Subcloning, characterization, and affinity labeling of Escherichia coli glycinamide ribonucleotide transformylase

Glycinamide ribonucleotide transformylase (GAR TFase; EC 2.1.2.2) has been purified 70-fold to apparent homogeneity from Escherichia coli harboring an expression vector encoding the purN gene product, GAR TFase. The protein is a monomer of Mr 23,241 and catalyzes a single reaction. Steady-state kinetic parameters for the enzyme have been obtained. The structural requirements for cofactor utilization have been investigated and found to parallel those of the multifunctional avian enzyme. The enzyme was inactivated with the affinity label N10-(bromoacetyl)-5,8-dideazafolate in a stoichiometric and active-site-specific manner. The ionization state of the cofactor analogue in the enzyme-cofactor complex appears to require the dissociation of the proton at N3 of the pyrimidine within the complex.     

Active enzyme sedimentation, sedimentation velocity, and sedimentation equilibrium studies of succinyl-CoA synthetases of porcine heart and Escherichia coli

Succinyl-CoA synthetases from Escherichia coli and porcine heart muscle have been viewed as prototypes of two classes of the enzyme. The bacterial enzyme has been reported to be an alpha 2 beta 2 tetramer, with many suggestions in the literature for cooperative interactions between active sites that may contribute to its catalytic efficacy. In contrast, gel filtration experiments of others have indicated that the heart enzyme is a simple alpha beta dimer, with no evidence of dimerization or interaction between like sites. All previous estimates of molecular size of these enzymes have been carried out at concentrations that are much higher than those that are used during activity measurements. The present study was carried out to confirm the differences in the quaternary structures of these two species of succinyl-CoA synthetase and to extend our knowledge of these structures to very low concentrations to enable correlation of their subunit structures with their catalytic properties. Conventional sedimentation velocity centrifugation with both enzymes indicates behavior typical of noninteracting globular proteins with no evidence of size heterogeneity. The sedimentation coefficients at infinite dilution (s20,w) have been determined to be 7.04 S and 4.55 S for the E. coli and porcine heart enzymes, respectively. Sedimentation velocity measurements have been extended to very low enzyme concentrations (typical of those used in activity measurements) by active enzyme centrifugation experiments, in which we have determined the rate of sedimentation of a zone of active enzyme through a chromogenic substrate solution.(ABSTRACT TRUNCATED AT 250 WORDS)     

Expression, purification and characterization of enoyl-ACP reductase II, FabK, from Porphyromonas gingivalis

The rapid rise in bacterial drug resistance coupled with the low number of novel antimicrobial compounds in the discovery pipeline has led to a critical situation requiring the expedient discovery and characterization of new antimicrobial drug targets. Enzymes in the bacterial fatty acid synthesis pathway, FAS-II, are distinct from their mammalian counterparts, FAS-I, in terms of both structure and mechanism. As such, they represent attractive targets for the design of novel antimicrobial compounds. Enoyl-acyl carrier protein reductase II, FabK, is a key, rate-limiting enzyme in the FAS-II pathway for several bacterial pathogens. The organism, Porphyromonas gingivalis, is a causative agent of chronic periodontitis that affects up to 25% of the US population and incurs a high national burden in terms of cost of treatment. P. gingivalis expresses FabK as the sole enoyl reductase enzyme in its FAS-II cycle, which makes this a particularly appealing target with potential for selective antimicrobial therapy. Herein we report the molecular cloning, expression, purification and characterization of the FabK enzyme from P. gingivalis, only the second organism from which this enzyme has been isolated. Characterization studies have shown that the enzyme is a flavoprotein, the reaction dependent upon FMN and NADPH and proceeding via a Ping-Pong Bi-Bi mechanism to reduce the enoyl substrate. A sensitive assay measuring the fluorescence decrease of NADPH as it is converted to NADP(+) during the reaction has been optimized for high-throughput screening. Finally, protein crystallization conditions have been identified which led to protein crystals that diffract x-rays to high resolution.     

Cloning, sequence analysis, and characterization of the 'lysyl oxidase' from Pichia pastoris

Lysyl oxidase from Pichia pastoris has been successfully overexpressed. EPR and resonance Raman experiments have shown that copper and TPQ are present, respectively. Lysyl oxidase from P. pastoris has a similar substrate specificity to the mammalian enzyme (both have been shown to oxidize peptidyl lysine residues) and is 30% identical to the human kidney diamine oxidase (the highest of any non-mammalian source). This enzyme also has a relatively broad substrate specificity compared to other amine oxidases. Molecular modeling data suggest that the substrate channel in lysyl oxidase from P. pastoris permits greater active site access than observed in structurally-characterized amine oxidases. This larger channel may account for the diversity of substrates that are turned over by this enzyme.     

