Purification, characterization, and metabolic function of tungsten-containing aldehyde ferredoxin oxidoreductase from the hyperthermophilic and proteolytic archaeon Thermococcus strain ES-1.

Thermococcus strain ES-1 is a strictly anaerobic, hyperthermophilic archaeon that grows at temperatures up to 91 degrees C by the fermentation of peptides. It is obligately dependent upon elemental sulfur (S(o)) for growth, which it reduces to H2S. Cell extracts contain high aldehyde oxidation activity with viologen dyes as electron acceptors. The enzyme responsible, which we term aldehyde ferredoxin oxidoreductase (AOR), has been purified to electrophoretic homogeneity. AOR is a homodimeric protein with a subunit M(r) of approximately 67,000. It contains molybdopterin and one W, four to five Fe, one Mg, and two P atoms per subunit. Electron paramagnetic resonance analyses of the reduced enzyme indicated the presence of a single [4Fe-4S]+ cluster with an S = 3/2 ground state. While AOR oxidized a wide range of aliphatic and aromatic aldehydes, those with the highest apparent kcat/Km values (> 10 microM-1S-1) were acetaldehyde, isovalerylaldehyde, and phenylacetaldehyde (Km values of < 100 microM). The apparent Km value for Thermococcus strain ES-1 ferredoxin was 10 microM (with crotonaldehyde as the substrate). Thermococcus strain ES-1 AOR also catalyzed the reduction of acetate (apparent Km of 1.8 mM) below pH 6.0 (with reduced methyl viologen as the electron donor) but at much less than 1% of the rate of the oxidative reaction (with benzyl viologen as the electron acceptor at pH 6.0 to 10.0). The properties of Thermococcus strain ES-1 AOR are very similar to those of AOR previously purified from the saccharolytic hyperthermophile Pyrococcus furiosus, in which AOR was proposed to oxidize glyceraldehyde as part of a novel glycolytic pathway (S. Mukund and M. W. W. Adams, J. Biol. Chem. 266:14208-14216, 1991). However, Thermococcus strain ES-1 is not known to metabolize carbohydrates, and glyceraldehyde was a very poor substrate (kcat/Km of < 0.2 microM-1S-1) for its AOR. The most efficient substrates for Thermococcus strain ES-1 AOR were the aldehyde derivatives of transaminated amino acids. This suggests that the enzyme functions to oxidize aldehydes generated during amino acid catabolism, although the possibility that AOR generates aldehydes from organic acids produced by fermentation cannot be ruled out.     

Purification and properties of protoporphyrinogen oxidase from an anaerobic bacterium, Desulfovibrio gigas.

Protoporphyrinogen oxidase has been solubilized from plasma membranes of Desulfovibrio gigas. The enzyme was purified to apparent homogeneity with single silver-stained protein bands on isoelectric focusing and sodium dodecyl sulfate-polyacrylamide gels. This protoporphyrinogen oxidase has a molecular weight (Mr) of 148,000 and is composed of three dissimilar subunits of Mrs 12,000, 18,500, and 57,000, which are held together by sulfhydryl bonds. Unlike other protoporphyrinogen oxidases, which use molecular oxygen as an electron acceptor, this enzyme does not couple to oxygen. The protoporphyrinogen oxidase donates electrons to 2,6-dichlorophenol-indophenol but not to NAD+, NADP+, flavin adenine dinucleotide, or flavin mononucleotide. The natural physiological electron acceptor of the protoporphyrinogen oxidase from D. gigas is unknown. By using 2,6-dichlorophenol-indophenol as the electron acceptor, the Km and Vmax values for oxidation of protoporphyrinogen were determined to be 21 microM and 8.38 nmol/min per 70 micrograms of protein, respectively. The catalytic rate constant, Kcat, was calculated to be 17.7 mol of protoporphyrin formed per mole of enzyme per min of incubation, and the Kcat/Km was 0.84. Energies of activation were calculated from Arrhenius plots with 7,429 cal (ca. 31,080 J)/mol per degree below 10 degrees C and 1,455 cal (ca. 6,088, J)/mol per degree above 10 degrees C. Optimum enzyme activity was at 23 degrees C, and inhibition was observed with both N-ethylmaleimide and iodoacetamide.     

