Induction of thymidine kinase and DNase in varicella-zoster virus-infected cells and kinetic properties of the virus-induced thymidine kinase.

Thymidine kinase (TK), DNA polymerase, and DNase activities were induced in human foreskin fibroblasts after varicella-zoster virus infection. The induced TK and DNase activities have electrophoretic mobilities different from the corresponding host enzymes. Varicella-zoster virus-induced TK was purified and separated from the host enzyme by affinity column chromatography. This enzyme has been shown to have a broader substrate specificity with respect to either the phosphate donor or acceptor as compared with human cytoplasmic and mitochondrial TKs. The best phosphate donor is ATP, with a Km of 16 microM. The Km values of thymidine, deoxycytidine, and 5-propyl deoxyuridine were estimated to be 0.4, 180, and 0.8 microM, respectively. The Ki values for several analogs of thymidine such as 5-iododeoxyuridine, arabinofuranosylthymine, 5-ethyl deoxyuridine, and 5-cyanodeoxyuridine were also examined. TTP acted as a noncompetitive inhibitor with respect to thymidine with a Ki of 5 microM. The kinetic behavior of varicella-zoster virus-induced TK is different from human cytoplasmic, human mitochondrial, and herpes simplex virus type 1- and 2-induced TKs.     

Human thymidine kinase: purification and some properties of the TK1 isoenzyme from placenta

Summary Human thymidine kinase TK1 isoenzyme has been purified 1 800-fold from placenta to a specific activity of 2.9 nmoles/min/mg of protein. The rapid purification procedure includes affinity chromatography on a thymidine-Sepharose column. At all stages of purification, the enzyme showed irreversible lability. The native molecular weight was determined to be 45 000. Human placental TK1 exhibited specificity for ATP and thymidine as substrates, and significant inhibition was found only with thymidine nucleotides. TTP was the most effective inhibitor.     

Identification of the ATP-binding domain of vaccinia virus thymidine kinase

Although small in size (20 kDa), the vaccinia virus (VV) thymidine kinase protein (EC 2.7.1.21 TK) is a relatively complex enzyme which must contain domains involved in binding both substrates (ATP and thymidine) and a feedback inhibitor (dTTP), as well as sequences directing the association of individual protein monomers into a functional tetrameric enzyme. Alignment of predicted amino acid sequences of the thymidine kinase genes from a variety of sources was used to identify highly conserved regions as a first step toward locating potential regions housing essential domains. A conserved domain (domain I) near the amino terminus of VV TK protein had characteristics consistent with a nucleotide-binding site. Analysis of the nucleotide substrate specificity of VV TK indicated that ATP acts as the major phosphate donor for thymidine phosphorylation while GTP, CTP, and UTP were inefficient substrates. Site-directed mutagenesis was performed on domain I to generate 11 mutant enzymes. Comparison of the wild-type and mutant proteins with regard to enzyme activity revealed that two of the mutant enzymes, T18 and S19, exhibited enhanced enzyme activity (3.73-fold and 1.35-fold, respectively) relative to the control. The other mutations introduced led to greatly reduced levels of enzyme activity which correlated with a reduced or altered ability of the mutant enzymes to bind ATP as determined by ATP-agarose affinity chromatography. Wild-type VV TK bound to an ATP affinity column could also be eluted with dTTP. Glycerol gradient separation of wild-type TK in the presence or absence of dTTP indicated that dissociation of the tetrameric complex was not the means by which enzymatic inhibition was achieved. Taken together, these results suggest that (i) domain I (amino acids 11-22) of the VV TK corresponds to the ATP-binding site, and (ii) that dTTP is able to interfere with ATP binding, either directly or indirectly, and thereby inhibit enzymatic activity without dissociating the native enzyme.     

Conservative mutations of glutamine-125 in herpes simplex virus type 1 thymidine kinase result in a ganciclovir kinase with minimal deoxypyrimidine kinase activities

