Crystal structure of calf spleen purine nucleoside phosphorylase complexed to a novel purine analogue

The combined use of a rapid virtual screen of a small fragment library together with a single point enzyme assay has been used for the discovery of novel PNP inhibitors. The availability of readily soakable crystals of bovine PNP has allowed the approach to be experimentally validated by determining the crystal structure of one of the inhibitor-PNP complexes. Comparison of the experimentally determined binding mode with that predicted by the virtual screening shows them to be similar. This represents a starting point for the growth of the ligand into a higher affinity inhibitor.     

Docking and small angle X-ray scattering studies of purine nucleoside phosphorylase

Docking simulations have been used to assess protein complexes with some success. Small angle X-ray scattering (SAXS) is a well-established technique to investigate protein spatial configuration. This work describes the integration of geometric docking with SAXS to investigate the quaternary structure of recombinant human purine nucleoside phosphorylase (PNP). This enzyme catalyzes the reversible phosphorolysis of N-ribosidic bonds of purine nucleosides and deoxynucleosides. A genetic deficiency due to mutations in the gene encoding for PNP causes gradual decrease in T-cell immunity. Inappropriate activation of T-cells has been implicated in several clinically relevant human conditions such as transplant rejection, rheumatoid arthritis, lupus, and T-cell lymphomas. PNP is therefore a target for inhibitor development aiming at T-cell immune response modulation and has been submitted to extensive structure-based drug design. The present analysis confirms the trimeric structure observed in the crystal. The potential application of the present procedure to other systems is discussed.     

Overexpression, purification and characterization of functional calf purine nucleoside phosphorylase (PNP)

Calf PNP is a ubiquitous enzyme of the salvage metabolic pathway. The procedure for this enzyme production in large quantities is described. The coding sequence of bovine PNP was amplified from the calf spleen cDNA library and was inserted into an expression vector pET28a(+). The construct was transformed into Escherichia coli BL21(DE3) strain. The protein expression efficiencies in the presence and the absence of IPTG were compared. It was found that IPTG is not necessary for obtaining a large quantity of recombinant calf PNP: 35 mg from 1 L cell culture. The enzyme was purified to 92% homogeneity by a two-step procedure consisting of gel filtration and ion exchange chromatography. The purity of recombinant enzyme is sufficient to form well diffracting single crystals.The basic kinetic parameters of recombinant PNP were determined and compared with the parameters of commercially available PNP from calf spleen. The specific activity in 50 mM phosphate buffer with inosine as a variable substrate (30.7 μmol min⁻¹ mg⁻¹) and other kinetic parameters: Michaelis constants, maximal velocities, dissociation and inhibition constants, determined for several typical PNP ligands, are similar to the values published previously for non-recombinant calf spleen PNP. As expected for mammalian PNP, recombinant calf PNP was found to have no substrate activity vs adenosine. The overexpression and purification method of the recombinant calf PNP provides significant amounts of the enzyme, which can successfully replace the non-recombinant PNP.     

Guanosine triphosphate catabolism in purine nucleoside phosphorylase deficient human B lymphoblastoid cells

GTP catabolism induced by sodium azide or deoxyglucose was studied in purine nucleoside phosphorylase (PNP) deficient human B lymphoblastoid cells. In PNP deficient cells, as in control cells, guanylate was both dephosphorylated and deaminated but dephosphorylation was the major pathway. Only nucleosides were excreted during GTP catabolism by PNP deficient cells and the main product was guanosine. The level of nucleoside excretion was largely affected by intracellular orthophosphate (P_i) level. In contrast, normal cells excreted nucleosides only at low P_i level while at high P_i levels, purine bases (guanine and hypoxanthine) were exclusively excreted. PNP deficiency had no effect on the extent of GMP deamination.     

Kinetics and crystal structure of human purine nucleoside phosphorylase in complex with 7-methyl-6-thio-guanosine

Purine nucleoside phosphorylase (PNP) catalyzes the reversible phosphorolysis of nucleosides and deoxynucleosides, generating ribose 1-phosphate and the purine base, which is an important step of purine catabolism pathway. The lack of such an activity in humans, owing to a genetic disorder, causes T-cell impairment, and drugs that inhibit this enzyme may have the potential of being utilized as modulators of the immunological system to treat leukemia, autoimmune diseases, and rejection in organ transplantation. Here, we describe kinetics and crystal structure of human PNP in complex with 7-methyl-6-thio-guanosine, a synthetic substrate, which is largely used in activity assays. Analysis of the structure identifies different protein conformational changes upon ligand binding, and comparison of kinetic and structural data permits an understanding of the effects of atomic substitution on key positions of the synthetic substrate and their consequences to enzyme binding and catalysis. Such knowledge may be helpful in designing new PNP inhibitors.     