Purification and characterization of the hydrogen uptake hydrogenase from the hyperthermophilic archaebacterium Pyrodictium brockii.

Pyrodictium brockii is a hyperthermophilic archaebacterium with an optimal growth temperature of 105 degrees C. P. brockii is also a chemolithotroph, requiring H2 and CO2 for growth. We have purified the hydrogen uptake hydrogenase from membranes of P. brockii by reactive red affinity chromatography and sucrose gradient centrifugation. The molecular mass of the holoenzyme was 118,000 +/- 19,000 Da in sucrose gradients. The holoenzyme consisted of two subunits by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The large subunit had a molecular mass of 66,000 Da, and the small subunit had a molecular mass of 45,000 Da. Colorometric analysis of Fe and S content in reactive red-purified hydrogenase revealed 8.7 +/- 0.6 mol of Fe and 6.2 +/- 1.2 mol of S per mol of hydrogenase. Growth of cells in 63NiCl2 resulted in label incorporation into reactive red-purified hydrogenase. Growth of cells in 63NiCl2 resulted in label incorporation into reactive red-purified hydrogenase. Temperature stability studies indicated that the membrane-bound form of the enzyme was more stable than the solubilized purified form over a period of minutes with respect to temperature. However, the membranes were not able to protect the enzyme from thermal inactivation over a period of hours. The artificial electron acceptor specificity of the pure enzyme was similar to that of the membrane-bound form, but the purified enzyme was able to evolve H2 in the presence of reduced methyl viologen. The Km of membrane-bound hydrogenase for H2 was approximately 19 microM with methylene blue as the electron acceptor, whereas the purified enzyme had a higher Km value.     

Long-chain alcohol and aldehyde dehydrogenase activities in Acinetobacter calcoaceticus strain HO1-N.

Three alcohol dehydrogenases have been identified in Acinetobacter calcoaceticus sp. strain HO1-N: an NAD(+)-dependent enzyme and two NADP(+)-dependent enzymes. One of the NADP(+)-dependent alcohol dehydrogenases was partially purified and was specific for long-chain substrates. With tetradecanol as substrate an apparent Km value of 5.2 microM was calculated. This enzyme has a pI of 4.5 and a molecular mass of 144 kDa. All three alcohol dehydrogenases were constitutively expressed. Three aldehyde dehydrogenases were also identified: an NAD(+)-dependent enzyme, an NADP(+)-dependent enzyme and one which was nucleotide independent. The NAD(+)-dependent enzyme represented only 2% of the total activity and was not studied further. The NADP(+)-dependent enzyme was strongly induced by growth of cells on alkanes and was associated with hydrocarbon vesicles. With tetradecanal as substrate an apparent Km value of 0.2 microM was calculated. The nucleotide-independent aldehyde dehydrogenase could use either Würster's Blue or phenazine methosulphate (PMS) as an artificial electron acceptor. This enzyme represents approximately 80% of the total long-chain aldehyde oxidizing activity within the cell when the enzymes were induced by growing the cells on hexadecane. It is particulate but can be solubilized using Triton X-100. The enzyme has an apparent Km of 0.36 mM for decanal.     

Pyruvate:quinone oxidoreductase from Corynebacterium glutamicum: purification and biochemical characterization

Pyruvate:quinone oxidoreductase catalyzes the oxidative decarboxylation of pyruvate to acetate and CO2 with a quinone as the physiological electron acceptor. So far, this enzyme activity has been found only in Escherichia coli. Using 2,6-dichloroindophenol as an artificial electron acceptor, we detected pyruvate:quinone oxidoreductase activity in cell extracts of the amino acid producer Corynebacterium glutamicum. The activity was highest (0.055 +/- 0.005 U/mg of protein) in cells grown on complex medium and about threefold lower when the cells were grown on medium containing glucose, pyruvate, or acetate as the carbon source. From wild-type C. glutamicum, the pyruvate:quinone oxidoreductase was purified about 180-fold to homogeneity in four steps and subjected to biochemical analysis. The enzyme is a flavoprotein, has a molecular mass of about 232 kDa, and consists of four identical subunits of about 62 kDa. It was activated by Triton X-100, phosphatidylglycerol, and dipalmitoyl-phosphatidylglycerol, and the substrates were pyruvate (kcat=37.8 +/- 3 s(-1); Km=30 +/- 3 mM) and 2-oxobutyrate (kcat=33.2 +/- 3 s(-1); Km=90 +/- 8 mM). Thiamine pyrophosphate (Km=1 microM) and certain divalent metal ions such as Mg2+ (Km=29 microM), Mn2+ (Km=2 microM), and Co2+ (Km=11 microM) served as cofactors. In addition to several dyes (2,6-dichloroindophenol, p-iodonitrotetrazolium violet, and nitroblue tetrazolium), menadione (Km=106 microM) was efficiently reduced by the purified pyruvate:quinone oxidoreductase, indicating that a naphthoquinone may be the physiological electron acceptor of this enzyme in C. glutamicum.     