The herpes simplex virus type 1 thymidine kinase (HSV-1 TK) is the major anti-herpes virus pharmacological target, and it is being utilized in combination with the prodrug ganciclovir as a toxin gene therapeutic for cancer. One active-site amino acid, glutamine-125 (Gln-125), has been shown to form hydrogen bonds with bound thymidine, thymidylate, and ganciclovir in multiple X-ray crystal structures. To examine the role of Gln-125 in HSV-1 TK activity, three site-specific mutations of this residue to an aspartic acid, an asparagine, or a glutamic acid were introduced. These three mutants and wild-type HSV-1 TK were expressed in E. coli and partially purified and their enzymatic properties compared. In comparison to the Gln-125 HSV-1 TK, thymidylate kinase activity of all three mutants was decreased by over 90%. For thymidine kinase activity relative to Gln-125 enzyme, the K(m) of thymidine increased from 0.9 microM for the parent Gln-125 enzyme to 3 microM for the Glu-125 mutant, to 6000 microM for the Asp-125 mutant, and to 20 microM for the Asn-125 mutant. In contrast, the K(m) of ganciclovir decreased from 69 microM for the parent Gln-125 enzyme to 50 microM for the Asn-125 mutant and increased to 473 microM for the Glu-125 mutant. The Asp-125 enzyme was able to poorly phosphorylate ganciclovir, but with nonlinear kinetics. Molecular simulations of the wild-type and mutant HSV-1 TK active sites predict that the observed activities are due to loss of hydrogen bonding between thymidine and the mutant amino acids, while the potential for hydrogen bonding remains intact for ganciclovir binding. When expressed in two mammalian cell lines, the Glu-125 mutant led to GCV-mediated killing of one cell line, while the Asn-125 mutant was equally as effective as wild-type HSV-1 TK in metabolizing GCV and causing cell death in both cell lines.     

Purification and characterization of thymidine kinase from regenerating rat liver

Thymidine kinase (EC 2.7.1.21) from regenerating rat liver has been purified 70,000-fold to apparent homogeneity by affinity chromatography. Molecular weight of the native enzyme was found to be about 54,000, as determined by gel filtration. Electrophoresis in polyacrylamide gels containing sodium dodecyl sulfate yielded a single band with a molecular weight of 26,000, suggesting that thymidine kinase is a dimer of very similar or identical subunits. The Michaelis constant for thymidine is 2.2 microM. ATP acts as a sigmoidal substrate with a 'Km' of 0.2 mM. Reaction kinetics and product inhibition studies reveal the enzymatic mechanism to be sequential.     

Human herpesvirus 8 open reading frame 21 is a thymidine and thymidylate kinase of narrow substrate specificity that efficiently phosphorylates zidovudine but not ganciclovir

Human herpesvirus 8 (HHV8) open reading frame (ORF) 21 is predicted to encode a protein similar to the thymidine kinase (TK) enzyme of other herpesviruses. Expressed in mammalian cells, ORF 21 was found to have low TK activity, based on poor growth in media containing hypoxanthine-aminopterin-thymidine (HAT) and low incorporation of [(3)H]thymidine into high-molecular-weight DNA. Kinetic analysis using HHV8 TK as a purified glutathione S-transferase (GST) fusion protein showed that the enzyme has a comparatively high K(m) for thymidine (dThd) of approximately 33.2 microM. Nearly 50% of the phosphorylated product of the reaction with dThd was thymidylate. This monophosphate kinase activity was more pronounced with 3'-azido-3'-deoxythymidine (AZT), in which 78% of the reaction product was AZT diphosphate. Thymidine analogs competitively inhibited dThd phosphorylation by HHV8 TK, while 2'-deoxyguanosine, 2'-deoxyadenosine, 2'-deoxycytidine, and corresponding analogs did not. Further competition experiments revealed that the nucleoside analog ganciclovir (GCV), at up to 1,000-fold molar excess, could not significantly inhibit dThd phosphorylation by the enzyme. In support of these data, 143B TK(-) cells expressing HHV8 TK phosphorylated GCV very poorly and were not susceptible to GCV toxicity compared to parental cells. Phosphorylation of [(3)H]GCV by a purified GST-HHV8 TK fusion protein was not detected by high-pressure liquid chromatography analysis. Structural features of HHV8 TK substrate recognition were investigated. Therapeutic implications of these findings are discussed.     

