 g/g and 1.03 g/g, representing significant improvement over the starting strain JG207.ConclusionsThis is the first report of detailed investigation of intracellular cellobiose catabolism in cellulolytic fungus M. thermophila. These results provide insights that can be applied to industrial fungi for production of biofuels and biochemicals from cellobiose and cellulose.

Dynamic Modelling of Phosphorolytic Cleavage Catalyzed by Pyrimidine-Nucleoside Phosphorylase

Pyrimidine-nucleoside phosphorylases (Py-NPases) have a significant potential to contribute to the economic and ecological production of modified nucleosides. These can be produced via pentose-1-phosphates, an interesting but mostly labile and expensive precursor. Thus far, no dynamic model exists for the production process of pentose-1-phosphates, which involves the equilibrium state of the Py-NPase catalyzed reversible reaction. Previously developed enzymological models are based on the understanding of the structural principles of the enzyme and focus on the description of initial rates only. The model generation is further complicated, as Py-NPases accept two substrates which they convert to two products. To create a well-balanced model from accurate experimental data, we utilized an improved high-throughput spectroscopic assay to monitor reactions over the whole time course until equilibrium was reached. We examined the conversion of deoxythymidine and phosphate to deoxyribose-1-phosphate and thymine by a thermophilic Py-NPase from Geobacillus thermoglucosidasius. The developed process model described the reactant concentrations in excellent agreement with the experimental data. Our model is built from ordinary differential equations and structured in such a way that integration with other models is possible in the future. These could be the kinetics of other enzymes for enzymatic cascade reactions or reactor descriptions to generate integrated process models.

Crystal structure of Escherichia coli purine nucleoside phosphorylase in complex with 7-deazahypoxanthine.

Purine nucleoside phosphorylases (EC 2.4.2.1; PNPs) reversibly catalyze the phosphorolytic cleavage of glycosidic bonds in purine nucleosides to generate ribose 1-phosphate and a free purine base, and are key enzymes in the salvage pathway of purine biosynthesis. They also catalyze the transfer of pentosyl groups between purine bases (the transglycosylation reaction) and are widely used for the synthesis of biologically important analogues of natural nucleosides, including a number of anticancer and antiviral drugs. Potent inhibitors of PNPs are used in chemotherapeutic applications. The detailed study of the binding of purine bases and their derivatives in the active site of PNPs is of particular interest in order to understand the mechanism of enzyme action and for the development of new enzyme inhibitors. Here, it is shown that 7-deazahypoxanthine (7DHX) is a noncompetitive inhibitor of the phosphorolysis of inosine by recombinant Escherichia coli PNP (EcPNP) with an inhibition constant Ki of 0.13 mM. A crystal of EcPNP in complex with 7DHX was obtained in microgravity by the counter-diffusion technique and the three-dimensional structure of the EcPNP-7DHX complex was solved by molecular replacement at 2.51 Å resolution using an X-ray data set collected at the SPring-8 synchrotron-radiation facility, Japan. The crystals belonged to space group P6122, with unit-cell parameters a = b = 120.370, c = 238.971 Å, and contained three subunits of the hexameric enzyme molecule in the asymmetric unit. The 7DHX molecule was located with full occupancy in the active site of each of the three crystallographically independent enzyme subunits. The position of 7DHX overlapped with the positions occupied by purine bases in similar PNP complexes. However, the orientation of the 7DHX molecule differs from those of other bases: it is rotated by ∼180° relative to other bases. The peculiarities of the arrangement of 7DHX in the EcPNP active site are discussed.

Crystal structure of pyrimidine-nucleoside phosphorylase from Bacillus subtilis in complex with imidazole and sulfate.

Pyrimidine-nucleoside phosphorylase catalyzes the phosphorolytic cleavage of thymidine and uridine with equal activity. Investigation of this protein is essential for anticancer drug design. Here, the structure of this protein from Bacillus subtilis in complex with imidazole and sulfate is reported at 1.9 Å resolution, which is an improvement on the previously reported structure at 2.6 Å resolution. The localization and position of imidazole in the nucleoside-binding site reflects the possible binding of ligands that possess an imidazole ring.