Purification, characterization, cloning, and amino acid sequence of the bifunctional enzyme 5,10-methylenetetrahydrofolate dehydrogenase/5,10-methenyltetrahydrofolate cyclohydrolase from Escherichia coli.

We have purified the enzyme 5,10-methylenetetrahydrofolate dehydrogenase (EC 1.5.1.5) from Escherichia coli to homogeneity by a newly devised procedure. The enzyme has been purified at least 2,000-fold in a 31% yield. The specific activity of the enzyme obtained is 7.4 times greater than any previous preparation from this source. The purified enzyme is specific for NADP. The protein also contains 5,10-methenyltetrahydrofolate cyclohydrolase (EC 3.5.4.9) activity. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and behavior on a molecular sieving column suggest that the enzyme is a dimer of identical subunits. We have cloned the E. coli gene coding for the enzyme through the use of polymerase chain reaction based on primers designed from the NH2 terminal analysis of the isolated enzyme. We sequenced the gene. The derived amino acid sequence of the enzyme contains 287 amino acids of Mr 31,000. The sequence shows 50% identity to two bifunctional mitochondrial enzymes specific for NAD, and 40-45% identity to the presumed dehydrogenase/cyclohydrolase domains of the trifunctional C1-tetrahydrofolate synthase of yeast mitochondria and cytoplasm and human and rat cytoplasm. An identical sequence of 14 amino acids with no gaps is present in all 7 sequences.     

Carbamoyl-phosphate synthetases from Neurospora crassa. Immunological relatedness of the enzymes from Neurospora, bacteria, yeast, and mammals

Neurospora crassa contains two carbamoyl-phosphate synthetases: a mitochondrial enzyme (CPS-A) which supplies carbamoyl phosphate for arginine biosynthesis, and a nuclear enzyme whose product is used for the synthesis of pyrimidines. We have prepared antiserum against a highly purified preparation of the large subunit of CPS-A and have used the antiserum to demonstrate that the large subunit is, like most mitochondrially localized proteins, initially synthesized as a higher molecular weight precursor. The CPS-A antiserum cross-reacts with the nuclear enzyme, allowing us to identify the product of the complex N. crassa pyr-3 genetic locus as a protein with a subunit molecular weight of 180,000. Finally, we have found that the CPS-A antiserum also cross-reacts with carbamoyl-phosphate synthetases from bacteria, yeast, and mammals. The immunological relatedness of carbamoyl-phosphate synthetases from such diverse species suggests that the protein sequences required for carbamoyl phosphate production have been highly conserved during the course of evolution.     

Pyruvate decarboxylase of Zymomonas mobilis: isolation, properties, and genetic expression in Escherichia coli.

Pyruvate decarboxylase (EC 4.1.1.1) from Zymomonas mobilis purified to homogeneity by using dye-ligand and ion-exchange chromatography. Antibodies produced against the enzyme and the amino-terminal sequence obtained for the pure enzyme were used to select and confirm the identity of a genomic clone encoding the enzyme selected from a genomic library of Z. mobilis DNA cloned into pUC9. The genomic fragment encoding the enzyme expressed high levels of pyruvate decarboxylase in Escherichia coli. Possible RNA polymerase and ribosome-binding sites have been identified in the 5'-untranslated region of the pyruvate decarboxylase gene.     

Microsomal enzymes of cholesterol biosynthesis from lanosterol. Characterization, solubilization, and partial purification of NADPH-dependent delta 8,14-steroid 14-reductase

