Purification and properties of NADH-dependent 5, 10-methylenetetrahydrofolate reductase (MetF) from Escherichia coli

A K-12 strain of Escherichia coli that overproduces methylenetetrahydrofolate reductase (MetF) has been constructed, and the enzyme has been purified to apparent homogeneity. A plasmid specifying MetF with six histidine residues added to the C terminus has been used to purify histidine-tagged MetF to homogeneity in a single step by affinity chromatography on nickel-agarose, yielding a preparation with specific activity comparable to that of the unmodified enzyme. The native protein comprises four identical 33-kDa subunits, each of which contains a molecule of noncovalently bound flavin adenine dinucleotide (FAD). No additional cofactors or metals have been detected. The purified enzyme catalyzes the reduction of methylenetetrahydrofolate to methyltetrahydrofolate, using NADH as the reductant. Kinetic parameters have been determined at 15 degreesC and pH 7.2 in a stopped-flow spectrophotometer; the Km for NADH is 13 microM, the Km for CH2-H4folate is 0.8 microM, and the turnover number under Vmax conditions estimated for the reaction is 1,800 mol of NADH oxidized min-1 (mol of enzyme-bound FAD)-1. NADPH also serves as a reductant, but exhibits a much higher Km. MetF also catalyzes the oxidation of methyltetrahydrofolate to methylenetetrahydrofolate in the presence of menadione, which serves as an electron acceptor. The properties of MetF from E. coli differ from those of the ferredoxin-dependent methylenetetrahydrofolate reductase isolated from the homoacetogen Clostridium formicoaceticum and more closely resemble those of the NADH-dependent enzyme from Peptostreptococcus productus and the NADPH-dependent enzymes from eukaryotes.     

Stability of recombinant yeast protoporphyrinogen oxidase: effects of diphenyl ether-type herbicides and diphenyleneiodonium

Protoporphyrinogen oxidase catalyzes the oxygen-dependent aromatization of protoporphyrinogen IX to protoporphyrin IX and is the molecular target of diphenyl ether-type herbicides. Structural features of yeast protoporphyrinogen oxidase were assessed by circular dichroism studies on the enzyme purified from E. coli cells engineered to overproduce the protein. Coexpression of the bacterial gene ArgU that encodes tRNAAGA,AGG and a low induction temperature for protein synthesis were critical for producing protoporphyrinogen oxidase as a native, active, membrane-bound flavoprotein. The secondary structure of the protoporphyrinogen oxidase was 40.0 +/- 1. 5% alpha helix, 23.5 +/- 2.5% beta sheet, 18.0 +/- 2.0% beta turn, and 18.5 +/- 2.5% random-coil. Purified protoporphyrinogen oxidase appeared to be a monomeric protein that was relatively heat-labile (Tm of 44 +/- 0.5 degreesC). Acifluorfen, a potent inhibitor that competes with the tetrapyrrole substrate, and to a lower extent FAD, the cofactor of the enzyme, protected the protein from thermal denaturation, raising the Tm to 50.5 +/- 0.5 degreesC (acifluorfen) and 46.5 +/- 0.5 degreesC (FAD). However, diphenyleneiodonium, a slow tight-binding inhibitor that competes with dioxygen, did not protect the enzyme from heat denaturation. Acifluorfen binding to the protein increased the activation energy for the denaturation from 15 to 80 kJ.mol-1. The unfolding of the protein was a two-step process, with an initial fast reversible unfolding of the native protein followed by slow aggregation of the unfolded monomers. Functional analysis indicated that heat denaturation caused a loss of enzyme activity and of the specific binding of radiolabeled inhibitor. Both processes occurred in a biphasic manner, with a transition temperature of 45 degreesC.     

