Substrate specificity of E. coli uridine phosphorylase. Further evidences of high-syn conformation of the substrate in uridine phosphorolysis

Twenty five uridine analogues have been tested and compared with uridine with respect to their potency to bind to E. coli uridine phosphorylase. The kinetic constants of the phosphorolysis reaction of uridine derivatives modified at 2′-, 3′- and 5′-positions of the sugar moiety and 2-, 4-, 5- and 6-positions of the heterocyclic base were determined. The absence of the 2′- or 5′-hydroxyl group is not crucial for the successful binding and phosphorolysis. On the other hand, the absence of both the 2′- and 5′-hydroxyl groups leads to the loss of substrate binding to the enzyme. The same effect was observed when the 3′-hydroxyl group is absent, thus underlining the key role of this group. Our data shed some light on the mechanism of ribo- and 2′-deoxyribonucleoside discrimination by E. coli uridine phosphorylase and E. coli thymidine phosphorylase. A comparison of the kinetic results obtained in the present study with the available X-ray structures and analysis of hydrogen bonding in the enzyme-substrate complex demonstrates that uridine adopts an unusual high-syn conformation in the active site of uridine phosphorylase.     

Substrate Specificity of Thymidine Phosphorylase of E. Coli: Role of Hydroxyl Groups

Substrate specificity of E. coli thymidine phosphorylase to pyrimidine nucleoside modified at 5 ′-, 3 ′-, and 2 ′-positions of sugar moiety has been studied. Equilibrium (K_eq) and kinetics constants of phosphorolysis reaction of nucleosides were measured. The most important hydrogen bonds in enzyme-substrate complex have been determined.     

Stabilization of thymidine phosphorylase from Escherichia coli by immobilization and post immobilization techniques

Homodimeric thymidine phosphorylase from Escherichia coli (TP, E.C. 2.4.2.4) was immobilized on solid support with the aim to have a stable and recyclable biocatalyst for nucleoside synthesis. Immobilization by ionic adsorption on amine-functionalized agarose and Sepabeads^® resulted in a very high activity recovery (>85%). To prevent undesirable leakage of immobilized enzyme away from the support, the ionic preparations were cross-linked with aldehyde dextran (MW 20 kDa) and the influence of the dextran oxidation degree on the resulting biocatalyst activity was evaluated. Although in all cases the percentage of expressed activity after immobilization drastically decreased (≤25%), this procedure allowed to obtain an active catalyst which resulted up to 6-fold and 3-fold more stable than the soluble (non immobilized) enzyme and the just adsorbed (non cross-linked) counterpart, respectively, at pH 10 and 37 °C. No release of the enzyme from the support could be observed. Covalent immobilization on aldehyde or epoxy supports was generally detrimental for enzyme activity. Optimal TP preparation, achieved by immobilization onto Sepabeads^® coated with polyethyleneimine and cross-linked, was successfully used for the one-pot synthesis of 5-fluoro-2′-deoxyuridine starting from 2′-deoxyuridine or thymidine (20 mM) and 5-fluorouracil (10 mM). In both cases, the reaction proceeded at the same rate (3 μmol min⁻¹) affording 62% conversion in 1 h.     

Engineering the acceptor specificity of trehalose phosphorylase for the production of trehalose analogs

Trehalose (α‐D ‐glucopyranosyl‐(1,1)‐α‐D ‐glucopyranoside) is widely used in the food industry, thanks to its protective effect against freezing and dehydration. Analogs of trehalose have the additional benefit that they are not digested and thus do not contribute to our caloric intake. Such trehalose analogs can be produced with the enzyme trehalose phosphorylase, when it is applied in the reverse, synthetic mode. Despite the enzyme's broad acceptor specificity, its catalytic efficiency for alternative monosaccharides is much lower than for glucose. For galactose, this difference is shown here to be caused by a lower K_m whereas the k_cat for both substrates is equal. Consequently, increasing the affinity was attempted by enzyme engineering of the trehalose phosphorylase from Thermoanaerobacter brockii, using both semirational and random mutagenesis. While a semirational approach proved unsuccessful, high‐throughput screening of an error‐prone PCR library resulted in the discovery of three beneficial mutations that lowered K_m two‐ to three‐fold. In addition, it was found that mutation of these positions also leads to an improved catalytic efficiency for mannose and fructose, suggesting their involvement in acceptor promiscuity. Combining the beneficial mutations did not further improve the affinity, and even resulted in a decreased catalytic activity and thermostability. Therefore, enzyme variant R448S is proposed as new biocatalyst for the industrial production of lactotrehalose (α‐D ‐glucopyranosyl‐(1,1)‐α‐D ‐galactopyranoside). © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2012     

