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.     

Cloning, expression, purification, and some properties of calf purine nucleoside phosphorylase

Calf spleen purine nucleoside phosphorylase (PNP) is considered a model enzyme for the trimeric PNPs subfamily. PCR amplification of the calf phosphorylase from the calf spleen library, cloning, overexpression of the recombinant PNP, its enzymatic activity and interactions with typical ligands of mammalian wild type PNP are described. Relative activity of the recombinant phosphorylase versus several substrates is similar to the respective values obtained for the enzyme isolated from calf spleen. As for the nonrecombinant calf PNP, the unusual fluorescence properties of the PNP/guanine complex were observed and characterized.     

Cloning,Expression, Purification,and Some Properties of Calf Purine Nucleoside Phosphorylase

Calf spleen purine nucleoside phosphorylase (PNP) is considered a model enzyme for the trimeric PNPs subfamily. PCR amplification of the calf phosphorylase from the calf spleen library, cloning, overexpression of the recombinant PNP, its enzymatic activity and interactions with typical ligands of mammalian wild type PNP are described. Relative activity of the recombinant phosphorylase versus several substrates is similar to the respective values obtained for the enzyme isolated from calf spleen. As for the nonrecombinant calf PNP, the unusual fluorescence properties of the PNP/guanine complex were observed and characterized.     

Synthesis and biological evaluation of 9-deazaguanine derivatives connected by a linker to difluoromethylene phosphonic acid as multi-substrate analogue inhibitors of PNP

9-(5',5'-difluoro-5'-phosphonopentyl)-9-deazaguanine (DFPP-DG) was designed as a multi-substrate analogue inhibitor against purine nucleoside phosphorylase (PNP) on the basis of X-ray crystallographic data obtained for a binary complex of 9-(5',5'-difluoro-5'-phosphonopentyl)guanine (DFPP-G) with calf spleen PNP. DFPP-DG and its analogous compounds were adjusted by length of the linker achieved by the Sonogashira-coupling reaction between a 9-deaza-9-iodoguanine derivative and omega-alkynyldifluoromethylene phosphonates as a key reaction. DFPP-DG is a very potent PNP inhibitor with apparent inhibition constants (in the presence of 1 mM phosphate) of 4.4 and 8.1 nM versus calf spleen and human erythrocyte PNPs, respectively. One of its analogues, homo-DFPP-DG, with longer chain linking phosphonate and 9-deazaguanine is even more potent versus human enzyme, with an apparent inhibition constant of 5.3 nM (in the presence of 1mM phosphate).     

Trimeric purine nucleoside phosphorylase: Exploring postulated one-third-of-the-sites binding in the transition state

Transition-state analogue inhibitors, immucillins, were reported to bind to trimeric purine nucleoside phosphorylase (PNP) with the stoichiometry of one molecule per enzyme trimer [Miles, R. W.; Tyler, P. C.; Furneaux, R. H.; Bagdassarian, C. K.; Schramm, V. L. Biochem. 1998, 37, 8615]. In attempts to observe and better understand the nature of this phenomenon we have conducted calorimetric titrations of the recombinant calf PNP complexed with immucillin H. However, by striking contrast to the earlier reports, we have not observed negative cooperativity and we got the stoichiometry of three immucillin molecules per enzyme trimer. Similar results were obtained from fluorimetric titrations, and for other inhibitors bearing features of the transition state. However, we observed apparent cooperativity between enzyme subunits and apparent lower stoichiometry when we used the recombinant enzyme not fully purified from hypoxanthine, which is moped from Escherichia coli cells. Results presented here prove that one-third-of-the-sites binding does not occur for trimeric PNP, and give the highly probable explanation why previous experiments were interpreted in terms of this phenomenon.     

Fluorescence studies of calf spleen purine nucleoside phosphorylase (PNP) complexes with guanine and 9-deazaguanine