Valine, not methionine, is amino acid 106 in human cytosolic thymidine kinase (TK1). Impact on oligomerization, stability, and kinetic properties

Cytosolic thymidine kinase (TK1) cDNA from human lymphocytes was cloned, expressed in Escherichia coli, purified, and characterized with respect to the ATP effect on thymidine affinity and oligomerization. Sequence analysis of this lymphocyte TK1 cDNA and 21 other cDNAs or genomic TK1 DNAs from healthy cells or leukemic or transformed cell lines revealed a valine at amino acid position 106. The TK1 sequence in NCBI GenBank(TM) has methionine at this position. The recombinant lymphocyte TK1(Val-106) (rLy-TK1(Val-106)) has the same enzymatic and oligomerization properties as endogenous human lymphocyte TK1 (Ly-TK1); ATP exposure induces an enzyme concentration-dependent reversible transition from a dimer to a tetramer with 20-30-fold higher thymidine affinity (K(m) about 15 and 0.5 microm, respectively). Substitution of Val-106 with methionine to give rLy-TK1(Met-106) results in a permanent tetramer with the high thymidine affinity (K(m) about 0.5 microm), even without ATP exposure. Furthermore, rLy-TK1(Met-106) is considerably less stable than rLy-TK1(Val-106) (t(12) at 15 degrees C is 41 and 392 min, respectively). Because valine with high probability is the naturally occurring amino acid at position 106 in human TK1 and because this position has high impact on the enzyme properties, the Val-106 form should be used in future investigations of recombinant human TK1.     

Characterization of three essential residues in the conserved ATP-binding region of Epstein-Barr virus thymidine kinase

The thymidine kinase encoded by Epstein-Barr virus (EBV TK) is an important target for antiviral therapy and the treatment of EBV-associated malignancies. Through computer-assisted alignment with other human herpesviral TK proteins, EBV TK was shown to contain a conserved ATP-binding motif as for the other TK enzymes. To investigate functional roles of three highly conserved residues (G294, K297, T298) within this region, site-directed mutagenesis was employed to generate various mutants. The TK enzyme activity and ATP-binding ability of these mutant TK enzymes were determined and compared with EBV wild-type TK (wtTK). Mutant G294V lost its ATP-binding ability and was inactive in enzyme activity assay. As the enzyme activity of G294A was reduced to 20% of that of wtTK, the K(m) for ATP binding of G294A was 48.7 microM as compared with 30.0 microM of EBV wtTK. These results suggested that G294 participates in ATP binding and contributes to maintenance of structure. EBV TK mutants K297E, K297Q, and K297R lost their ATP-binding ability and enzyme activity. However, K297R was shown to have a preference for usage of GTP (K(m): 43.0 microM) instead of ATP (K(m): 87.6 microM) as the phosphate donor. This implies that, in addition to nucleotide binding, K297 was involved in the selection of phosphate donor. While EBV TK mutant T298S retained approximately 80% of wtTK enzyme activity, T298A lost its enzyme activity, suggesting that a hydroxyl group at this position is important for the enzyme activity. Interestingly, T298A retained its ATP-binding ability, suggesting a role of T298 in the catalytic process but not in the coordination of ATP. This study demonstrated that amino acid residues G294, K297, and T298 in the ATP-binding motif of EBV TK enzyme are essential for the enzymatic activity but are involved in different aspects of its action.     

Chemotherapy of varicella-zoster virus by a novel class of highly specific anti-VZV bicyclic pyrimidine nucleosides

(E)-5-(2-bromovinyl)-2'-deoxyuridine (BVDU) is a potent inhibitor of herpes simplex virus type 1 (HSV-1) and varicella-zoster virus (VZV). Its mechanism of action is based on a specific conversion to its 5'-mono- and 5'-diphosphate derivative by HSV-1- and VZV-encoded thymidine kinase, and after further conversion to its 5'-triphosphate derivative, inhibition of the viral DNA polymerase and eventual incorporation into the viral DNA. Recently, a new structural class of bicyclic pyrimidine nucleoside analogues (designated BCNAs) with highly specific and selective anti-VZV activity in cell culture has been discovered. The compounds need a long alkyl or alkylaryl side-chain at the base moiety for pronounced biological activity. This property makes these compounds highly lipophilic. They are also endowed with fluorescent properties when exposed to light with short UV wavelength. In striking contrast to BVDU, the members of this class of compounds are active only against VZV, but not against any other virus, including the closely related HSV-1, HSV-2 and cytomegalovirus. The most active compounds inhibit VZV replication at subnanomolar concentrations and are not toxic at high micromolar concentrations. The compounds lose their antiviral activity against thymidine kinase (TK)-deficient VZV strains, pointing to a pivotal role of the viral TK in their activation (phosphorylation). Kinetic studies with purified enzymes revealed that the compounds were recognized by VZV TK as a substrate, but not by HSV-1 TK, nor by cytosolic or mitochondrial TK. VZV TK is able to phosphorylate the test compounds not only to their corresponding 5'-mono- but also to their 5'-diphosphate derivatives. These data may readily explain and rationalize the anti-VZV selectivity of the BCNAs. There is no clear-cut correlation between the antiviral potency of the compounds and their affinity for VZV TK, pointing to a different structure/activity relationship of the eventual antiviral target of these compounds. The compounds are stable in solution and, in contrast to BVDU, not susceptible to degradation by thymidine phosphorylase. The bicyclic pyrimidine nucleoside analogues represent an entirely new class of highly specific anti-VZV compounds that should be further pursued for clinical development.     