Detection of l-alanylaminopeptidase activity in microorganisms using fluorogenic self-immolative enzyme substrates

A series of fluorogenic enzymatic substrates that incorporate a self-immolative spacer were synthesised for the purpose of identifying l-alanylaminopeptidase activity in microorganisms in agar media. These substrates resulted in the generation of fluorescent microorganism colonies with Gram-negative microorganisms.     

The evolution of catalytic residues and enzyme mechanism within the bacterial nucleoside phosphorylase superfamily 1

Nucleoside phosphorylases are essential for the salvage and catabolism of nucleotides in bacteria and other organisms, and members of this enzyme superfamily have been of interest for the development of antimicrobial and cancer therapies. The nucleotide phosphorylase superfamily 1 encompasses a number of different enzymes which share a general superfold and catalytic mechanism, while they differ in the nature of the nucleophiles used and in the nature of characteristic active site residues. Recently, one subfamily, the uridine phosphorylases, has been subdivided into two types which differ with respect to the mechanism of transition state stabilization, as dictated by differences in critical amino acid residues. Little is known about the phylogenetic distribution and relationship of the two different types, as well as the relationship to other NP-1 superfamily members. Here comparative genomic analysis illustrates that UP-1s and UP-2s fall into monophyletic groups and are biased with respect to species representation. UP-1 evolved in Gram negative bacteria, while Gram positive species tend to predominantly contain UP-2. PNP (a sister clade to all UPs) contains both Gram positive and Gram negative species. The findings imply that the nucleoside phosphorylase superfamily 1 evolved through a series of three important duplications, leading to the separate, monophyletic enzyme families, coupled to individual lateral transfer events. Extensive horizontal transfer explains the occurrence of unexpected uridine phosphorylases in some genomes. This study provides a basis for understanding the evolution of uridine and purine nucleoside phosphorylases with respect to DNA/RNA metabolism and with potential utility in the design of antimicrobial and anti-tumor drugs.     

Characterization of the adenine nucleoside specific phosphorylase of Bacillus cereus

Adenosine phosphorylase, a purine nucleoside phosphorylase endowed with high specificity for adenine nucleosides, was purified 117-fold from vegetative forms of Bacillus cereus. The purification procedure included ammonium sulphate fractionation, pH 4 treatment, ion exchange chromatography on DEAE-Sephacel, gel filtration on Sephacryl S-300 HR and affinity chromatography on N⁶-adenosyl agarose. The enzyme shows a good stability to both temperature and pH. It appears to be a homohexamer of 164 ± 5 kDa. Kinetic characterization confirmed the specificity of this phosphorylase for 6-aminopurine nucleosides. Adenosine was the preferred substrate for nucleoside phosphorolysis (k_cat/K_m 2.1 × 10⁶ s^− 1 M^− 1), followed by 2′-deoxyadenosine (k_cat/K_m 4.2 × 10⁵ s^− 1 M^− 1). Apparently, the low specificity of adenosine phosphorylase towards 6-oxopurine nucleosides is due to a slow catalytic rate rather than to poor substrate binding.     

Effect of the phosphate substrate on drug-inhibitor binding to human purine nucleoside phosphorylase

The thermodynamics of the drug-inhibitors acyclovir, ganciclovir, and 9-benzylguanine binding to human purine nucleoside phosphorylase (hsPNP) were determined from isothermal titration calorimetry as a function of the substrate phosphate ion (Pi) concentration from 0 to 0.125 M and temperature from 15 °C to 35 °C. At 25 °C and with an increase in the Pi concentration from 0 to 50 mM, acyclovir binding becomes more entropically-driven and ganciclovir binding becomes more enthalpically-driven. At 25 °C, the tighter 9-benzylguanine binding reaction goes from an enthalpically-driven reaction in the absence of Pi to an entropically-driven reaction at 10 mM Pi, and the enthalpically-driven nature of the binding reaction is restored at 75 mM Pi. Since the dependencies of the driving-nature of the binding reactions on Pi concentration can be simulated by Pi binding to its catalytic site, it is believed that bound Pi affects the interactions of the side-chains with the ribose catalytic site. However, the binding constants are unaffected by change in the bound Pi concentration because of enthalpy-entropy compensation. The enzymatic activity of hsPNP was determined by an ITC-based assay employing 7-methylguanosine and Pi as the substrates. The heat of reaction determined from the assay increased by 7.5 kJ mol⁻¹ with increase in Pi concentration from 50 to 100 mM and is attributed to weak binding of the Pi to a secondary regulatory site. Although the binding constants of acyclovir and ganciclovir at 20 μM hsPNP were in agreement with the inverse inhibition constants determined from the ITC enzyme inhibition assays at 60 nM, the binding constant of 9-benzylguanine, which interacts with Phe159 from an adjacent subunit, decreased from 5.62 × 10⁵ M⁻¹ to 1.14 × 10⁵ M⁻¹. This reduction in the 9-benzylguanine binding affinity along with a 7-fold increase in the specific activity of hsPNP at 14.5 nM results from partial dissociation of the hsPNP trimer into monomers below the 60 nM level.     