A comparison of human prothrombin, factor IX (Christmas factor), factor X (Stuart factor), and protein S

Human prothrombin, factor IX, and factor X have been idolated in high yield and characterized as the their amino-terminal sequence, molecular weight, amino acid composition, and migration in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. An additional human plasma protein, called protein S, has also been purified and its properties have been compared with those of prothrombin, factor IX, and factor X. Prothrombin (mol wt 72 000), factor IX (mol wt 57 000), and protein S (mol wt 69 000) are single-chain glycoproteins, while factor X (mol wt 59 000) is a glycoprotein composed of two polypeptide chains held together by a disulfide bond(s). The amino-terminal sequence of the light chain of human factor X is homologous with prothrombin, factor IX, and protein S. The heavy chain of human factor X is slightly larger than the heavy chain of bovine factor X and differs from bovine factor X in its amino-terminal sequence.     

Calmodulin-dependent protein kinase II from bovine cardiac muscle: purification and differential activation by calcium

Calmodulin-dependent protein kinase II was purified to apparent homogeneity with a high yield from the total calmodulin-binding protein fraction of bovine cardiac muscle in a single step by gel filtration column chromatography. This procedure is simple and suitable for adaptation to large scale preparations. The purified calmodulin-dependent protein kinase has a specific enzymic activity of 2.4 mumol/min/mg when mixed histone was used as a substrate. The preparation of enzyme appears to be homogeneous when examined by SDS-PAGE. The molecular weight of the enzyme was determined to be 570 kDa by gel filtration. SDS-PAGE of the enzyme subunit showed a single protein band with an apparent molecular weight of 56 kDa. These results suggest that the calmodulin-dependent protein kinase II from bovine heart is composed of 10 identical subunits. Anti-peptide antibody raised against multifunctional calmodulin-dependent protein kinase II from rat brain showed a single immunoreactive band of 56 kDa on Western blot. These results suggested that bovine cardiac muscle calmodulin-dependent protein kinase could resemble the brain isozyme. Calmodulin-dependent protein kinase II undergoes autophosphorylation with a maximal incorporation of 1 mol of phosphate per mol of the subunit of the enzyme and the autophosphorylated enzyme remains active in the absence of Ca2+ and calmodulin. The concentration of Ca2+ required for the activation of calmodulin-dependent protein kinase II depends on the level of calmodulin in the reaction.     

Human protoporphyrinogen oxidase: relation between the herbicide binding site and the flavin cofactor

Protoporphyrinogen IX oxidase (protox) catalyzes the oxidation of protoporphyrinogen IX to protoporphyrin IX in the penultimate step of heme and chlorophyll biosynthesis in animals and plants. Protox is the target of light-dependent peroxidizing herbicides and is inhibited at nanomolar levels by several chemical classes including tetrahydrophthalimides (discussed below) and diphenyl ethers (e.g., acifluorfen) usually with little selectivity between the mammalian and plant enzymes. The herbicide binding site is examined here with a photoaffinity radioligand optimized on the basis of structure-activity relationships. A radiosynthetic procedure is described for this new herbicidal probe, N-(5-azido-4-chloro-2-fluorophenyl)-3,4,5, 6-[3H]tetrahydrophthalimide ([3H]AzTHP), resulting in high specific activity (2.6 TBq/mmol). Human protox expressed in Escherichia coli and purified by affinity chromatography is used with [3H]AzTHP to characterize the herbicide/substrate binding site. Specific binding of [3H]AzTHP to human protox is rapid, completely reversible in the absence of light with a Kd of 93 nM, and competitively inhibited by the 5-propargyloxy analogue and by acifluorfen, which are known to bind at the substrate (protoporphyrinogen) site. The Bmax establishes one [3H]AzTHP binding site per FAD. Diphenyleneiodonium, proposed to inhibit protox by interaction with the FAD cofactor, inhibits enzyme activity by 48% at 100 micro M without affecting [3H]AzTHP binding in the presence or absence of substrate, suggesting that the herbicide binding site may not be proximal to FAD. The first step has been taken in photoaffinity labeling the herbicide/substrate site with [3H]AzTHP resulting in apparent covalent derivatization of 13% of the herbicide binding site.     