A Rapid Purification of L-Glutamic Acid Decarboxylase from the Brain of the Locust Schistocerca gregaria

L-Glutamic acid decarboxylase (GAD; EC 4.1.1.15) was purified to apparent homogeneity from the brain of the locust Schistocerca gregaria using a combination of chromatofocusing (Mono P) and gel filtration (Superose 12) media. The homogeneity of the enzyme preparation was established by native polyacrylamide gel electrophoresis (PAGE) with silver staining. The molecular weight of the purified enzyme was estimated from native gradient gel electrophoresis and gel filtration chromatography to be 97,000 +/- 4,000 and 93,000 +/- 5,000, respectively. When analysed by sodium dodecyl sulphate-PAGE, the enzyme was found to be composed of two distinct subunits of Mr 51,000 +/- 1,000 and 44,000 +/- 1,500. Tryptic peptide maps of iodinated preparations of these two subunits showed considerable homology, suggesting that the native enzyme is a dimer of closely related subunits. The purified enzyme had a pH optimum of 7.0-7.4 in 100 mM potassium phosphate buffer and an apparent Km for glutamate of 5.0 mM. The enzyme was strongly inhibited by the carbonyl-trapping reagent aminooxyacetic acid with an I50 value of 0.2 microM.     

Mammalian thymidine kinase 2. Direct photoaffinity labeling with [32P]dTTP of the enzyme from spleen, liver, heart and brain.

Thymidine kinase 2 (TK2), also called mitochondrial thymidine kinase, is a pyrimidine deoxyribonucleoside kinase expressed in all cells and tissues. It was recently purified to apparent homogeneity from human leukemic spleen and the active enzyme was shown to be a monomer of a 29-kDa polypeptide. The enzyme is feedback-inhibited by both end products, dCTP and dTTP. Here we show that TK2 purified from several different sources, including purified beef heart mitochondria, could be directly photoaffinity labeled with radioactive dTTP (approximately 18% of all TK2 molecules were cross-linked to dTTP after 20 min of ultraviolet irradiation) or to a lower extent with dCTP. Photo-incorporation was inhibited by the presence of the other effector but also the phosphate donor ATP blocked photolabeling, with dTTP. Addition of nucleoside substrates gave only a marginal inhibition of photo-incorporation. There were no detectable difference in the molecular size of photolabeled TK2 isolated from human spleen, brain or placenta, monkey liver, beef heart and beef heart mitochondria. Nor was there any significant differences in the enzyme kinetic properties of these enzymes. Cleavage of labeled TK2 with cyanogen bromide showed that dTTP was incorporated into a single 3-kDa peptide. TK2 was the only pyrimidine deoxynucleoside kinase expressed in liver, heart and brain. A detailed characterization of the subunit structure and substrate specificity of this enzyme is of importance for the design of new antiviral and cytostatic therapies based on nucleoside analogs.     

Purification and characterization of herpes simplex virus (type 1) thymidine kinase produced in Escherichia coli by a high efficiency expression plasmid utilizing a lambda PL promoter and cI857 temperature-sensitive repressor