Dynamic Optimization of the PyNP/PNP Phosphorolytic Enzymatic Process Using MOSAICmodeling

AbstractPyrimidine and purine nucleoside phosphorylases catalyze the reversible phosphorolytic cleavage and formation of the glycosidic bond of purine and pyrimidine nucleosides, respectively, and are thus, key catalysts for the synthesis of new compounds. The selection of the best combination of enzyme and reaction conditions is not trivial, as each of the two enzymes can perform the reaction in two directions and thus, also competes with the other one for the reaction intermediates. A generic approach to the solution of this problem based on the formulation of a mixed integer dynamic optimization program using MOSAICmodeling is presented.

Structural investigation of the thymidine phosphorylase from Salmonella typhimurium in the unliganded state and its complexes with thymidine and uridine.

Highly specific thymidine phosphorylases catalyze the phosphorolytic cleavage of thymidine, with the help of a phosphate ion, resulting in thymine and 2-deoxy-α-D-ribose 1-phosphate. Thymidine phosphorylases do not catalyze the phosphorolysis of uridine, in contrast to nonspecific pyrimidine nucleoside phosphorylases and uridine phosphorylases. Understanding the mechanism of substrate specificity on the basis of the nucleoside is essential to support rational drug-discovery investigations of new antitumour and anti-infective drugs which are metabolized by thymidine phosphorylases. For this reason, X-ray structures of the thymidine phosphorylase from Salmonella typhimurium were solved and refined: the unliganded structure at 2.05 Å resolution (PDB entry 4xr5), the structure of the complex with thymidine at 2.55 Å resolution (PDB entry 4yek) and that of the complex with uridine at 2.43 Å resolution (PDB entry 4yyy). The various structural features of the enzyme which might be responsible for the specificity for thymidine and not for uridine were identified. The presence of the 2'-hydroxyl group in uridine results in a different position of the uridine furanose moiety compared with that of thymidine. This feature may be the key element of the substrate specificity. The specificity might also be associated with the opening/closure mechanism of the two-domain subunit structure of the enzyme.

Increasing ATP conservation in maltose consuming yeast, a challenge for industrial organic acid production in non-aerated reactors

Anaerobic fermentation processes are economically attractive for industry, as costs for aeration and stirring are greatly reduced. An example of such a process is (bio)ethanol production by Saccharomyces cerevisiae, in which a low but positive ATP gain leads to elevated product yields. However, for other fermentative pathways, such as in an engineered homolactic strain of S. cerevisiae, the net ATP gain for the fermentation of glucose to lactate is null due to the requirement of ATP for product export. Therefore, increasing the conservation of ATP is of major importance for such 'zero-ATP pathways'. One opportunity to increase ATP conservation arises when disaccharides are used as a substrate. In many industrial microorganisms, disaccharides are cleaved by hydrolysis, which results in the dissipation of energy that is available in this cleaving reaction. However, phosphorolytic cleavage could be used to replace the hydrolysis of disaccharides, thereby increasing the ATP yield. In this study, growth of S. cerevisiae on maltose was used as a model. All known native maltose metabolism genes were removed and replaced by a maltose phosphorylase (Lactobacillus sanfranciscensis) and a single overexpressed copy of the native MAL11 maltose transporter. Because maltose phosphorylase cleaves maltose into glucose and β-glucose-1-phosphate, additionally a β-phosphoglucomutase (Lactococcus lactis) was co-expressed in this strain. Anaerobic maltose-limited chemostat cultures showed that replacement of maltose hydrolysis by phosphorolysis increased the biomass yield of the mutant strain by 26% over the wild type strain, demonstrating the potential of phosphorolysis to improve ATP conservation of disaccharide metabolism in industrial microorganisms [1].