Purification and properties of an NADPH-linked aldehyde reductase from rat kidney

Rat kidney was shown to contain two NADPH-linked aldehyde reductases (alcohol:NADP+) oxidoreductase, EC 1.1.1.2) with different substrate affinities. The high-Km aldehyde reductase, which was purified to apparent homogeneity, had a molecular weight of 32 000 as determined by Sephadex G-100 gel filtration, and of 37 000 by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The purified enzyme reduced various aliphatic aldehydes of different carbon-chain lengths besides many chemicals containing aldehyde groups. The Km values for n-hexadecanal and n-octadecanal were 8 microM and 4 microM, respectively. Bovine serum albumin (1.8 mM) stimulated the reduction of n-hexadecanal and n-octadecanal, and increased the Vmax values by about 15-fold without changing the Km values. The kidney enzyme was not distinguishable from the brain and liver high-Km aldehyde reductases in mobility on polyacrylamide gel electrophoresis, immunological properties, peptide maps or substrate specificity.     

Purification and catalytic properties of Ech hydrogenase from Methanosarcina barkeri.

Methanosarcina barkeri has recently been shown to produce a multisubunit membrane-bound [NiFe] hydrogenase designated Ech (Escherichia coli hydrogenase 3) hydrogenase. In the present study Ech hydrogenase was purified to apparent homogeneity in a high yield. The enzyme preparation obtained only contained the six polypeptides which had previously been shown to be encoded by the ech operon. The purified enzyme was found to contain 0.9 mol of Ni, 11.3 mol of nonheme-iron and 10.8 mol of acid-labile sulfur per mol of enzyme. Using the purified enzyme the kinetic parameters were determined. The enzyme catalyzed the H2 dependent reduction of a M. barkeri 2[4Fe-4S] ferredoxin with a specific activity of 50 U x mg protein-1 at pH 7.0 and exhibited an apparent Km for the ferredoxin of 1 microM. The enzyme also catalyzed hydrogen formation with the reduced ferredoxin as electron donor at a rate of 90 U x mg protein-1 at pH 7.0. The apparent Km for the reduced ferredoxin was 7.5 microM. Reduction or oxidation of the ferredoxin proceeded at similar rates as the reduction or oxidation of oxidized or reduced methylviologen, respectively. The apparent Km for H2 was 5 microM. The kinetic data strongly indicate that the ferredoxin is the physiological electron donor or acceptor of Ech hydrogenase. Ech hydrogenase amounts to about 3% of the total cell protein in acetate-grown, methanol-grown or H2/CO2-grown cells of M. barkeri, as calculated from quantitative Western blot experiments. The function of Ech hydrogenase is ascribed to ferredoxin-linked H2 production coupled to the oxidation of the carbonyl-group of acetyl-CoA to CO2 during growth on acetate, and to ferredoxin-linked H2 uptake coupled to the reduction of CO2 to the redox state of CO during growth on H2/CO2 or methanol.     