The structural gene for herpes simplex virus (type 1) thymidine kinase was cloned downstream from the lambda phage high efficiency leftward promotor in a plasmid (pHETK2) also containing the gene for the lambda cI857 temperature-sensitive repressor. Thymidine kinase is synthesized as a run-on product containing the NH2 terminus of the lambda N protein. Heat inactivation of the lambda repressor by growth at 42 degrees C results in the accumulation of thymidine kinase as approximately 4% of the total soluble cellular protein. Thymidine kinase has been purified to greater than 95% homogeneity by high speed centrifugation, ammonium sulfate fractionation, and Sephadex G-100 and hydroxylapatite column chromatography. Thymidine kinase has a subunit Mr = 42,000 determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and behaves as a dimer during Sephadex G-100 chromatography and glycerol gradient centrifugation. Thymidine kinase is enzymatically active from pH 6 to 10 with maximum activity at pH 8.5. The enzyme is protected from heat inactivation by thymidine and has a half-life at 40 degrees C of 30 min in the presence of thymidine and 3 min in its absence. Thymidine kinase displays Michaelis-Menten kinetics with apparent Michaelis constants of 0.6 and 118 microM for thymidine and ATP, respectively. Iododeoxycytidine is a competitive inhibitor of thymidine with an apparent Ki of 14 microM. The anti-herpes drug acyclovir (9-[(2-hydroxyethoxy)methyl]guanine) also appears to be a competitive inhibitor of thymidine (Ki of approximately 300 microM) but requires 3,000-fold higher concentrations than thymidine to give 50% inhibition. Other nucleoside triphosphates can substitute for ATP in the kinase reaction with the exception of dTTP which appears to inhibit thymidine kinase activity by about 50% when present in concentrations equal to that of thymidine.     

Immunoaffinity purification of rat liver transketolase: evidence for multiple forms of the enzyme

Rat liver transketolase (TK) has been purified, in a single step, by immunoaffinity chromatography on specific TK antibodies covalently linked to Sepharose 4B. The procedure described also involves the raising and isolation of rabbit TK antibodies to the conventionally purified enzyme [F. Paoletti (1983) Arch. Biochem. Biophys. 222, 489-496]. Affinity chromatography allows a 100-fold purification of TK from the cell cytosol and a recovery of about 70% of the original activity. The TK isolated has a specific activity of 2.7-3.2 at 25 degrees C and migrates as a single band on polyacrylamide gel electrophoresis at pH 9.1. Multiple forms of the enzyme, with distinct pI values in the range 7-8, have been detected in purified preparations by means of analytical isoelectric focusing and staining for TK. No addition of either Mg2+ or thiamine pyrophosphate is required for the activity of the enzyme which, in the native form, exhibits a molecular weight of about 139,000. Two moles of thiamine pyrophosphate can be resolved for each mole of enzyme. Affinity TK preparations are virtually free of glyceraldehyde-3-phosphate dehydrogenase, pentose-phosphate epimerase, and isomerase, although slight contamination by phosphohexose isomerase may occur.     

Purification and characterization of Streptomyces griseus catechol O-methyltransferase

A soluble (100,000 x g supernatant) methyltransferase catalyzing the transfer of the methyl group of S-adenosyl-L-methionine to catechols was present in cell extracts of Streptomyces griseus. A simple, general, and rapid catechol-based assay method was devised for enzyme purification and characterization. The enzyme was purified 141-fold by precipitation with ammonium sulfate and successive chromatography over columns of DEAE-cellulose, DEAE-Sepharose, and Sephacryl S-200. The purified cytoplasmic enzyme required 10 mM magnesium for maximal activity and was catalytically optimal at pH 7. 5 and 35 degrees C. The methyltransferase had an apparent molecular mass of 36 kDa for both the native and denatured protein, with a pI of 4.4. Novel N-terminal and internal amino acid sequences were determined as DFVLDNEGNPLENNGGYXYI and RPDFXLEPPYTGPXKARIIRYFY, respectively. For this enzyme, the K(m) for 6,7-dihydroxycoumarin was 500 +/- 21.5 microM, and that for S-adenosyl-L-methionine was 600 +/- 32.5 microM. Catechol, caffeic acid, and 4-nitrocatechol were methyltransferase substrates. Homocysteine was a competitive inhibitor of S-adenosyl-L-methionine, with a K(i) of 224 +/- 20.6 microM. Sinefungin and S-adenosylhomocysteine inhibited methylation, and the enzyme was inactivated by Hg(2+), p-chloromercuribenzoic acid, and N-ethylmaleimide.     