Interactions of trimeric calf spleen purine nucleoside phosphorylase (PNP) with guanine (Gua) and its analogue, 9-deazaguanine (9-deaza-Gua), were studied by means of the steady-state fluorescence. The aim was to test the hypothesis that the enzyme stabilizes the anionic form of purine, inferred previously from the unusual increase of fluorescence observed after binding of guanine by calf spleen PNP. We have found that the dissociation constants obtained form titration experiments are in fact pH-independent in the range 7.0-10.25 for both PNP/Gua and PNP/9-deaza-Gua complexes. In particular, at pH 7.0 we found K _d = 0.12 ± 0.02 μ M for Gua and 0.16 ± 0.01 μ M for 9-deaza-Gua, while at the conditions where there is more than 40% of the anionic form the respective values were K _d = 0.15 ± 0.01 μ M for Gua (pH 9.0) and 0.25 ± 0.02 μ M for 9-deaza-Gua (pH 10.25). Hence, the enzyme does not prefer binding of anionic forms of these ligands in respect to the neutral ones. This result questions the involvement of the anionic forms in the reaction catalyzed by trimeric PNPs, and contradicts the hypothesis of a strong hydrogen bond formation between the enzyme Asn 243 residue and the purine N(7) position.     

Fluorescence studies of calf spleen purine nucleoside phosphorylase (PNP) complexes with guanine and 9-deazaguanine

Interactions of trimeric calf spleen purine nucleoside phosphorylase (PNP) with guanine (Gua) and its analogue, 9-deazaguanine (9-deaza-Gua), were studied by means of the steady-state fluorescence. The aim was to test the hypothesis that the enzyme stabilizes the anionic form of purine, inferred previously from the unusual increase of fluorescence observed after binding of guanine by calf spleen PNP. We have found that the dissociation constants obtained form titration experiments are in fact pH-independent in the range 7.0-10.25 for both PNP/Gua and PNP/9-deaza-Gua complexes. In particular, at pH 7.0 we found Kd = 0.12 +/- 0.02 micro M for Gua and 0.16 +/- 0.01 micro M for 9-deaza-Gua, while at the conditions where there is more than 40% of the anionic form the respective values were Kd = 0.15 +/- 0.01 micro M for Gua (pH 9.0) and 0.25 +/- 0.02 micro M for 9-deaza-Gua (pH 10.25). Hence, the enzyme does not prefer binding of anionic forms of these ligands in respect to the neutral ones. This result questions the involvement of the anionic forms in the reaction catalyzed by trimeric PNPs, and contradicts the hypothesis of a strong hydrogen bond formation between the enzyme Asn 243 residue and the purine N7 position.     

Oligomeric structure of mammalian purine nucleoside phosphorylase in solution determined by analytical ultracentrifugation

The influence of phosphate, ionic strength, temperature and enzyme concentration on the oligomeric structure of calf spleen purine nucleoside phosphorylase (PNP) in solution was studied by analytical ultracentrifugation methods. Sedimentation equilibrium analysis used to directly determine the enzyme molecular mass revealed a trimeric molecule with Mr = (90.6 +/- 2.1) kDa, regardless the conditions investigated: protein concentration in the range 0.02-1.0 mg/ml, presence of up to 100 mM phosphate and up to 200 mM NaCl, temperature in the range 4-25 degrees C. The sedimentation coefficient (6.04 +/- 0.02) S, together with the diffusion coefficient (6.15 +/- 0.11) 10(-7) cm2/s, both values obtained from the classic sedimentation velocity method at 1.0 mg/ml PNP concentration in 20 mM Hepes, pH 7.0, yielded a molecular mass of (90.2 +/- 1.6) kDa as expected for the trimeric enzyme molecule. Moreover, as shown by active enzyme sedimentation, calf spleen PNP remained trimeric even at low protein concentrations (1 microg/ml). Hence in solution, similar like in the crystalline state, calf spleen PNP is a homotrimer and previous suggestions for dissociation of this enzyme into more active monomers, upon dilution of the enzyme or addition of phosphate, are incorrect.     

Interactions of Calf Spleen Purine Nucleoside Phosphorylase with Formycin B and its Aglycone—Spectroscopic and Kinetic Studies

Phosphorolysis of 7-methylguanosine by calf spleen purine nucleoside phosphorylase (PNP) is weakly inhibited, uncompetitively, by Formycin B (FB) with K _i = 100 μ M and more effectively by its aglycone (7KPP), IC₅₀ 35–100 μ M. In striking contrast, 7KPP inhibits the reverse reaction (synthesis of 8-azaguanosine from 8-azaguanine) competitively, with K _i ～ 2–4 μ M. Formycin B forms only a weakly fluorescent complex with PNP, and 7KPP even less so, indicating that both ligands bind as the neutral, not anionic, forms. 7KPP is a rare example of a PNP non-substrate inhibitor of both the phosphorolytic and reverse synthetic pathways.     