Validation of the catalytic mechanism of Escherichia coli purine nucleoside phosphorylase by structural and kinetic studies

The catalytic mechanism of Escherichia coli purine nucleoside phosphorylase (PNP) is revised using site-directed mutagenesis, kinetic studies and structure determinations.The experimental evidence on the role of the particular catalytic amino acid during catalysis has not been available. Therefore, the active site mutants Arg24Ala, Asp204Ala, Asp204Asn, Arg217Ala and Asp204Ala/Arg217Ala were prepared and their kinetics and thermodynamic studies were carried out. The activity tests with natural substrates and 7-methylguanosine confirmed the earlier hypothesis, that catalysis involves protonation of the purine base at position N7 by Asp204, which is triggered by Arg217.The crystal structures of the wild type in complexes with phosphate and sulphate, respectively, and of the Arg24Ala mutant in complex with phosphate/sulphate were determined. The structural data show that previously observed conformational change is a result of the phosphate binding and its interaction with Arg24.As E. coli PNP is a promising candidate for the tumour-directed gene therapy, our results may also help to design efficient mutants useful in gene therapy.     

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。     

Characterization of the subunits of purine nucleoside phosphorylase from cultured normal human fibroblasts

In previous communications we have demonstrated that the subunits of normal human erythrocyte purine nucleoside phosphorylase can be resolved into four major (1–4) and two minor (1p and 2p) components with the same molecular weight but different apparent isoelectric points (and net ionic charge). The existence of subunits with different charge results in a complex isoelectric focusing pattern of the native erythrocytic enzyme. In contrast, the isoelectric focusing pattern of the native enzyme obtained from cultured human fibroblasts is simpler. The multiple native isoenzymes obtained from human erythrocytes and human brain have isoelectric points ranging from 5.0 to 6.4 and from 5.2 to 5.8, respectively, whereas cultured human fibroblasts have two major native isoenzymes with apparent isoelectric points of 5.1 and 5.6.Purine nucleoside phosphorylase has been purified at least a hundredfold from ³⁵S-labeled cultured human fibroblasts. A two-dimensional electrophoretic analysis of the denatured purified normal fibroblast enzyme revealed that it consists mainly of subunit 1 (90%) with small amounts of subunits 2 (10%) and 3 (1%). This accounts for the observed differences between the native isoelectric focusing and the electrophoretic patterns of the erythrocyte and fibroblast enzymes. The purine nucleoside phosphorylase subunit 1 is detectable in the autoradiogram from a two-dimensional electrophoretic analysis of a crude, unpurified extract of ³⁵S-labeled cultured normal human fibroblasts. The fibroblast phosphorylase coincides with the erythrocytic subunit 1 of the same enzyme, and the cultured fibroblasts of a purine nucleoside phosphorylase deficient patient (patient I) lack this protein component, genetically confirming the identity of the purine nucleoside phosphorylase subunit in cultured fibroblasts.This work was supported by a grant from the National Institute of Arthritis, Metabolism, and Digestive Diseases, National Institutes of Health, United States Public Health Service. L. J. G. is supported by a fellowship from the National Institute of Child Health and Human Development. D. W. M. is an Investigator, Howard Hughes Medical Institute.     

Role of ionization of the phosphate cosubstrate on phosphorolysis by purine nucleoside phosphorylase (PNP) of bacterial (<Emphasis Type="Italic">E. coli</Emphasis>) and mammalian (human) origin