Construction of cellobiose phosphorylase variants with broadened acceptor specificity towards anomerically substituted glucosides

The general application of glycoside phosphorylases such as cellobiose phosphorylase (CP) for glycoside synthesis is hindered by their relatively narrow substrate specificity. We have previously reported on the creation of Cellulomonas uda CP enzyme variants with either modified donor or acceptor specificity. Remarkably, in this study it was found that the donor mutant also displays broadened acceptor specificity towards several β‐glucosides. Triple mutants containing donor (T508I/N667A) as well as acceptor mutations (E649C or E649G) also display a broader acceptor specificity than any of the parent enzymes. Moreover, further broadening of the acceptor specificity has been achieved by site‐saturation mutagenesis of residues near the active site entrance. The best enzyme variant contains the additional N156D and N163D mutations and is active towards various alkyl β‐glucosides, methyl α‐glucoside and cellobiose. In comparison with the wild‐type C. uda CP enzyme, which cannot accept anomerically substituted glucosides at all, the obtained increase in substrate specificity is significant. The described CP enzyme variants should be useful for the synthesis of cellobiosides and other glycosides with prebiotic and pharmaceutical properties. Biotechnol. Bioeng. 2010;107: 413–420. © 2010 Wiley Periodicals, Inc.     

Enzymatic activities of uridine and thymidine phosphorylase in normal and cancerous uterine cervical tissues

In this study, the preliminary analyses were conducted of enzymatic activities of uridine phosphorylase (UP) and thymidine phosphorylase (TP) in normal tissues and cancer tissues of the uterine cervix. The study was performed on 27 patients of cervical cancer, treated first in our hospital. Normal cervical tissues obtained from 15 patients undergoing hysterectomy for benign diseases were used as controls. The supernatant of the homogenated cervical tissues and the stroma (5-FU and ribose-1-P or deoxyribose-1-P) were analyzed by high performance liquid chromatography, and then the UP and TP activities calculated. TP activity was significantly greater than UP activity (P < 0.0001). Both UP and TP showed significantly greater activity in cancer tissues than in normal tissues (P < 0.0001). In the TP activity of the cancer tissues, there was no significant difference among the histological types, while the TP activity tended to be significantly higher in the cases with lymph node metastasis. These results showed that the TP-mediated route seemed important as the 5FU metabolic pathway in the uterine cervical tissues, and TP enzymatic activity might be associated with lymph node metastasis.     

纤维素酶的底物专一性

天然纤维素的有效酶解取决于外切葡聚糖纤维二糖水解酶（ＣＢＨ）和内切葡聚糖水解酶（ＥＧ）的协同作用。ＥＧ随机水解纤维素无定形区分子链内的β－１,４－糖苷键;ＣＢＨ则由分子链的还原性末端水解出纤维二糖。这种底物专一性差别的原因在于ＣＢＨ呈“桶状”的活性部痊表面存在２个“ｌｏｏｐ”结构,只能容许纤维素分子链的末端伸入到活性裂隙中。ＥＧ无“ｌｏｏｐ”结构在存在,对底物是充分可及的。ＥＧ催化结构域中底物结合     

Rational design of bis-indolylmethane-oxadiazole hybrids as inhibitors of thymidine phosphorylase

Inhibition of Thymidine phosphorylase (TP) is continuously studied for the design and development of new drugs for the treatment of neoplastic diseases. As a part of our effort to identify TP inhibitors, we performed a structure-based virtual screening (SBVS) of our compound collection. Based on the insights gained from structures of virtual screening hits, a scaffold was designed using 1,3,4-oxadiazole as the basic structural feature and SAR studies were carried out for the optimization of this scaffold. Twenty-five novel bis-indole linked 1,3,4-oxadiazoles (7–31) were designed, synthesized and tested in vitro against E. coli TP (EcTP). Compound 7 emerged as potent TP inhibitor with an IC₅₀ value of 3.50?±?0.01?μM. Docking studies were carried out using GOLD software on thymidine phosphorylase from human (hTP) and E. coli (EcTP). Various hydrogen bonding, hydrophobic interactions, and π-π stacking were observed between designed molecules and the active site amino acid residues of the studied enzymes.     