Factor X activating enzyme from Russell''s viper venom: isolation and characterization

The protease from Russell's viper venom that activates factor X (Stuart factor), factor IX (Christmas factor), and protein C was purified by gel filtration on Sephadex G-150 and QAE-Sephadex A-50 column chromatography. The purified enzyme migrated as a single band in sodium dodecyl sulfate-polyacrylamide gel electrophoresis with an apparent molecular weight of 79 000. A minimal molecular weight of 78 500 +/- 800 was determined by sedimentation equilibrium in the presence of 6 M guanidine hydrochloride. Upon reduction with 2-mercaptoethanol, a heavy chain (mol wt 59 000) and a light chain were observed. The light chain migrated as a single band (mol wt 19 000) in 7.5% polyacrylamide-sodium dodecyl sulfate gels but appeared as a doublet (mol wt 18 000 and 20 000) in 10% polyacrylamide-sodium dodecyl sulfate gels. The amino-terminal end of the heavy chain was heterogeneous and contained isoleucine, valine and serine. The amino-terminal sequence of the light chain was Val-Leu-Asp. The factor X activator contained 13% carbohydrate including 6.0% hexose, 1.7% N-acetyleneuraminic acid, and 5.3% galactosamine. Most of the carbohydrate was found to be present in the heavy chain, although some was also observed in both forms of the light chain. The factor X activator had no esterase activity toward benzoyl-Phe-Val-Arg-p-nitroanilide or benzoylarginine ethyl ester and was not inhibited by 0.05 M diisopropyl phosphorofluoridate. These data indicate that factor X activator from Russell's viper venom is a highly specific protease composed of one heavy chain and one light chain, and these chains are held together by a disulfide bond(s).     

Purification and characterization of a neutral, bile salt-independent retinyl ester hydrolase from rat liver microsomes. Relationship To rat carboxylesterase ES-2

A neutral, bile salt-independent retinyl ester hydrolase (NREH) has been purified from a rat liver microsomal fraction. The purification procedure involved detergent extraction, DEAE-Sepharose ion exchange, Phenyl-Sepharose hydrophobic interaction, Sephadex G-100 and Sephacryl S-200 gel filtration chromatographies, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The isolated enzyme has an apparent molecular mass of approximately 66 kDa under denaturing conditions on SDS-PAGE. Analysis of the amino acid sequences of four peptides isolated after proteolytic digestion revealed that the enzyme is highly homologous with other rat liver carboxylesterases. In particular, the sequences of the four peptides of the NREH (60 amino acids total) were identical to those of a rat carboxylesterase expressed in the liver (Alexson, S. E. H., Finlay, T. H., Hellman, U., Svensson, L. T., Diczfalusy, U., and Eggertsen, G. (1994) J. Biol. Chem. 269, 17118-17124). Antibodies against this enzyme also react with the purified NREH. Purified NREH shows a substrate preference for retinyl palmitate over triolein and did not catalyze the hydrolysis of cholesteryl oleate. With retinyl palmitate as substrate, the enzyme had a pH optimum of 7 and showed apparent saturation kinetics, with half-maximal activity achieved at substrate concentrations (Km) of approximately 70 microM.     

A small, thermostable, and monofunctional chorismate mutase from the archaeon Methanococcus jannaschii

The gene for chorismate mutase (CM) from the archaeon Methanococcus jannaschii, an extreme thermophile, was subcloned and expressed in Escherichia coli. This gene, which belongs to the aroQ class of CMs, encodes a monofunctional enzyme (AroQf) able to complement the CM deficiency of an E. coli mutant strain. The purified protein follows Michaelis-Menten kinetics (kcat = 5.7 s-1 and Km = 41 microM at 30 degreesC) and displays pH-independent activity in the range of pH 5-9. Its activation parameters [Delta H = 16.2 kcal/mol, Delta S = -1. 7 cal/(mol.K)] are similar to those of another well characterized AroQ class CM, the mesophilic AroQp domain from E. coli. Like AroQp, the thermophilic CM is an alpha-helical dimer, but approximately 5 kcal/mol more stable than its mesophilic counterpart as judged from equilibrium denaturation studies. The possible origins of the thermostability of M. jannaschii AroQf, the smallest natural CM characterized to date, are discussed in light of available sequence and tertiary structural information.     