The DsbA signal peptide-mediated secretion of a highly efficient raw-starch-digesting,recombinant α-amylase from Bacillus licheniformis ATCC 9945a

In this study, a new approach for extracellular production of recombinant α-amylase in Escherichia coli was investigated. A gene encoding a highly efficient raw-starch-digesting α-amylase from Bacillus licheniformis ATCC 9945a was cloned and expressed in E. coli. The gene encoding mature α-amylase was cloned into the pDAss expression vector, and secretion of the gene product was regulated by fusion to the signal peptide of DsbA, a well-characterized E. coli periplasmic protein. E. coli BL21 (DE3) carrying pDAss vector containing amylase gene had approximately 2.5-fold higher volumetric enzyme productivity than the natural system. The recombinant enzyme showed higher efficiency for digesting diverse raw starches when compared with the native enzyme and was similar to commercial α-amylase in its ability to hydrolyze raw starches. The properties of the recombinant enzyme demonstrate the potential of the DsbA signal peptide approach for the secretory production of the fully active, industrially important recombinant enzyme.     

SPECTROSCOPIC AND KINETIC STUDIES OF INTERACTIONS OF CALF SPLEEN PURINE NUCLEOSIDE PHOSPHORYLASE WITH 8-AZAGUANINE,AND ITS 9-(2-PHOSPHONYLMETHOXYETHYL) DERIVATIVE

Spectroscopic and kinetic studies of interactions of calf spleen purine nucleoside phosphorylase with 8-azaguanine, an excellent fluorescent/fluorogenic substrate for the synthetic pathway of the reaction, and its 9-(2-phosphonylmethoxyethyl) derivative, a bisubstrate analogue inhibitor, were carried out. The goal was to clarify the catalytic mechanism of the enzymatic reaction by identification of ionic/tautomeric forms of these ligands in the complex with PNP.     

Two fluorogenic substrates for purine nucleoside phosphorylase,selective for mammalian and bacterial forms of the enzyme

Two nontypical nucleosides, 7-β-d-ribosyl-2,6-diamino-8-azapurine and 8-β-d-ribosyl-2,6-diamino-8-azapurine, have been found to exhibit moderately good, and selective, substrate properties toward calf and bacterial (Escherichia coli) forms of purine nucleoside phosphorylase (PNP). The former compound is effectively phosphorolysed by calf PNP and the latter by PNP from E. coli. Both compounds are fluorescent with λ_max ∼ 425 to 430 nm, but the reaction product, 2,6-diamino-8-azapurine, emits in a different spectral region (λ_max ∼ 363 nm) with nearly 40% yield, providing a strong fluorogenic effect at 350 to 360 nm.     

Toxoplasma gondii purine nucleoside phosphorylase biochemical characterization, inhibitor profiles, and comparison with the Plasmodium falciparum ortholog

Purine nucleoside phosphorylase (PNP) is an important component of the nucleotide salvage pathway in apicomplexan parasites and a potential target for drug development. The intracellular pathogen Toxoplasma gondii was therefore tested for sensitivity to immucillins, transition state analogs that exhibit high potency against PNP in the malaria parasite Plasmodium falciparum. Growth of wild-type T. gondii is unaffected by up to 10 microm immucillin-H (ImmH), but mutants lacking the (redundant) purine salvage pathway enzyme adenosine kinase are susceptible to the drug, with an IC50 of 23 nm. This effect is rescued by the reaction product hypoxanthine, but not the substrate inosine, indicating that ImmH acts via inhibition of T. gondii PNP. The primary amino acid sequence of TgPNP is >40% identical to PfPNP, and recombinant enzymes exhibit similar kinetic parameters for most substrates. Unlike the Plasmodium enzyme, however, TgPNP cannot utilize 5'-methylthio-inosine (MTI). Moreover, TgPNP is insensitive to methylthio-immucillin-H (MT-ImmH), which inhibits PfPNP with a Ki* of 2.7 nm. MTI arises through the deamination of methylthio-adenosine, a product of the polyamine biosynthetic pathway, and its further metabolism to hypoxanthine involves PfPNP in purine recycling (in addition to salvage). Remarkably, analysis of the recently completed T. gondii genome indicates that polyamine biosynthetic machinery is completely lacking in this species, obviating the need for TgPNP to metabolize MTI. Differences in purine and polyamine metabolic pathways among members of the phylum Apicomplexa and these parasites and their human hosts are likely to influence drug target selection strategies. Targeting T. gondii PNP alone is unlikely to be efficacious for treatment of toxoplasmosis.     