The membrane-bound enzyme of microsomes that catalyzes NADPH-dependent reduction of the 14-double bond of conjugated delta 8,14- and delta 7,14-sterols has been studied both as collected in microsomes from broken cell preparations of rat liver and after solubilization. Optimal incubation conditions for assay of the membrane-bound enzyme have been determined, and properties of the microsomal enzyme have been established with respect to cofactor requirements, kinetics, pH, addition of inhibitors, addition of glycerol phosphatides, and sterol substrate specificity. The 14-reductase is readily solubilized with a mixture of octylglucoside and taurodeoxycholic acid. The solubilized enzyme has been enriched by precipitation with polyethylene glycol and chromatography on DEAE-Sephacel and hydroxylapatite columns. The resulting partially purified enzyme has been obtained free of other microsomal enzymes of cholesterol biosynthesis: 4-methyl sterol oxidase, delta 5,7-sterol 7-reductase, delta 8,24-sterol 24-reductase, 3-ketosteroid reductase, and steroid 8----7-ene isomerase, plus microsomal cytochrome P-450, cytochrome P-450 reductase, cytochrome b5 reductase, and cytochrome b5. The partially purified enzyme is stimulated by addition of phospholipids. All of the properties exhibited by partially purified 14-reductase are consistent with the suggestion that the solubilized and enriched enzyme catalyzes the microsomal reduction of the 14-double bond of the sterol-conjugated dienes. However, presence of the enzyme does not prove that the sterol-conjugated dienes are obligatory precursors of cholesterol.     

Activation energy, relative substrate specificity and optimum pH of guanase from humanrat, mouse and guinea pig sera and tissues.

Some properties of serum and tissue guanase from man, rat, mouse and guinea pig have been studied. Serum guanase differs from tissue guanase in activation energy in the human species and in guinea pig. The mouse and rat brain enzyme differs from the liver and kidney enzyme. The guanase of each species differs from the enzyme of the other species in one or more properties.     

Characterization of botryococcene synthase enzyme activity, a squalene synthase-like activity from the green microalga Botryococcus braunii, Race B

The extracellular matrix of the alga Botryococcus braunii, Race B, consists mainly of botryococcenes, which have potential as a hydrocarbon fuel. Botryococcenes are structurally similar to squalene raising the possibility of a common enzyme for the biosynthesis of both. While B. braunii squalene synthase (SS) enzyme activity has been documented, botryococcene synthase (BS) enzyme activity has not been. In the current study, an assay for BS activity has been developed and used to show that many of the assay conditions for BS enzyme activity are similar to those of SS. However, SS enzyme activity is stimulated by Tween 80 while BS enzyme activity is inhibited. Moreover, BS enzyme activity was correlated with the accumulation of botryococcenes during a B. braunii culture growth cycle, which was distinctly different from the profile of SS enzyme activity. While the current results indicate a conservation of enzymological features amongst the BS and SS enzymes, raising the possibility of one enzyme capable of catalyzing both activities, they are also consistent with these two activities arising from separate and distinct enzymes.     

Typing of human, animal, food, and environmental isolates of Listeria monocytogenes by multilocus enzyme electrophoresis

In order to elucidate some aspects of the epidemiology of listeriosis in Switzerland, 181 strains of Listeria monocytogenes isolated from humans, animals, food, and the environment have been analyzed by multilocus enzyme electrophoresis at 21 enzyme loci. The clone responsible for several recent food-borne outbreaks in Switzerland and in North America (marked by electrophoretic type 1 and serovar 4b) has been found frequently among strains isolated from animals. Thus, animals may represent a major source of diffusion of this clone in the environment and in food, in which it has been found only sporadically, however. Two other unrelated clones (including strains belonging to serovars 1/2b and 1/2c) have often been isolated from meat but not from animals. These findings indicate that contamination of meat with L. monocytogenes might originate mainly from the environment in which it is processed rather than from animals themselves. This could explain the differences in the distribution of L. monocytogenes serovars isolated from meat and from animals.     

Cytochrome c oxidases: polypeptide composition, role of subunits, and location of active metal centers

The general structure of the enzyme, its polypeptide composition, and a proposal for a rational nomenclature are discussed. The mitochondrially coded and bacterial cytochrome c oxidase subunits have been analyzed with more attention focused on elucidating the number of metals present in the enzyme and the ligands available for their coordination. The picture of a 2 Cu/2 Fe enzyme has been compared with that of a 3 Cu/2 Fe enzyme and a new model is proposed for the location of the metal centers in the enzyme.     

Mouse-liver glutathione reductase. Purification, kinetics, and regulation.