Bacillus subtilis HemY is a peripheral membrane protein essential for protoheme IX synthesis which can oxidize coproporphyrinogen III and protoporphyrinogen IX

The hemY gene of the Bacillus subtilis hemEHY operon is essential for protoheme IX biosynthesis. Two previously isolated hemY mutations were sequenced. Both mutations are deletions affecting the hemY reading frame, and they cause the accumulation of coproporphyrinogen III or coproporphyrin III in the growth medium and the accumulation of trace amounts of other porphyrinogens or porphyrins intracellularly. HemY was found to be a 53-kDa peripheral membrane-bound protein. In agreement with recent findings by Dailey et al. (J. Biol. Chem. 269:813-815, 1994) B. subtilis HemY protein synthesized in Escherichia coli oxidized coproporphyrinogen III and protoporphyrinogen IX to coproporphyrin and protoporphyrin, respectively. The protein is not a general porphyrinogen oxidase since it did not oxidize uroporphyrinogen III. The apparent specificity constant, kcat/Km, for HemY was found to be about 12-fold higher with coproporphyrinogen III as a substrate compared with protoporphyrinogen IX as a substrate. The protoporphyrinogen IX oxidase activity is consistent with the function of HemY in a late step of protoheme IX biosynthesis, i.e., HemY catalyzes the penultimate step of the pathway. However, the efficient coproporphyrinogen III to coproporphyrin oxidase activity is unexplained in the current view of protoheme IX biosynthesis.     

Expression, characterization, and detection of human uridine phosphorylase and identification of variant uridine phosphorolytic activity in selected human tumors

Uridine phosphorylase (UPase) catalyzes the reversible phosphorolysis of uridine to uracil. We purified the enzyme from the murine colon 26 tumor using a two-step procedure through 5-amino-benzylacyclouridine affinity chromatography. Antibodies raised in rabbits against the purified protein revealed single bands in Western blots of normal human tissue and tumor extracts. The polyclonal antibody used to screen a human liver expression library allowed the isolation of a 1.2-kb clone that contained the entire open reading frame of the human UPase. The UPase cDNA has been expressed as a fusion protein in Escherichia coli using the pMal-C2 vector. The kinetic analysis demonstrated that the recombinant UPase preferentially uses uridine, 5-fluorouracil, and uracil as substrates, although lower levels of activity were observed with 2-deoxyuridine and thymidine. Clinical samples of human tumors and adjacent normal tissues were assayed for phosphorolytic activity and sensitivity to 5-benzylacyclouridine (BAU), a potent inhibitor of the enzyme presently in Phase I-II clinical trial. Activity in normal tissues appeared to be low but very sensitive to BAU (approximately 90% inhibition at 10 microM). Tumors had generally 2-3-fold greater activity compared with adjacent normal tissues. In breast cancer specimens and head-neck squamous carcinomas, however, uridine cleavage was only partially inhibited (40-60%) by 10 or 100 microM BAU. The BAU-insensitive activity requires phosphate and pH conditions similar to the normal enzyme, and the new phosphorolytic activity was independent from thymidine phosphorylase. The BAU-insensitive phosphorolytic activity in selected tumors, coupled with the potent inhibitory activity of BAU against the "classical" uridine phosphorylase in normal human tissues, provides the rationale for combining BAU with 5-fluorouracil in the treatment of breast and head-neck tumors.     

Purification of chondroitin 6-sulfotransferase secreted from cultured chick embryo chondrocytes

Chondroitin 6-sulfotransferase, which transfers sulfate from 3'-phosphoadenylyl sulfate to position 6 of N-acetylgalactosamine in chondroitin, was purified 1,430-fold to apparent homogeneity with a 22% yield from the serum-free culture medium of chick embryo chondrocytes by affinity chromatography on heparin-Sepharose CL-6B, wheat germ agglutinin-agarose, and 3',5'-ADP-agarose. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified enzyme showed a single broad protein band with an apparent molecular weight of 75,000. Since the purified enzyme has an apparent molecular weight of 160,000 as judged by gel chromatography on Superose 12, the active form of chondroitin 6-sulfotransferase may be a dimer. The purified enzyme transferred sulfate to chondroitin, chondroitin sulfate, and corneal keratan sulfate. Chondroitin sulfate E from squid cartilage, dermatan, sulfate, and heparan sulfate hardly served as acceptors of the sulfotransferase. The sulfated product derived from keratan sulfate was degraded by keratanase but not by chondroitinase ABC.     