Spectroscopic and kinetic studies of interactions of calf spleen purine nucleoside phosphorylase with 8-azaguanine, and its 9-(2-phosphonylmethoxyethyl) derivative

Spectroscopic and kinetic studies of interactions of calf spleen purine nucleoside phosphorylase with 8-azaguanine, an excellent fluorescent/fluorogenic substrate for the synthetic pathway of the reaction, and its 9-(2-phosphonylmethoxyethyl) derivative, a bisubstrate analogue inhibitor, were carried out. The goal was to clarify the catalytic mechanism of the enzymatic reaction by identification of ionic/tautomeric forms of these ligands in the complex with PNP.     

Interactions of calf spleen purine nucleoside phosphorylase with formycin B and its aglycone - spectroscopic and kinetic studies

Phosphorolysis of 7-methylguanosine by calf spleen purine nucleoside phosphorylase (PNP) is weakly inhibited, uncompetitively, by Formycin B (FB) with Ki = 100 micro M and more effectively by its aglycone (7KPP), IC50 35-100 micro M. In striking contrast, 7KPP inhibits the reverse reaction (synthesis of 8-azaguanosine from 8-azaguanine) competitively, with Ki approximately 2-4 micro M. Formycin B forms only a weakly fluorescent complex with PNP, and 7KPP even less so, indicating that both ligands bind as the neutral, not anionic, forms. 7KPP is a rare example of a PNP non-substrate inhibitor of both the phosphorolytic and reverse synthetic pathways.     

Crystal structure of the purine nucleoside phosphorylase (PNP) from Cellulomonas sp. and its implication for the mechanism of trimeric PNPs

The three-dimensional structure of the trimeric purine nucleoside phosphorylase (PNP) from Cellulomonas sp. has been determined by X-ray crystallography. The binary complex of the enzyme with orthophosphate was crystallized in the orthorhombic space group P212121 with unit cell dimensions a=64.1 A, b=108.9 A, c=119.3 A and an enzymatically active trimer in the asymmetric unit. X-ray data were collected at 4 degrees C using synchrotron radiation (EMBL/DESY, Hamburg). The structure was solved by molecular replacement, with the calf spleen PNP structure as a model, and refined at 2.2 A resolution. The ternary "dead-end" complex of the enzyme with orthophosphate and 8-iodoguanine was obtained by soaking crystals of the binary orthophosphate complex with the very weak substrate 8-iodoguanosine. Data were collected at 100 K with CuKalpha radiation, and the three-dimensional structure refined at 2.4 A resolution. Although the sequence of the Cellulomonas PNP shares only 33 % identity with the calf spleen enzyme, and almost no identity with the hexameric Escherichia coli PNP, all three enzymes have many common structural features, viz. the nine-stranded central beta-sheet, the positions of the active centres, and the geometrical arrangement of the ligands in the active centres. Some similarities of the surrounding helices also prevail. In Cellulomonas PNP, each of the three active centres per trimer is occupied by orthophosphate, and by orthophosphate and base, respectively, and small structural differences between monomers A, B and C are observed. This supports cooperativity between subunits (non-identity of binding sites) rather than existence of more than one binding site per monomer, as previously suggested for binding of phosphate by mammalian PNPs. The phosphate binding site is located between two conserved beta- and gamma-turns and consists of Ser46, Arg103, His105, Gly135 and Ser223, and one or two water molecules. The guanine base is recognized by a zig-zag pattern of possible hydrogen bonds, as follows: guanine N-1...Glu204 O(epsilon1)...guanine NH2...Glu204 O(epsilon2). The exocyclic O6 of the base is bridged via a water molecule to Asn246 N(delta), which accounts for the inhibitory, but lack of substrate, activity of adenosine. An alternative molecular mechanism for catalysis by trimeric PNPs is proposed, in which the key catalytic role is played by Glu204 (Glu201 in the calf and human enzymes), while Asn246 (Asn243 in the mammalian enzymes) supports binding of 6-oxopurines rather than catalysis. This mechanism, in contrast to that previously suggested, is consistent with the excellent substrate properties of N-7 substituted nucleosides, the specificity of trimeric PNPs versus 6-oxopurine nucleosides and the reported kinetic properties of Glu201/Ala and Asn243/Ala point variants of human PNP.     