Glutathione reductase from the liver of DBA/2J mice was purified to homogeneity by means of ammonium sulfate fractionation and two subsequent affinity chromatography steps using 8-(6-aminohexyl)-amino-2'-phospho-adenosine diphosphoribose and N6-(6-aminohexyl)-adenosine 2',5'-biphosphate-Sephadex columns. A facile procedure for the synthesis of 8-(6-aminohexyl)-amino-2'-phospho-adenosine diphosphoribose is also presented. The purified enzyme exhibits a specific activity of 158 U/mg and an A280/A460 of 6.8. It was shown to be a dimer of Mr 105000 with a Stokes radius of 4.18 nm and an isoelectric point of 6.46. Amino acid composition revealed some similarity between the mouse and the human enzyme. Antibodies against mouse glutathione reductase were raised in rabbits and exhibited high specificity. The catalytic properties of mouse liver glutathione reductase have been studied under a variety of experimental conditions. As with the same enzyme from other sources, the kinetic data are consistent with a 'branched' mechanism. The enzyme was stabilized against thermal inactivation at 80 degrees C by GSSG and less markedly by NADP+ and GSH, but not by NADPH or FAD. Incubation of mouse glutathione reductase in the presence of NADPH or NADH, but not NADP+ or NAD+, produced an almost complete inactivation. The inactivation by NADPH was time, pH and concentration dependent. Oxidized glutathione protected the enzyme against inactivation, which could also be reversed by GSSG or other electron acceptors. The enzyme remained in the inactive state even after eliminating the excess NADPH. The inactive enzyme showed the same molecular weight as the active glutathione reductase. The spectral properties of the inactive enzyme have also been studied. It is proposed that auto-inactivation of glutathione reductase by NADPH and the protection as well as reactivation by GSSG play in vivo an important regulatory role.     

Aspartokinase from wheat germ: isolation, characterization, and regulation

Aspartokinase has been isolated from wheat germ and a preliminary survey made of its properties in a partially purified extract. The enzyme has an absolute requirement for ATP and a divalent metal ion. The phosphate donor can be either ATP or GTP, but other nucleotides are ineffective. Both magnesium and manganese will activate the enzyme, whereas calcium shows a trace amount of activity. The enzyme has a Km of 16.7 mm for aspartate, 1.2 mm for ATP, and 3.3 mm for MgCl(2). Lysine inhibits the reaction at fairly low concentrations, and threonine inhibits at high concentrations. Other amino acids which are derived from aspartate (methionine, homoserine, threonine, and isoleucine) have little effect. When lysine and threonine are added together, they show a concerted inhibition of the reaction. The enzyme is also stabilized against heat inactivation by lysine and threonine together but not by either when added separately. It is suggested that aspartokinase from plants is a regulatory enzyme and exhibits a concerted feedback mechanism.     

Purification, crystallization, and preliminary X-ray data for porcine fumarase

Single crystals of fumarase purified from pig heart have been prepared from solutions containing polyethylene glycol. The crystals give diffraction data corresponding to Bragg spacings of 2.0 A and contain a single subunit of the enzyme in the asymmetric unit of the C222 unit cell. Therefore, the subunits of this tetrameric molecule are arranged with the point symmetry group 222. The present purification scheme and studies of the NH2-terminal amino acid sequences suggest that only a single form of the enzyme is present, and it is thought to be the mitochondrial enzyme.     

Typing of human, animal, food, and environmental isolates of Listeria monocytogenes by multilocus enzyme electrophoresis.

In order to elucidate some aspects of the epidemiology of listeriosis in Switzerland, 181 strains of Listeria monocytogenes isolated from humans, animals, food, and the environment have been analyzed by multilocus enzyme electrophoresis at 21 enzyme loci. The clone responsible for several recent food-borne outbreaks in Switzerland and in North America (marked by electrophoretic type 1 and serovar 4b) has been found frequently among strains isolated from animals. Thus, animals may represent a major source of diffusion of this clone in the environment and in food, in which it has been found only sporadically, however. Two other unrelated clones (including strains belonging to serovars 1/2b and 1/2c) have often been isolated from meat but not from animals. These findings indicate that contamination of meat with L. monocytogenes might originate mainly from the environment in which it is processed rather than from animals themselves. This could explain the differences in the distribution of L. monocytogenes serovars isolated from meat and from animals.     

Further inhibition studies on guanidinobenzoatase, a trypsin-like enzyme associated with tumour cells

Guanidinobenzoatase is a proteolytic enzyme capable of degrading fibronectin and is a tumour associated enzyme. Guanidinobenzoatase has been shown to be an arginine selective protease and is distinct from trypsin, plasminogen activator, plasmin, thrombin and a newly described tumour associated enzyme specific for guanidino phenylalanine residues. These conclusions have been derived from inhibition studies employing 4-methyl-p-guanidinobenzoate as substrate. Three active site titrants for trypsin have been shown to be good substrates for guanidinobenzoatase. A new active site titrant for trypsin, rhodamine bisguanidinobenzoate, can also be used to assay guanidinobenzoatase in a stoichiometric manner. This active site titrant can be employed to label guanidinobenzoate on the surface of leukaemia cells.     