Possible molecular mechanism of loss of homologous and heterologous gap junctional intercellular communication in rat liver epithelial cell lines

We have identified beta-galactosidase activity in purified bovine rod outer segments (ROS), using rho-nitrophenyl-beta-D-galactopyranoside (PNPG) and chlorophenol red-beta-D-galactopyranoside (CPRG) as substrates. This glycosylhydrolase activity did not appear to represent contamination from other retinal subcellular fractions, based upon the relative specific activities of beta-galactosidase vs. other hydrolases (N-acetyl-beta-glucosaminidase, alpha- and beta-mannosidase, alpha-fucosidase, and acid phosphatase) in bovine retina and ROS homogenates. Using PNPG as a substrate, two pH optima were observed (at 3.5 and 5.5), while the hydrolysis of CPRG exhibited a single, broad pH optimum centered at 5.5. In contrast, hydrolysis of PNPG and CPRG by retinal homogenates exhibited single pH optima, at 3.5 and 5.5., respectively. ROS beta-galactosidase activity increased linearly with time, temperature, and protein concentration, and obeyed Michaelis-Menten kinetics with both substrates. For PNPG, Vmax approximately 88 nmol/h/mg protein and the apparent Km approximately 147 microM. For CPRG, Vmax approximately 33 nmol/h/mg protein and the apparent Km approximately 50 microM. ROS beta-galactosidase activity was affected by carbohydrates and their derivatives: glucose, fucose, sucrose, maltose and N-acetyl-galactosamine were found to stimulate the activity, while D-galactono-gamma-lactone and, to a lesser extent, D-galactose were inhibitory. The enzyme activity also was slightly stimulated by [Cl-] and markedly by dithiothreitol (DTT), while rho-chloro-mercuribenzoic acid (PCMB) and rho-hydroxymercuribenzoic acid (PHMB) inactivated the enzyme. In addition, the enzymatic activity was also found to be differentially sensitive to various anionic and nonionic detergents. However, n-octyl-beta-D-glucoside was slightly stimulatory.(ABSTRACT TRUNCATED AT 250 WORDS)     

Isolation and characterization of a novel inositol hexakisphosphate binding protein from mammalian cell cytosol

Inositol hexakisphosphate (IP6) is present in most mammalian cells, although its intracellular function is as yet undefined. We find that the total protein fraction from bovine brain cytosol contains a significant level of specific binding for IP6 precipitable with 40% saturated ammonium sulfate. A protein complex has been isolated from this fraction that specifically binds IP6 and is purified about 500-fold over the cytosol. The IP6 binding protein (IP6BP) chromatographs as a single peak of binding activity on a gel exclusion column, with a Stokes radius equivalent to 266 +/- 14 kDa. The IP6BP is a heterooligomeric complex composed of a number of subunits with molecular weights varying from 23,000 to 60,000, as determined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS-PAGE). Scatchard analyses of IP6 binding of both the crude ammonium sulfate fraction and the purified complex show the presence of a similar high-affinity binding site (Kd approximately 6.0 nM). Bmax for the purified fraction is 1.8 nmol of IP6/mg of protein or 0.48 mol of IP6 bound/mol of complex. Other inositol polyphosphates, such as inositol 1,3,4,5,6-pentakisphosphate, inositol 1,3,4,5-tetrakisphosphate, and inositol 1,4,5-trisphosphate, are poor competitors for IP6 binding to the purified complex. The purification scheme, when applied to a rat liver cytosol fraction, yields a similar IP6BP. This complex has an apparent size of 512,000 using gel exclusion chromatography and contains an additional protein band with M(r) = 97,000 by SDS-PAGE.(ABSTRACT TRUNCATED AT 250 WORDS)     