Still a Long Way to Fully Understanding the Molecular Mechanism of Escherichia coli Purine Nucleoside Phosphorylase

The results of several decades of studying the catalytic mechanism of Escherichia colt purine nucleoside phosphorylases (PNP) by solution studies and crystal structure determinations are presented. Potentially PNPs can be used for enzyme-activating prodrug gene therapy against solid tumours because of the differences in specificity between human and E. coli PNPs. Biologically active form of PNP from E. coli is a homohexamer that catalyses the phosphorolytic cleavage of the glycosidic bond of purine nucleosides. Two conformations of the active site are possible after substrate(s) binding: open and closed. A series of determined 3D-structures of PNP binary and ternary complexes facilitated the prediction of the main steps in the catalytic mechanism. For their validation the active site mutants: Arg24Ala, Asp204Ala, Arg217Ala, Asp204Asn and double mutant Asp204Ala/Arg217Ala were prepared, The activity tests confirm that catalysis involves protonation of the purine base at position N7 and give better insight into the cooperativity between subunits in this oligomeric enzyme.

New phosphate binding sites in the crystal structure of Escherichia coli purine nucleoside phosphorylase complexed with phosphate and formycin A

Purine nucleoside phosphorylase (PNP) from Escherichia coli is a homohexamer that catalyses the phosphorolytic cleavage of the glycosidic bond of purine nucleosides. The first crystal structure of the ternary complex of this enzyme (with a phosphate ion and formycin A), which is biased by neither the presence of an inhibitor nor sulfate as a precipitant, is presented. The structure reveals, in some active sites, an unexpected and never before observed binding site for phosphate and exhibits a stoichiometry of two phosphate molecules per enzyme subunit. Moreover, in these active sites, the phosphate and nucleoside molecules are found not to be in direct contact. Rather, they are bridged by three water molecules that occupy the “standard” phosphate binding site. Structured summary of protein interactionsPNP and PNPbind by x-ray crystallography (View interaction)

Comparative investigations on thermostable pyrimidine nucleoside phosphorylases from Geobacillus thermoglucosidasius and Thermus thermophilus

The recombinant expression and biocatalytic characterization of two thermostable pyrimidine nucleoside phosphorylases (PyNP), isolated from Geobacillus thermoglucosidasius (Gt) and Thermus thermophilus (Tt) is described. Both enzymes are highly thermostable (half life of GtPyNP is 1.6 h at 70 °C, half life of TtPyNP is >24 h at 80 °C). Kinetic parameters for the phosphorolysis of natural substrates were determined for GtPyNP at 60 °C (Km for uridine 2.3 mM, Km for thymidine 1.3 mM) and TtPyNP at 80 °C (Km for uridine 0.15 mM, Km for thymidine 0.43 mM). The kcat values for uridine are almost identical for both enzymes (ca. 277 s−1), while the kcat value for thymidine is about 8 times higher for TtPyNP than for GtPyNP (679 s−1 vs. 83 s−1). Both enzymes were tested towards the ability to catalyze the phosphorolytic cleavage of 2′-fluorosubstituted pyrimidine nucleosides – a prerequisite for the efficient synthesis of a number of relevant purine nucleoside analogues. GtPyNP showed poor activity towards 2′-deoxy-2′-fluorouridine (dUrd2′F; 0.4% substrate conversion after 30 min), and the phosphorolysis of the epimeric counterpart 1-(2-deoxy-2-fluoro-β-d-arabinofuranosyl)uracil (dUrd2′F) could not be detected at all. By contrast, TtPyNP showed dramatically higher conversion rates (15.6% and 1.6% conversion in 30 min of both substrates, respectively). The amount of converted pyrimidine nucleosides increased significantly with time. After 17 h 65% of dUrd2′F and 46% of dUrd2′F was phosphorolytically cleaved. Our results demonstrate the potential of TtPyNP as a biocatalyst in transglycosylation reactions aiming at the production of 2′-fluorosubstituted purine nucleosides that are highly bioactive but hardly accessible by chemical methods.

Structural basis of the substrate specificity of Bacillus cereus adenosine phosphorylase.