Substrate specificity of potato lipoxygenase

The substrate specificity of potato lipoxygenase was examined using a partially purified enzyme preparation from tubers of a potato variety with low lipolytic acyl hydrolase activity. Potato lipoxygenase is fully active only on free linoleic acid or linolenic acid, and only acts directly on more complex glyceride moieties in the absence of any significant endogenous lipolytic acyl hydrolase activity.     

Crystal structures of Escherichia coli uridine phosphorylase in two native and three complexed forms reveal basis of substrate specificity, induced conformational changes and influence of potassium

Uridine phosphorylase (UP) is a key enzyme in the pyrimidine salvage pathway that catalyses the reversible phosphorolysis of uridine to uracil and ribose 1-phosphate. Inhibiting liver UP in humans raises blood uridine levels and produces a protective effect ("uridine rescue") against the toxicity of the chemotherapeutic agent 5-fluorouracil without reducing its antitumour activity. We have investigated UP-substrate interactions by determining the crystal structures of native Escherichia coli UP (two forms), and complexes with 5-fluorouracil/ribose 1-phosphate, 2-deoxyuridine/phosphate and thymidine/phosphate. These hexameric structures confirm the overall structural similarity of UP to E.coli purine nucleoside phosphorylase (PNP) whereby, in the presence of substrate, each displays a closed conformation resulting from a concerted movement that closes the active site cleft. However, in contrast to PNP where helix segmentation is the major conformational change between the open and closed forms, in UP more extensive changes are observed. In particular a swinging movement of a flap region consisting of residues 224-234 seals the active site. This overall change in conformation results in compression of the active site cleft. Gln166 and Arg168, part of an inserted segment not seen in PNP, are key residues in the uracil binding pocket and together with a tightly bound water molecule are seen to be involved in the substrate specificity of UP. Enzyme activity shows a twofold dependence on potassium ion concentration. The presence of a potassium ion at the monomer/monomer interface induces some local rearrangement, which results in dimer stabilisation. The conservation of key residues and interactions with substrate in the phosphate and ribose binding pockets suggest that ribooxocarbenium ion formation during catalysis of UP may be similar to that proposed for E.coli PNP.     

Enzymatic catalysis in crystals of Escherichia coli maltodextrin phosphorylase

The bacterial enzyme maltodextrin phosphorylase (MalP) catalyses the phosphorolysis of an alpha-1,4-glycosidic bond in maltodextrins, removing the non-reducing glucosyl residues of linear oligosaccharides as glucose-1-phosphate (Glc1P). In contrast to the well-studied muscle glycogen phosphorylase (GP), MalP exhibits no allosteric properties and has a higher affinity for linear oligosaccharides than GP. We have used MalP as a model system to study catalysis in the crystal in the direction of maltodextrin synthesis. The 2.0A crystal structure of the MalP/Glc1P binary complex shows that the Glc1P substrate adopts a conformation seen previously with both inactive and active forms of mammalian GP, with the phosphate group not in close contact with the 5'-phosphate group of the essential pyridoxal phosphate (PLP) cofactor. In the active MalP enzyme, the residue Arg569 stabilizes the negative-charged Glc1P, whereas in the inactive form of GP this key residue is held away from the catalytic site by loop 280s and an allosteric transition of the mammalian enzyme is required for activation. The comparison between MalP structures shows that His377, through a hydrogen bond with the 6-hydroxyl group of Glc1P substrate, triggers a conformational change of the 380s loop. This mobile region folds over the catalytic site and contributes to the specific recognition of the oligosaccharide and to the synergism between substrates in promoting the formation of the MalP ternary complex. The structures solved after the diffusion of oligosaccharides (either maltotetraose, G4 or maltopentaose, G5) into MalP/Glc1P crystals show the formation of phosphate and elongation of the oligosaccharide chain. These structures, refined at 1.8A and at 2.2A, confirm that only when an oligosaccharide is bound to the catalytic site will Glc1P bend its phosphate group down so it can contact the PLP 5' phosphate group and promote catalysis. The relatively large oligosaccharide substrates can diffuse quickly into the MalP/Glc1P crystals and the enzymatic reaction can occur without significant crystal damage. These structures obtained before and after catalysis have been used as frames of a molecular movie. This movie reveals the relative positions of substrates in the catalytic channel and shows a minimal movement of the protein, involving mainly Arg569, which tracks the substrate phosphate group.     