A fast method for obtaining highly pure recombinant herpes simplex virus type 1 thymidine kinase

Recombinant Herpes Simplex Virus Type 1 thymidine kinase (TK) was isolated in a fast and gentle two-step procedure from Escherichia coli as a thrombin cleavable fusion protein. The TK was expressed as an inducible glutathione S-acetyl transferase fusion protein and purified in a first step by glutathione affinity chromatography. Proteolytic cleavage of the column bound TK with thrombin led to a truncated enzyme, resulting from two new and hitherto unknown cleavage sites, determined by N-terminal sequencing. In a second step, the TK was further purified from the cleavage products by ATP affinity chromatography, yielding homogeneously pure TK as shown by SDS-PAGE and mass spectrometry. Both the fusion protein and the purified enzyme show enzymatic activity with the same Km value of 0.2 microM for the natural substrate thymidine. Determination of the native molecular weight indicated that the pure enzyme and the fusion protein are biologically active as homodimers. Therefore the recombinant enzyme has the same biochemical characteristics as the viral TK, expressed in infected cells.     

Isolation and properties of a new, soluble, hemoprotein (H-450) from pig liver

A new soluble hemoprotein, designated as H-450, has been purified from pig liver. The absolute absorption spectrum of H-450 shows maxima at 550 and 428 nm. The dithionite-reduced H-450 has absorption peaks at 572, 540, and 450 nm; the unique Soret band at 450 nm is the basis for our tentative designation of this new hemoprotein as H-450 (hemoprotein 450). The spectrum of dithionite-reduced H-450 at 77 K gives two alpha peaks (571 and 566 nm), three beta peaks (546, 537, and 529 nm), and a Soret band at 449 nm. The prosthetic group of H-450 has been identified as protoheme IX. Gel electrophoresis experiments show that H-450 is composed of two nonidentical subunits, alpha and beta (mol wts = 61 000 and 45 000). H-450 contains 1 mol of heme/alphabeta dimer of 106 000 molecular weight. Preliminary sedimentation equilibrium experiments suggest a minimum molecular weight of 218 000 for the native protein. This corresponds to a tetramer, alpha2beta2 containing two heme groups. H-450 is not reduced by reduced nicotinamide adenine dinucleotide (NADH), NADH phosphate, ascorbate, or ferrocyanide. Neither reduced nor oxidized H-450 binds CO, 1 mM cyahide, or 1 mM azide. Dithionite-reduced H-450 is autoxidizable. The molar extinction coefficient of native H-450 is 261 X 103 at 280 nm and 263 X 103 at 428 nm. The purification procedure involves homogenization, high-speed centrifugation, ammonium sulfate fractionation, diethylaminoethylcellulose chromatography, density gradient centrifugation, a calcium phosphate gel step, and a second density gradient centrifugation. The procedure yeilds approximately 2 mg of purified protein from 750 g of pig liver.     

Benzoyl-coenzyme A reductase (dearomatizing), a key enzyme of anaerobic aromatic metabolism. ATP dependence of the reaction, purification and some properties of the enzyme from Thauera aromatica strain K172