Purine nucleoside phosphorylases catalyze the phosphorolytic cleavage of the glycosidic bond of purine (2'-deoxy)nucleosides, generating the corresponding free base and (2'-deoxy)-ribose 1-phosphate. Two classes of PNPs have been identified: homotrimers specific for 6-oxopurines and homohexamers that accept both 6-oxopurines and 6-aminopurines. Bacillus cereus adenosine phosphorylase (AdoP) is a hexameric PNP; however, it is highly specific for 6-aminopurines. To investigate the structural basis for the unique substrate specificity of AdoP, the active-site mutant D204N was prepared and kinetically characterized and the structures of the wild-type protein and the D204N mutant complexed with adenosine and sulfate or with inosine and sulfate were determined at high resolution (1.2-1.4 Å). AdoP interacts directly with the preferred substrate through a hydrogen-bond donation from the catalytically important residue Asp204 to N7 of the purine base. Comparison with Escherichia coli PNP revealed a more optimal orientation of Asp204 towards N7 of adenosine and a more closed active site. When inosine is bound, two water molecules are interposed between Asp204 and the N7 and O6 atoms of the nucleoside, thus allowing the enzyme to find alternative but less efficient ways to stabilize the transition state. The mutation of Asp204 to asparagine led to a significant decrease in catalytic efficiency for adenosine without affecting the efficiency of inosine cleavage.

A corrected space group forSulfolobus sulfataricus5′-deoxy-5′-methylthioadenosine phosphorylase II

5'-deoxy-5'-methylthioadenosine phosphorylase (MTAP) catalyzes the phosphorolytic cleavage of 5'-deoxy-5'-methylthioadenosine (MTA), a byproduct of polyamine biosynthesis. The Sulfolobus sulfataricus genome encodes two MTAPs. SsMTAP I has broad substrate specifity, accepting guanosine, inosine, adenosine and MTA, while SsMTAP II is specific for MTA. SsMTAP I forms a donut-shaped hexamer, while SsMTAP II is a hexamer formed from trimers packed face to face. The structure of SsMTAP II was originally determined in space group P1 (PDB entry 2a8y) and showed R32 pseudosymmetry. Post-analysis using phenix.xtriage showed that the correct space group is C2. Here, the structure refined in space group C2 is reported and the factors that initially led to the incorrect space-group assignment are discussed.

Increasing free-energy (ATP) conservation in maltose-grown Saccharomyces cerevisiae by expression of a heterologous maltose phosphorylase

Increasing free-energy conservation from the conversion of substrate into product is crucial for further development of many biotechnological processes. In theory, replacing the hydrolysis of disaccharides by a phosphorolytic cleavage reaction provides an opportunity to increase the ATP yield on the disaccharide. To test this concept, we first deleted the native maltose metabolism genes in Saccharomyces cerevisiae. The knockout strain showed no maltose-transport activity and a very low residual maltase activity (0.03 μmol mg protein −1 min −1). Expression of a maltose phosphorylase gene from Lactobacillus sanfranciscensis and the MAL11 maltose-transporter gene resulted in relatively slow growth ( μ aerobic 0.09±0.03 h −1). Co-expression of Lactococcus lactis β-phosphoglucomutase accelerated maltose utilization via this route ( μ aerobic 0.21±0.01 h −1, μ anaerobic 0.10±0.00 h −1). Replacing maltose hydrolysis with phosphorolysis increased the anaerobic biomass yield on maltose in anaerobic maltose-limited chemostat cultures by 26%, thus demonstrating the potential of phosphorolysis to improve the free-energy conservation of disaccharide metabolism in industrial microorganisms.

Establishment of an alternative phosphoketolase-dependent pathway for fructose catabolism in Ralstonia eutropha H16