Substrate specificity of african oil palm tree peroxidase

The optimal conditions for catalysis by the peroxidase isolated from leaves of African oil palm tree (AOPTP) have been determined. The pH optimum for oxidation of the majority of substrates studied in the presence of AOPTP is in the interval of 4.5-5.5. A feature of AOPTP is low pH value (3.0) at which the peroxidase shows its maximal activity toward 2,2"-azino-bis(3-ethylbenz-thiazoline-6-sulfonic acid) (ABTS). Increasing the buffer concentration changes the AOPTP activity, the degree of the effect depending upon the chemical structure of the substrate. Under optimal conditions of AOPTP catalysis, the values of second order rate constant characterizing efficiency of enzymatic oxidation of substrates have been calculated. It was shown that among 12 peroxidase substrates studied, ABTS and ferulic acid are the best substrates for AOPTP. The results show that substrate specificities of AOPTP and royal palm tree peroxidase are similar, but different from substrate specificity of other plant peroxidases.     

Identification of 1,2,4-triazoles as new thymidine phosphorylase inhibitors: Future anti-tumor drugs

Thymidine phosphorylase (TP) is over expressed in several solid tumors and its inhibition can offer unique target suitable for drug discovery in cancer. A series of 1,2,4-triazoles 3a–3l has been synthesized in good yields and subsequently inhibitory potential of synthesized triazoles 3a–3l against thymidine phosphorylase enzyme was evaluated. Out of these twelve analogs five analogues 3b, 3c, 3f, 3l and 3l exhibited a good inhibitory potential against thymidine phosphorylase. Inhibitory potential in term of IC₅₀ values were found in the range of 61.98 ± 0.43 to 273.43 ± 0.96 μM and 7-Deazaxanthine was taken as a standard inhibitor with IC₅₀ = 38.68 ± 4.42 μM. Encouraged by these results, more analogues 1,2,4-triazole-3-mercaptocarboxylic acids 4a–4g were synthesized and their inhibitory potential against thymidine phosphorylase was evaluated. In this series, six analogues 4b–4g exhibited a good inhibitory potential in the range of 43.86 ± 1.11–163.43 ± 2.03 μM. Angiogenic response of 1,2,4-triazole acid 4d was estimated using the chick chorionic allantoic membrane (CAM) assay. In the light of these findings, structure activity relationship and molecular docking studies of selected triazoles to determine the key binding interactions was discussed. Docking studies demonstrate that synthesized analogues interacted with active site residues of thymidine phosphorylase enzyme through π-π stacking, thiolate and hydrogen bonding interactions.     

嘌呤核苷磷酸化酶基因的克隆及原核表达载体的构建

通过PCR方法从产气肠杆菌、胡萝卜软腐欧文氏菌、大肠杆菌扩增嘌呤核苷磷酸化酶(PNPase)基因,然后将扩增的约720bp的基因片段克隆到pET-28b表达载体上,构建重组PNPase的表达载体。核苷酸及推导的氨基酸序列分析表明,该基因在三个菌株之间有很高的同源性。SDS-PAGE电泳结果显示出明显的特异性蛋白质条带,其分子量约为29.8kDa.该载体的构建为进一步研究核苷及其类似物的生物合成奠定基础。     

Substrate specificity of polyphenol oxidase

Abstract

The ubiquitous type-3 copper enzyme polyphenol oxidase (PPO) has found itself the subject of profound inhibitor research due to its role in fruit and vegetable browning and mammalian pigmentation. The enzyme itself has also been applied in the fields of bioremediation, biocatalysis and biosensing. However, the nature of PPO substrate specificity has remained elusive despite years of study. Numerous theories have been proposed to account for the difference in tyrosinase and catechol oxidase activity. The “blocker residue” theory suggests that bulky residues near the active site cover CuA, preventing monophenol coordination. The “second shell” theory suggests that residues distant (～8?Å) from the active site, guide and position substrates within the active site based on their properties e.g., hydrophobic, electrostatic. It is also hypothesized that binding specificity is related to oxidation mechanisms of the catalytic cycle, conferred by coordination of a conserved water molecule by other conserved residues. In this review, we highlight recent developments in the structural and mechanistic studies of PPOs and consolidate key concepts in our understanding toward the substrate specificity of PPOs.     