Anoxic metabolism of many aromatic compounds proceeds via the common intermediate benzoyl-CoA. Benzoyl-CoA is dearomatized by benzoyl-CoA reductase (dearomatizing) in a two-electron reduction step, possibly yielding cyclohex-1,5-diene-1-carboxyl-CoA. This process has to overcome a high activation energy and is considered a biological Birch reduction. The central, aromatic-ring-reducing enzyme was investigated for the first time in the denitrifying bacterium Thauera aromatica strain K172. A spectrophotometric assay was developed which was strictly dependent on MgATP, both with cell extract and with purified enzyme. The oxygen-sensitive new enzyme was purified 35-fold with 20% yield under anaerobic conditions in the presence of 0.25 mM dithionite. It had a native molecular mass of approximately 170 kDa and consisted of four subunits a,b,c,d of 48, 45, 38 and 32 kDa. The oligomer composition of the protein most likely is abcd. The ultraviolet/visible spectrum of the protein as isolated, but without dithionite, was characteristic for an iron-sulfur protein with an absorption maximum at 279 nm and a broad shoulder at 390 nm. The estimated molar absorption coefficient at 390 nm was 35,000 M-1 cm-1. Reduction of the enzyme by dithionite resulted in a decrease of absorbance at 390 nm, and the colour turned from greenish-brown to red-brown. The enzyme contained 10.8 +/- 1.5 mol Fe and 10.5 +/- 1.5 mol acid-labile sulfur/mol. Besides zinc (0.5 mol/mol protein) no other metals nor selenium could be detected in significant amounts. The enzyme preparation contained a flavin or flavin-like compound; the estimated content was 0.3 mol/mol enzyme. The enzyme reaction required MgATP and a strong reductant such as Ti(III). The reaction catalyzed is: benzoyl-CoA + 2 Ti(III) + n ATP-->non-aromatic acyl-CoA + 2 Ti(IV) + n ADP + n Pi. The estimated number n of ATP molecules hydrolyzed/two electrons transferred in benzoyl-CoA reduction is 2-4. In the absence of benzoyl-CoA the enzyme exhibited oxygen-sensitive ATPase activity. The enzyme was specific for Mg(2+)-ATP, other nucleoside triphosphates being inactive (< 1%). Mg2+ could be substituted to some extent by Mn2+, Fe2+ and less efficiently by Co2+. Benzoate was not reduced, whereas some fluoro, hydroxy, amino and methyl analogues of the activated benzoic acid were reduced, albeit at much lower rate; the products remain to be identified. The specific activity with reduced methyl viologen as the electron donor was 0.55 mumol min-1 mg-1 corresponding to a catalytic number of 1.6 s-1. The apparent Km values under the assay conditions (0.5 mM for both reduced and oxidized methyl viologen) of benzoyl-CoA and ATP were 15 microM and 0.6 mM, respectively. The enzyme was inactivated by ethylene, bipyridyl and, in higher concentrations, by acetylene. Benzoyl-CoA reductase also catalyzed the ATP-dependent two-electron reduction of hydroxylamine (Km 0.15 mM) and azide. Some of the properties of the enzyme are reminiscent of those of nitrogenase which similarly overcomes the high activation energy for dinitrogen reduction by coupling electron transfer to the hydrolysis of ATP.     

Purification and some properties of maleylpyruvate hydrolase and fumarylpyruvate hydrolase from Pseudomonas alcaligenes

Hydrolysis of the gentisate ring-cleavage product, maleylpyruvate (cis-2,4-diketohept-5-enedioic acid), was shown to be catalyzed by an enzyme, maleylpyruvate hydrolase 11, in Pseudomonas alcaligenes (P25X1) after growth with 3-hydroxybenzoate. This activity was separated from fumarylpyruvate hydrolase activity during the course of its purification which accomplished an approximately 50-fold increase in specific activity. An apparent molecular weight of 77,000 was assigned on the basis of Sephadex G-200 chromatography. Despite the presence of up to three similarly migrating bands of protein on polyacrylamide-gel electrophoresis of the purified enzyme, at least two of these bands possessed maleylpyruvate hydrolase activity. Electrophoresis on sodium dodecyl sulfate-polyacrylamide before and after reduction with mercaptoethanol gave a principal band of molecular weight of 33,000 (and a minor band of molecular weight 50,000). A number of substituted maleylpyruvates also served as substrates for maleylpyruvate hydrolase 11, but maleylacetoacetate and fumarylpyruvate were not attacked. Fumarylpyruvate hydrolase was purified approximately 40-fold to give a single band on polyacrylamide gels and with an apparent molecular weight of 73,000 by Sephadex G-200 chromatography. Upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis before or after reduction with mercaptoethanol, a subunit molecular weight of 25,000 was obtained. Neither maleylpyruvate nor fumarylacetoacetate served as substrates for fumarylpyruvate hydrolase. The activities of both maleyl- and fumarylpyruvate hydrolases were stimulated by Mn(2+) ions. Reasons are discussed for the presence of both enzyme activities, one of which appears to be redundant.     