Purification, Characterization, and Comparison of the DNA Polymerases from Two Primate RNA Tumor Viruses

The DNA polymerase from the Mason-Pfizer monkey virus (M-PMV), an RNA tumor virus not typical type-C or type-B, has been purified a thousand-fold over the original crude viral suspension. This purified enzyme is compared to a similarly purified DNA polymerase from the primate woolly monkey virus, a type-C virus. The two enzymes have similar template specificities but differ in their requirements for optimum activity. Both DNA polymerases have a pH optimum of 7.3 in Tris buffer. M-PMV enzyme has maximum activity with 5 mM Mg(2+) and 40 mM potassium chloride, whereas the woolly monkey virus optima are 100 mM potassium chloride with 0.8 mM Mn(2+). The apparent molecular weight of the M-PMV enzyme is approximately 110,000, whereas the woolly monkey virus polymerase is approximately 70,000. The biochemical properties of these two enzymes were also compared to a similarly purified enzyme from a type-C virus from a lower mammal (Rauscher murine leukemia virus). The results show that more similarity exists between the DNA polymerases from viruses of the same type (type-C), than between the polymerases from viruses of different types but from closely related species.     

Purification, characterisation and mutagenesis of highly expressed recombinant yeast pyruvate kinase.

Recombinant yeast pyruvate kinase has been purified from a strain of Saccharomyces cerevisiae expressing the enzyme to very high levels. Expression was from a multicopy plasmid under the control of the yeast phosphoglycerate kinase promoter. The gene was expressed in the absence of the genomically encoded pyruvate kinase, using a strain of yeast in which the pyruvate kinase gene has been disrupted by the insertion of the yeast Ura3 gene. The purification procedure minimised proteolytic artefacts and enabled the convenient purification of 15-20 mg enzyme from 11 culture. The purified enzyme was characterised by a high specific activity and by a lack of proteolytic degradation. Two active-site mutants of yeast pyruvate kinase have been produced, expressed and characterised in this system and preliminary results are described.     

Catalytic activity and substrate specificity of the flavin-containing monooxygenase in microsomal systems: characterization of the hepatic, pulmonary and renal enzymes of the mouse, rabbit, and rat

Inhibitory antibodies against NADPH-cytochrome P-450 reductase, detergent solubilization to dissociate functional interaction between the reductase and cytochrome P-450, and selective trypsin degradation have been used to characterize flavin-containing monooxygenase activity in microsomes from different tissues and species. A comparison of assay methods is reported. The native microsome-bound flavin-containing monooxygenase of mouse, rabbit, and rat liver, lung, and kidney can metabolize compounds containing thiol, sulfide, thioamide, secondary and tertiary amine, hydrazine, and phosphine substituents. Therefore, this enzyme from these common experimental animals has catalytic capabilities similar to those of the well-characterized porcine liver enzyme. True allosteric activation by n-octylamine does not appear to be a property of either the mouse, rabbit, or rat liver enzymes, but is a property of the pig liver and mouse lung enzymes. The microsomal pulmonary flavin-containing monooxygenase of the rabbit has some unique substrate preferences which differ from the mouse lung enzyme. Both the rabbit and mouse pulmonary enzymes have recently been shown to be distinct enzyme forms. However, the rat pulmonary flavin-containing monooxygenase appears to be catalytically identical to the rat liver enzyme, and does not have any of the unusual catalytic properties of either the rabbit or mouse lung enzymes. Enzyme activity of mouse, rabbit, and rat kidney microsomes is qualitatively similar to the hepatic activities. Substrates which saturate the microsome-bound flavin-containing monooxygenase at 1.0 mM, including thiourea, thioacetamide, methimazole, cysteamine, and thiobenzamide, are metabolized at common maximal velocities. This suggests that the kinetic mechanism of the native enzyme is similar to that established for the isolated porcine liver enzyme in that the rate-limiting step of catalysis occurs after substrate binding, and that all substrates capable of saturating the microsomal enzyme should be metabolized at a common maximal velocity.