代谢工程方法改造大肠杆菌生产胸苷

胸苷是抗艾滋病药物司他夫定(3′-脱氧-2′,3′-双脱氢胸苷)和叠氮胸苷的重要前体物质。应用代谢工程方法对大肠杆菌Escherichia coli BL21(DE3)生物合成胸苷进行了研究。通过敲除E.coli BL21嘧啶回补途径的deo A、tdk和udp三个基因,BS03工程菌株能够积累21.6 mg/L胸苷。为了增加合成胸苷前体物核糖-5-磷酸和NADPH的供给,进一步敲除pgi和pyr L使工程菌BS05胸苷的产量提高到90.5 mg/L。而通过过表达胸苷合成途径的ush A、thy A、dut、ndk、nrd A和nrd B六个基因,菌株BS08胸苷的产量能达到272 mg/L。通过分批补料发酵,BS08最终可以积累1 248.8 mg/L的胸苷。本研究结果表明经过代谢工程改造的E.coli BL21具有良好的胸苷合成能力和应用潜力。     

Substrate specificity and kinetic mechanism of purine nucleoside phosphorylase from Mycobacterium tuberculosis

Purine nucleoside phosphorylase from Mycobacterium tuberculosis (MtPNP) is numbered among targets for persistence of the causative agent of tuberculosis. Here, it is shown that MtPNP is more specific to natural 6-oxopurine nucleosides and synthetic compounds, and does not catalyze the phosphorolysis of adenosine. Initial velocity, product inhibition and equilibrium binding data suggest that MtPNP catalyzes 2′-deoxyguanosine (2dGuo) phosphorolysis by a steady-state ordered bi bi kinetic mechanism, in which inorganic phosphate (P_i) binds first followed by 2dGuo, and ribose 1-phosphate dissociates first followed by guanine. pH-rate profiles indicated a general acid as being essential for both catalysis and 2dGuo binding, and that deprotonation of a group abolishes P_i binding. Proton inventory and solvent deuterium isotope effects indicate that a single solvent proton transfer makes a modest contribution to the rate-limiting step. Pre-steady-state kinetic data indicate that product release appears to contribute to the rate-limiting step for MtPNP-catalyzed reaction.     

Thymidine esters as substrate analogue inhibitors of angiogenic enzyme thymidine phosphorylase in vitro

Thymidine phosphorylase (TP) catalyzes the cleavage of thymidine into thymine and 2-deoxy-α-d-ribose-1-phosphate. Elevated activity of TP prevents apoptosis, and induces angiogenesis which ultimately leads to tumor growth and metastasis. Critical role of TP in cancer progression makes it a valid target in anti-cancer research. Discovery of small molecules as TP inhibitors is vigorously pursued in cancer therapy. In the present study, we functionalized thymidine as benzoyl ester to synthesize compounds 3–16. In vitro evaluation of thymidine esters for their thymidine phosphorylase inhibition activity was subsequently carried out. Compounds 4, 10, 14, and 15 showed good activities with lower IC₅₀ values than the standard, 7-deazaxanthine (IC₅₀ = 41.0 ± 1.63 μM). Among them, compound 14 showed five folds higher activity (IC₅₀ = 7.5 ± 0.8 μM), while 4 (IC₅₀ = 18.5 ± 1.0 μM) and 10 (IC₅₀ = 18.8 ± 1.2 μM) showed two folds higher activity than the standard. Compound 15 showed slightly better activity (IC₅₀ = 33.3 ± 1.5 μM) to the standard. Potent compounds were further subjected to kinetic and molecular docking studies to identify their mode of inhibition, and to study their interactions with the protein at atomic level, respectively. All active compounds were non-cytotoxic to mouse fibroblast 3T3 cell line. These results identify thymidine esters as substrate analogue (substrate-like) inhibitors of angiogenic enzyme thymidine phosphorylase for further studies.     

Substrate Shape Preference of Escherichia coli Ribonuclease P Ribozyme and Holo Enzyme Using Bottom-Half Part-Shifting Variants of Pre-tRNA

We showed previously that the bacterial ribonuclease P (RNase P) ribozyme has substrate shape preference depending on the concentrations of catalytically important magnesium ions. The ribozyme discriminates a canonical cloverleaf precursor tRNA from a hairpin RNA with a CCA-tag sequence at low concentrations of magnesium ions. By detailed analysis of the shape preference using the bottom-half part-shifting variants of a tRNA precursor, we showed that the RNAs in a T-shape structure can be substrates for the ribozyme reactions even at low concentrations of magnesium ions, and that the RNA in a natural L-shape is the best substrate for both the ribozyme and the holo enzyme. The results also showed that the position of the bottom-half part did not affect the cleavage site selection of a substrate by the enzyme. Our results are the first kinetic evidence to show the importance of the bottom-half part of tRNA molecule, and our result also showed that the holo enzyme can discriminate substrate shape as well as the ribozyme at low concentrations of metal ions.