Molecular cloning and functional expression of rat liver glutathione-dependent dehydroascorbate reductase

We have isolated a cDNA clone for a novel glutathione-dependent dehydroascorbate reductase from a rat liver cDNA library in lambdagt11 by immunoscreening. The authenticity of the clone was confirmed as follows: first, the antibody that had been purified through affinity for the protein expressed by the cloned lambdagt11 phage recognized only the enzyme in a crude extract from rat liver; and second, two internal amino acid sequences of purified enzyme were identified in the protein sequence predicted from the cDNA. The predicted protein consists of 213 amino acids with a molecular weight of 24,929, which is smaller by approximately 3,000 than the value obtained by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. This discrepancy of the molecular weight was explained by post-translational modification because the recombinant protein expressed by a mammalian system (Chinese hamster ovary cells) was of the same size as rat liver enzyme but larger than the protein expressed by a bacterial system (Escherichia coli). Chinese hamster ovary cells, originally devoid of glutathione-dependent dehydroascorbate reductase activity, was made to elicit the enzyme activity (1.5 nmol/min/mg of cytosolic protein) by expression of the recombinant protein. Additionally, the cells expressing the enzyme were found to accumulate 1.7 times as much ascorbate as the parental cells after incubation with dehydroascorbate. This result points to the importance of the dehydroascorbic acid reductase in maintaining a high concentration of ascorbate in the cell.     

The delta-subunit of pyruvate ferredoxin oxidoreductase from Pyrococcus furiosus is a redox-active, iron-sulfur protein: evidence for an ancestral relationship with 8Fe-type ferredoxins

Pyruvate ferredoxin oxidoreductase (POR) from the hyperthermophilic archaeon Pyrococcus furiosus (Pf) catalyzes the final oxidative step in carbohydrate fermentation in which pyruvate is oxidized to acetyl-CoA and CO2, coupled to the reduction of ferredoxin (Fd). POR is composed of two 'catalytic units' of molecular mass approximately 120 kDa. Each unit consists of four subunits, alpha beta gamma delta, with masses of approximately 44, 36, 20, and 12 kDa, respectively, and contains at least two [4Fe-4S] clusters. The precise mechanism of catalysis and the role of the individual subunits are not known. The gene encoding the delta-subunit of Pf POR has been expressed in E. coli, and the protein was purified after reconstitution with iron and sulfide. The reconstituted delta-subunit (recPOR-delta) is monomeric with a mass of 11 879 +/- 1.2 Da as determined by mass spectrometry, in agreement with that predicted from the gene sequence. Purified recPOR-delta contains 8 Fe mol/mol and remained intact when incubated at 85 degreesC for 2 h, as judged by its visible absorption properties. The reduced form of the protein exhibited an EPR spectrum characteristic of two, spin-spin interacting [4Fe-4S]1+ clusters. When compared with the EPR properties of the reduced holoenzyme, the latter was shown to contain a third [4Fe-4S]1+ cluster in addition to the two within the delta-subunit. The reduction potential of the two 4Fe clusters in isolated recPOR-delta (-403 +/- 8 mV at pH 8.0 and 24 degreesC) decreased linearly with temperature (-1.55 mV/ degreesC) up to 82 degreesC. RecPOR-delta replaced Pf Fd as an in vitro electron carrier for two oxidoreductases from Pf, POR and Fd:NADP oxidoreductase, and the POR holoenzyme displayed a higher apparent affinity for its own subunit (apparent Km = 1.0 microM at 80 degreesC) than for Fd (apparent Km = 4.4 microM). The molecular and spectroscopic properties and amino acid sequence of the isolated delta-subunit suggest that it evolved from an 8Fe-type Fd by the addition of approximately 40 residues at the N-terminus, and that this extension enabled it to interact with additional subunits within POR.