The β-proteobacterium Ralstonia eutropha H16 utilizes fructose and gluconate as carbon sources for heterotrophic growth exclusively via the Entner-Doudoroff pathway with its key enzyme 2-keto-3-deoxy-6-phosphogluconate (KDPG) aldolase. By deletion of the responsible gene eda, we constructed a KDPG aldolase-negative strain, which is disabled to supply pyruvate for energy metabolism from fructose or gluconate as sole carbon sources. To restore growth on fructose, an alternative pathway, similar to the fructose-6-phosphate shunt of heterofermentative bifidobacteria, was established. For this, the xfp gene from Bifidobacterium animalis, coding for a bifunctional xylulose-5-phosphate/fructose-6-phosphate phosphoketolase (Xfp; Meile et al. in J Bacteriol 183:2929-2936, 2001), was expressed in R. eutropha H16 PHB(-)4 Δeda. This Xfp catalyzes the phosphorolytic cleavage of fructose 6-phosphate to erythrose 4-phosphate and acetylphosphate as well as of xylulose 5-phosphate to glyceralaldehyde 3-phosphate and acetylphosphate. The recombinant strain showed phosphoketolase (PKT) activity on either substrate, and was able to use fructose as sole carbon source for growth, because PKT is the only enzyme that is missing in R. eutropha H16 to establish the artificial fructose-6-phosphate shunt. The Xfp-expressing strain R. eutropha H16 PHB(-)4 Δeda (pBBR1MCS-3::xfp) should be applicable for a novel variant of a plasmid addiction system to stably maintain episomally encoded genetic information during fermentative production processes. Plasmid addiction systems are often used to ensure plasmid stability in many biotechnology relevant microorganisms and processes without the need to apply external selection pressure, like the addition of antibiotics. By episomal expression of xfp in a R. eutropha H16 mutant lacking KDPG aldolase activity and cultivation in mineral salt medium with fructose as sole carbon source, the growth of this bacterium was addicted to the constructed xfp harboring plasmid. This novel selection principle extends the applicability of R. eutropha H16 as production platform in biotechnological processes.

Crystal Structures of Phosphoketolase: THIAMINE DIPHOSPHATE-DEPENDENT DEHYDRATION MECHANISM*

Thiamine diphosphate (ThDP)-dependent enzymes are ubiquitously present in all organisms and catalyze essential reactions in various metabolic pathways. ThDP-dependent phosphoketolase plays key roles in the central metabolism of heterofermentative bacteria and in the pentose catabolism of various microbes. In particular, bifidobacteria, representatives of beneficial commensal bacteria, have an effective glycolytic pathway called bifid shunt in which 2.5 mol of ATP are produced per glucose. Phosphoketolase catalyzes two steps in the bifid shunt because of its dual-substrate specificity; they are phosphorolytic cleavage of fructose 6-phosphate or xylulose 5-phosphate to produce aldose phosphate, acetyl phosphate, and H(2)O. The phosphoketolase reaction is different from other well studied ThDP-dependent enzymes because it involves a dehydration step. Although phosphoketolase was discovered more than 50 years ago, its three-dimensional structure remains unclear. In this study we report the crystal structures of xylulose 5-phosphate/fructose 6-phosphate phosphoketolase from Bifidobacterium breve. The structures of the two intermediates before and after dehydration (α,β-dihydroxyethyl ThDP and 2-acetyl-ThDP) and complex with inorganic phosphate give an insight into the mechanism of each step of the enzymatic reaction.

Phosphorolytic cleavage of sucrose by sucrose-grown ruminal bacterium Pseudobutyrivibrio ruminis strain k3

Rumen bacterium Pseudobutyrivibrio ruminis strain k3 utilized over 90% sucrose added to the growth medium as a sole carbon source. Zymographic studies of the bacterial cell extract revealed the presence of a single enzyme involved in sucrose digestion. Thin layer chromatography showed fructose and glucose-1-phosphate (Glc1P) as end products of the digestion of sucrose by identified enzyme. The activity of the enzyme depended on the presence of inorganic phosphate and was the highest at the concentration of phosphate 56 mmol/L. The enzyme was identified as the sucrose phosphorylase (EC 2.4.1.7) of molar mass approximately 54 kDa and maximum activity at pH 6.0 and 45 degrees C. The calculated Michaelis constant (Km) for Glc1P formation and release of fructose by partially purified enzyme were 4.4 and 8.56 mmol/L while the maximum velocities of the reaction (Vlim) were 1.19 and 0.64 micromol/L per mg protein per min, respectively.