Kinetics of the reactions of purine nucleoside phosphorylases (PNP) from E. coli (PNP-I, the product of the deoD gene) and human erythrocytes with their natural substrates guanosine (Guo), inosine (Ino), a substrate analogue N(7)-methylguanosine (m⁷Guo), and orthophosphate (P_i, natural cosubstrate) and its thiophosphate analogue (SP_i), found to be a weak cosubstrate, have been studied in the pH range 5–8. In this pH range Guo and Ino exist predominantly in the neutral forms (pK_a 9.2 and 8.8); m⁷Guo consists of an equilibrium mixture of the cationic and zwitterionic forms (pK_a 7.0); and P_i and SP_i exhibit equilibria between monoanionic and dianionic forms (pK_a 6.7 and 5.4, respectively). The phosphorolysis of m⁷Guo (at saturated concentration) with both enzymes exhibits Michaelis kinetics with SP_i, independently of pH. With P_i, the human enzyme shows Michaelis kinetics only at pH ∼5. However, in the pH range 5–8 for the bacterial enzyme, and 6–8 for the human enzyme, enzyme kinetics with P_i are best described by a model with high- and low-affinity states of the enzymes, denoted as enzyme-substrate complexes with one or two active sites occupied by P_i, characterized by two sets of enzyme-substrate dissociation constants (apparent Michaelis constants, K _m1 and K _m2) and apparent maximal velocities (V _max1 and V _max2). Their values, obtained from non-linear least-squares fittings of the Adair equation, were typical for negative cooperativity of both substrate binding (K _m1 < K _m2) and enzyme kinetics (V _max1/K _m1 > V _max2/K _m2). Comparison of the pH-dependence of the substrate properties of P_i versus SP_i points to both monoanionic and dianionic forms of P_i as substrates, with a marked preference for the dianionic species in the pH range 5–8, where the population of the P_i dianion varies from 2 to 95%, reflected by enzyme efficiency three orders of magnitude higher at pH 8 than that at pH 5. This is accompanied by an increase in negative cooperativity, characterized by a decrease in the Hill coefficient from n _H ∼1 to n _H ∼0.7 for Guo with the human enzyme, and to n _H ∼0.7 and 0.5 for m⁷Guo with the E. coli and human enzymes, respectively. Possible mechanisms of cooperativity are proposed. Attention is drawn to the substrate properties of SP_i in relation to its structure.     

Development of a capillary electrophoresis method for analyzing adenosine deaminase and purine nucleoside phosphorylase and its application in inhibitor screening

A novel capillary electrophoresis (CE) method was developed for simultaneous analysis of adenosine deaminase (ADA) and purine nucleoside phosphorylase (PNP) in red blood cells (RBCs). The developed method considered and took advantage of the natural conversion from the ADA product, inosine to hypoxanthine. The transformation ratio was introduced for ADA and PNP analysis to obtain more reliable results. After optimizing the enzymatic incubation and electrophoresis separation conditions, the determined activities of ADA and PNP in 12 human RBCs were 0.237–0.833 U/ml and 9.013–10.453 U/ml packed cells, respectively. The analysis of ADA in mice RBCs indicated that there was an apparent activity difference between healthy and hepatoma mice. In addition, the proposed method was also successfully applied in the inhibitor screening from nine traditional Chinese medicines, and data showed that ADA activities were strongly inhibited by Rhizoma Chuanxiong and Angelica sinensis. The inhibition effect of Angelica sinensis on ADA is first reported here and could also inhibit PNP activity.     

假交替单胞菌XM2107嘌呤核苷磷酸化酶基因克隆表达、重组蛋白纯化及酶学性质

【目的】嘌呤核苷磷酸化酶(PNP,EC.2.4.2.1)在酶法合成核苷类药物及中间体中具有广泛应用。本文研究的目标是,获得极地嗜冷菌假交替单胞菌Pseudoa lteromonas sp.XM2107嘌呤核苷磷酸化酶编码基因,并对该酶酶学性质进行研究,以考察该酶在核苷类中间体及药物合成中的潜在应用价值。【方法】利用同源序列PCR技术从Pseudoa lteromonas sp.XM2107基因组DNA中扩增出其编码嘌呤核苷磷酸化酶基因,测序获得编码序列。将该基因在大肠杆菌BL21(DE3)中进行重组表达以及金属螯合层析纯化,对其酶学性质进行初步研究。【结果】经过测序获得了该酶编码基因序列,全长702 bp,共编码233个氨基酸,大小为25 kDa,Genbank登录号为GQ475485。酶学性质研究发现,该重组酶最适反应温度为50℃,最适酶促反应pH为7.6(25 mmol/L磷酸盐缓冲液),最适酶促反应底物为肌苷(Km值0.389 mmol/L,37℃),且对底物腺苷和鸟苷也有磷酸解活性,在普通温度下具有较高催化活性和较好热稳定性。【结论】来源于Pseudoa lteromonas sp.XM2107的嘌呤核苷磷酸化酶在普通温度条件下具有较高的催化活性及良好热稳定性性质,在核苷类中间体和药物合成中具有较广泛的应用价值。     