Characteristics of 92 kDa type IV collagenase/gelatinase produced by granulocytic leukemia cells: structure,expression of cDNA in E. coli and enzymatic properties

Human neutrophils can be triggered to release the collagenolytic metalloenzymes, interstitial collagenase and 92 kDa type IV collagenase/gelatinase. We have isolated and sequenced a 2.3 kb cDNA from a chronic granulocytic leukemia cDNA library that encodes for human neutrophil type IV collagenase. With the exception of one amino-acid substitution at position 280 (Arg → Gln), the deduced amino-acid sequences of neutrophil gelatinase are identical to the amino-acid sequences of the enzyme isolated from fibrosarcoma cells. Expression of the cDNA in E. coli yielded a 72 kDa protein having a gelatinolytic activity on zymogram gel. The recombinant enzyme was activated with APMA and trypsin. The activation was accompanied by a reduction in molecular weight of ≈ 10 kDa; such a reduction is characteristic of matrix metalloproteinases. The recombinant gelatinase cleaved native type V and XI collagens. Native type I collagen was not a substrate for the enzyme. These data suggest that native and recombinant 92 kDa type IV collagenase produced in E. coli have similar biochemical properties. The successful expression of the collagenase in a prokaryotic system will greatly facilitate the structure-function characterization of the enzyme and allow a more precise analysis of its physiological and pathological roles.     

Purine Nucleoside Phosphorylase Targeted by Annexin V to Breast Cancer Vasculature for Enzyme Prodrug Therapy

Background and Purpose

The targeting of therapeutics is a promising approach for the development of new cancer treatments that seek to reduce the devastating side effects caused by the systemic administration of current drugs. This study evaluates a fusion protein developed as an enzyme prodrug therapy targeted to the tumor vasculature. Cytotoxicity would be localized to the site of the tumor using a protein fusion of purine nucleoside phosphorylase (PNP) and annexin V. Annexin V acts as the tumor-targeting component of the fusion protein as it has been shown to bind to phosphatidylserine expressed externally on cancer cells and the endothelial cells of the tumor vasculature, but not normal vascular endothelial cells. The enzymatic component of the fusion, PNP, converts the FDA-approved cancer therapeutic, fludarabine, into a more cytotoxic form. The purpose of this study is to determine if this system has a good potential as a targeted therapy for breast cancer.

Methods

A fusion of E. coli purine nucleoside phosphorylase and human annexin V was produced in E. coli and purified. Using human breast cancer cell lines MCF-7 and MDA-MB-231 and non-confluent human endothelial cells grown in vitro, the binding strength of the fusion protein and the cytotoxicity of the enzyme prodrug system were determined. Endothelial cells that are not confluent expose phosphatidylserine and therefore mimic the tumor vasculature.

Results

The purified recombinant fusion protein had good enzymatic activity and strong binding to the three cell lines. There was significant cell killing (p<0.001) by the enzyme prodrug treatment for all three cell lines, with greater than 80% cytotoxicity obtained after 6 days of treatment.

Conclusion

These results suggest that this treatment could be useful as a targeted therapy for breast cancer.     

Cloning of the Authentic Bovine Gene Encoding Pepsinogen A and Its Expression in Microbial Cells

Bovine pepsin is the second major proteolytic activity of rennet obtained from young calves and is the main protease when it is extracted from adult animals, and it is well recognized that the proteolytic specificity of this enzyme improves the sensory properties of cheese during maturation. Pepsin is synthesized as an inactive precursor, pepsinogen, which is autocatalytically activated at the pH of calf abomasum. A cDNA coding for bovine pepsin was assembled by fusing the cDNA fragments from two different bovine expressed sequence tag libraries to synthetic DNA sequences based on the previously described N-terminal sequence of pepsinogen. The sequence of this cDNA clearly differs from the previously described partial bovine pepsinogen sequences, which actually are rabbit pepsinogen sequences. By cloning this cDNA in different vectors we produced functional bovine pepsinogen in Escherichia coli and Saccharomyces cerevisiae. The recombinant pepsinogen is activated by low pH, and the resulting mature pepsin has milk-clotting activity. Moreover, the mature enzyme generates digestion profiles with α-, β-, or κ-casein indistinguishable from those obtained with a natural pepsin preparation. The potential applications of this recombinant enzyme include cheese making and bioactive peptide production. One remarkable advantage of the recombinant enzyme for food applications is that there is no risk of transmission of bovine spongiform encephalopathy.