Uridine kinase from Ehrlich ascites carcinoma. Purification and properties of homogeneous enzyme

Uridine kinase from Ehrlich ascites tumor cells has been purified about 60,000-fold to apparent homogeneity and with an overall recovery of about 40%. This purification was achieved using phosphocellulose and adenosine 5'-triphosphate-agarose affinity chromatography. The subunit molecular mass as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis was 31,000 daltons. With two-dimensional electrophoresis, only one spot was observed, indicating the absence of isoenzymes. Multiple peaks of activity are routinely observed on ion exchange chromatography or gel filtration, for both crude preparations or homogeneous uridine kinase, in agreement with our earlier results that this enzyme exists as multiple interconvertible oligomeric forms (Payne, R. C., and Traut, T. W. (1982) J. Biol. Chem. 257, 12485-12488). The purified enzyme has a specific activity of 283 mumol/min/mg of protein at 22 degrees C. Initial velocity studies using uridine and ATP are consistent with a sequential mechanism. Km values for uridine, cytidine, and ATP are 40, 57, and 450 microM, respectively. CTP and UTP are competitive inhibitors with respect to ATP, with Ki values for CTP and UTP of 10 and 61 microM, respectively. The enzyme was active with several nucleoside analogs, the Km values being 69 microM (5-fluorouridine), 200 microM (3-deazauridine), and 340 microM (6-azauridine). The pure enzyme is very sensitive to freezing, but can be maintained at O degrees C for 8 weeks with only 20% loss of activity. For long-term storage, enzyme in 50% glycerol can be maintained at -20 degrees C for many months with no detectable loss of activity.     

Mutant varicella-zoster virus thymidine kinase: correlation of clinical resistance and enzyme impairment.

Varicella-zoster virus (VZV) encodes a thymidine kinase (EC 2.7.2.21) which phosphorylates several antiviral nucleoside analogs, including acyclovir (ACV). A mutation in the VZV thymidine kinase coding sequence, resulting in an arginine-to-glutamine substitution at amino acid residue 130 (R130Q), is associated with clinical resistance to ACV. We have expressed the wild-type and the mutant enzymes in bacteria and have studied the kinetic characteristics of the purified enzymes. The arginine-to-glutamine substitution resulted in decreased catalytic activity and altered substrate specificity. The most striking effect was a decrease in the rates of nucleoside phosphorylation to less than 2% of the rates with the wild-type enzyme. This was accompanied by increased apparent Km values for thymidine and deoxycytidine. ACV was not detectably phosphorylated by the R130Q enzyme but still competed with thymidine for the enzyme. The inability of the R130Q enzyme to catalyze the phosphorylation of ACV correlates with resistance to ACV noted with a clinical isolate of VZV.     

The CTP:phosphocholine cytidylyltransferase encoded by the licC gene of Streptococcus pneumoniae: cloning, expression, purification, and characterization.

Streptococcus pneumoniae is a member of a small group of bacteria that display phosphocholine on the cell surface, covalently attached to the sugar groups of teichoic acid and lipoteichoic acid. The putative pathway for this phosphocholine decoration is, in its first two enzymes, functionally similar to the CDP-choline pathway used for phosphatidylcholine biosynthesis in eukaryotes. We show that the licC gene encodes a functional CTP:phosphocholine cytidylyltransferase (CCT). The enzyme has been expressed and purified to homogeneity. Assay conditions were optimized, particularly with respect to linearity with time, pH, Mg(2+), and ammonium sulfate concentration. The pure enzyme has K(M) values of 890+/-240 microM for CTP, and 390+/-170 microM for phosphocholine. The k(cat) is 17.5+/-4.0 s(-1). S. pneumoniae CTP:phosphocholine cytidylyltransferase (SpCCT) is specific for CTP or dCTP as the nucleotide substrate. SpCCT is strongly inhibited by Ca(2+). The IC(50) values for recombinant and native SpCCT are 0.32+/-0.04 and 0.27+/-0.03 mM respectively. The enzyme is also inhibited by all other tested divalent cations, including Mg(2+) at high concentrations. The cloning and expression of this enzyme sets the stage for design of inhibitors as possible antipneumococcal drugs.     

Expression and purification of human tryptophan hydroxylase from Escherichia coli and Pichia pastoris

Tryptophan hydroxylase (TPH) from several mammalian species has previously been cloned and expressed in bacteria. However, due to the instability of wild type TPH, most successful attempts have been limited to the truncated forms of this enzyme. We have expressed full-length human TPH in large amounts in Escherichia coli and Pichia pastoris and purified the enzyme using new purification protocols. When expressed as a fusion protein in E. coli, the maltose-binding protein-TPH (MBP-TPH) fusion protein was more soluble than native TPH and the other fusion proteins and had a 3-fold higher specific activity than the His-Patch-thioredoxin-TPH and 6xHis-TPH fusion proteins. The purified MBP-TPH had a V(max) of 296 nmol/min/mg and a K(m) for L-tryptophan of 7.5+/-0.7 microM, compared to 18+/-5 microM for the partially purified enzyme from P. pastoris. To overcome the unfavorable properties of TPH, the stabilizing effect of different agents was investigated. Both tryptophan and glycerol had a stabilizing effect, whereas dithiothreitol, (6R)-5,6,7,8,-tetrahydrobiopterin, and Fe(2+) inactivated the enzyme. Irrespective of expression conditions, both native TPH expressed in bacteria or yeast, or TPH fusion proteins expressed in bacteria exhibited a strong tendency to aggregate and precipitate during purification, indicating that this is an intrinsic property of this enzyme. This supports previous observations that the enzyme in vivo may be stabilized by additional interactions.     