大肠杆菌嘌呤核苷磷酸化酶基因的克隆与真核表达载体的构建

根据Ｇｅｎｂａｎｋ中大肠杆菌嘌呤核苷磷酸化酶（ＰＮＰ）基因的核苷酸序列,设计并合成了一对引物,以大肠杆菌基因组ＤＮＡ为模板,进行ＰＣＲ扩增,并将扩增产物定向连接到克隆、测序及真核表达载体ＰＣＤＮＡ３中,进行酶切鉴定、测序及序列分析。结果表明ＰＣＲ扩增出７４１ｂｐ大小的片段,通过酶切和序列分析证明含完整的ＰＮＰ基因序列且基因插入方向正确,此序列与文献报道的ＰＮＰ基因的同源性为９９．７％。说明克隆的ＰＮＰ基因与文献报道的基本一致,ｐｃＤＮＡ３－ＰＮＰ的构建成功为今后用其进行基因转染来研究ＰＮＰ／Ｍｅｐ－ｄＲ自杀基因系统在肿瘤基因治疗中的应用打下了基础。     

Crystal structure of purine nucleoside phosphorylase from Thermus thermophilus

The purine nucleoside phosphorylase from Thermus thermophilus crystallized in space group P4(3)2(1)2 with the unit cell dimensions a = 131.9 A and c = 169.9 A and one biologically active hexamer in the asymmetric unit. The structure was solved by the molecular replacement method and refined at a 1.9A resolution to an r(free) value of 20.8%. The crystals of the binary complex with sulfate ion and ternary complexes with sulfate and adenosine or guanosine were also prepared and their crystal structures were refined at 2.1A, 2.4A and 2.4A, respectively. The overall structure of the T.thermophilus enzyme is similar to the structures of hexameric enzymes from Escherichia coli and Sulfolobus solfataricus, but significant differences are observed in the purine base recognition site. A base recognizing aspartic acid, which is conserved among the hexameric purine nucleoside phosphorylases, is Asn204 in the T.thermophilus enzyme, which is reminiscent of the base recognizing asparagine in trimeric purine nucleoside phosphorylases. Isothermal titration calorimetry measurements indicate that both adenosine and guanosine bind the enzyme with nearly similar affinity. However, the functional assays show that as in trimeric PNPs, only the guanosine is a true substrate of the T.thermophilus enzyme. In the case of adenosine recognition, the Asn204 forms hydrogen bonds with N6 and N7 of the base. While in the case of guanosine recognition, the Asn204 is slightly shifted together with the beta(9)alpha(7) loop and predisposed to hydrogen bond formation with O6 of the base in the transition state. The obtained experimental data suggest that the catalytic properties of the T.thermophilus enzyme are reminiscent of the trimeric rather than hexameric purine nucleoside phosphorylases.     

基因工程菌酶法合成抗癌新药6-甲基嘌呤-2'-脱氧核苷

6-甲基嘌呤-2'-脱氧核苷(MePdR)是一种新型抗癌药物,它作为药物前体应用于PNP自杀基因治疗系统可以选择性杀伤肿瘤细胞.本实验构建了一个高效表达大肠杆菌来源的嘌呤核苷磷酸化酶重组质粒,并利用基因工程菌以15mmol/L 6-甲基嘌呤和60mmol/L 2'-脱氧尿苷为底物合成6-甲基嘌呤-2'-脱氧核苷,在40mmol/L pH7.0的磷酸缓冲液中,2%菌体在55℃反应2h,转化率可达83.78%.用硅胶制备薄层提纯得到白色针状晶体,收率为76.4%.HPLC测定该产物纯度99.3%,核磁共振鉴定该产物为MePdR.     

基因工程菌酶法合成抗癌新药6-甲基嘌呤-2′-脱氧核苷

6-甲基嘌呤-2′-脱氧核苷(MePdR)是一种新型抗癌药物,它作为药物前体应用于PNP自杀基因治疗系统可以选择性杀伤肿瘤细胞。本实验构建了一个高效表达大肠杆菌来源的嘌呤核苷磷酸化酶重组质粒,并利用基因工程菌以15mmol/L 6-甲基嘌呤和60mmol/L 2′-脱氧尿苷为底物合成6-甲基嘌呤-2′-脱氧核苷,在40mmol/L pH7.0的磷酸缓冲液中,2%菌体在55℃反应2h,转化率可达83.78%。用硅胶制备薄层提纯得到白色针状晶体,收率为76.4%。HPLC测定该产物纯度99.3%,核磁共振鉴定该产物为MePdR。