Expression and characterization of rat soluble catechol-O-methyltransferase fusion protein

Rat soluble catechol-O-methyltransferase cDNA was cloned into the pCAL-n-FLAG vector and expressed in Escherichia coli as a fusion protein with a calmodulin-binding peptide tag. The recombinant protein, comprising up to 30% of the total protein in the soluble fraction of E. coli, was purified by calmodulin affinity chromatography and gel filtration. Up to 16 mg of pure recombinant enzyme was recovered per liter of culture. Recombinant catechol-O-methyltransferase, in the bacterial soluble fraction, exhibited the same affinity for adrenaline as rat liver soluble catechol-O-methyltransferase (K(m) 428 [246, 609] microM and 531 [330, 732] microM, respectively), as well as the same affinity for the methyl donor, S-adenosyl-l-methionine (K(m) 27 [9, 45] microM and 38 [21, 55] microM, respectively). In addition, both the recombinant and the liver enzymes displayed the same sensitivity to the inhibitor 3,5-dinitrocatechol (IC(50) 132 [44, 397] nM and 74 [38, 143] nM, respectively), and both had the same catalytic number, respectively, 10.1 +/- 1.5 min(-1) and 8.3 +/- 0.3 min(-1). The purified recombinant enzyme also displayed the same affinity for the substrate as the purified rat liver catechol-O-methyltransferase (K(m) 336 [75, 597] microM and 439 [168, 711] microM, respectively) as well as the same inhibitor sensitivity (IC(50) 44 [19, 101] nM and 61 [33, 111] nM, respectively). This recombinant form of catechol-O-methyltransferase is kinetically identical to the rat liver enzyme. This system provides an easy and quick way of obtaining large amounts of soluble catechol-O-methyltransferase for both pharmacological and structural studies.     

Purification, properties, and oligomeric structure of glutathione reductase from the cyanobacterium Spirulina maxima

Glutathione reductase [NAD(P)H:GSSG oxidoreductase EC 1.6.4.2] from cyanobacterium Spirulina maxima was purified 1300-fold to homogeneity by a simple three-step procedure involving ammonium sulfate fractionation, ion exchange chromatography on DEAE-cellulose, and affinity chromatography on 2',5'-ADP-Sepharose 4B. Optimum pH was 7.0 and enzymatic activity was notably increased when the phosphate ion concentration was increased. The enzyme gave an absorption spectrum that was typical for a flavoprotein in that it had three peaks with maximal absorbance at 271, 370, and 460 nm and a E1%271 of 23.3 Km values were 120 +/- 12 microM and 3.5 +/- 0.9 microM for GSSG and NADPH, respectively. Mixed disulfide of CoA and GSH was also reduced by the enzyme under assay conditions, but the enzyme had a very low affinity (Km 3.3 mM) for this substrate. The enzyme was specific for NADPH. The isoelectric point of the native enzyme at 4 degrees C was 4.35 and the amino acid composition was very similar to that previously reported from other sources. The molecular weight of a subunit under denaturing conditions was 47,000 +/- 1200. Analyses of pure enzyme by a variety of techniques for molecular weight determination revealed that, at pH 7.0, the enzyme existed predominantly as a tetrameric species in equilibrium with a minor dimer fraction. Dissociation into dimers was achieved at alkaline pH (9.5) or in 6 M urea. However, the equilibrium at neutral pH was not altered by NADPH or by disulfide reducing reagents. The Mr and S20,w of the oligomeric enzyme were estimated to be 177,000 +/- 14,000 and 8.49 +/- 0.5; for the dimer, 99,800 +/- 7000 and 5.96 +/- 0.4, respectively. Low concentrations of urea increased the enzymatic activity, but this increase was not due to changes in the proportions of both forms.