Purification and properties of membrane-bound hydrogenase from Azotobacter vinelandii

Uptake hydrogenase (EC 1.12) from Azotobacter vinelandii has been purified 250-fold from membrane preparations. Purification involved selective solubilization of the enzyme from the membranes, followed by successive chromatography on DEAE-cellulose, Sephadex G-100, and hydroxylapatite. Freshly isolated hydrogenase showed a specific activity of 110 mumol of H2 uptake (min X mg of protein)-1. The purified hydrogenase still contained two minor contaminants that ran near the front on sodium dodecyl sulfate-polyacrylamide gels. The enzyme appears to be a monomer of molecular weight near 60,000 +/- 3,000. The pI of the protein is 5.8 +/- 0.2. With methylene blue or ferricyanide as the electron acceptor (dyes such as methyl or benzyl viologen with negative midpoint potentials did not function), the enzyme had pH optima at pH 9.0 or 6.0, respectively, It has a temperature optimum at 65 to 70 degrees C, and the measured half-life for irreversible inactivation at 22 degrees C by 20% O2 was 20 min. The enzyme oxidizes H2 in the presence of an electron acceptor and also catalyzes the evolution of H2 from reduced methyl viologen; at the optimal pH of 3.5, 3.4 mumol of H2 was evolved (min X mg of protein)-1. The uptake hydrogenase catalyzes a slow deuterium-water exchange in the absence of an electron acceptor, and the highest rate was observed at pH 6.0. The Km values varied widely for different electron acceptors, whereas the Km for H2 remained virtually constant near 1 to 2 microM, independent of the electron acceptors.     

Purine biosynthesis de novo in bovine retina: purification and characterization of amidophosphoribosyl transferase and phosphoribosyl pyrophosphate synthetase.

The ability of bovine retina to synthesize purines de novo is shown for the first time. Amidophosphoribosyl transferase (EC 2.4.2.14), the enzyme controlling the rate of the process, and phosphoribosyl pyrophosphate synthetase (EC 2.7.6.1), the enzyme regulating the intracellular contents of phosphoribosyl pyrophosphate (PRPP), were purified and characterized. The molecular masses of the enzyme subunits are similar to those of the purified enzyme from the liver. The molecular masses of amidophosphoribosyl transferase, PRPP synthetase catalytic subunit, and two PRPP synthetase-associated proteins are 50, 34, 39, and 41 kD, respectively. The apparent Km values of the enzymes and coenzymes are similar to those of the purified enzymes from the liver. For amidophosphoribosyl transferase, the apparent Km for Gln and PRPP are 0.75 +/- 0.05 and 0.66 +/- 0.09 mM, respectively (the corresponding Vmax values are 59 +/- 3 and 136 +/- 12 nmoles PPi/min per mg protein). For PRPP synthetase, the apparent Km for ribose-5-phosphate and ATP are 37.9 +/- 0.5 and 53 +/- 7 microM, respectively (the corresponding Vmax values are 61 +/- 4 and 52 +/- 3 nmoles PRPP/min per mg protein). The sensitivity of the retinal PRPP synthetase to inhibition by ADP and AMP was significantly lower than that of the enzyme from the liver.     

A new type of dihydroorotate dehydrogenase,type 1S,from the thermoacidophilic archaeon<Emphasis Type="Italic"> Sulfolobus solfataricus</Emphasis>

Dihydroorotate dehydrogenase (DHOD) (EC 1.3.3.1) from the thermoacidophilic archaeon Sulfolobus solfataricus P2 (DSM 1617) was partially purified 3,158-fold, characterized, and the encoding genes identified. Based on enzymological as well as phylogenetic methods, dihydroorotate dehydrogenase from S. solfataricus (DHODS) represents a new type of DHOD, type 1S. Furthermore, it is unable to use any of the (type-specific) natural electron acceptors employed by all other presently known DHODs. DHODS shows optimal activity at 70 degrees C in the pH range 7-8.5. It is capable of using ferricyanide, 2,6-dichlorophenolindophenol (DCIP), Q(0), and molecular oxygen as electron acceptor. Kinetic studies employing ferricyanide indicate a two-site ping-pong mechanism with K(M) values of 44.2+/-1.9 microM for the substrate dihydroorotate and 344+/-21 microM for the electron acceptor ferricyanide, as well as competitive product inhibition with a K(i) of 23.7+/-3.4 microM for the product orotate (OA). The specific activity, as determined from a partially purified sample, is approximately 20 micromol mg(-1) min(-1). DHODS is a heteromeric enzyme comprising a catalytic subunit encoded by pyrD (291 aa; MW=31.1 kDa) and an electron acceptor subunit (208 aa; MW=23.6 kDa), encoded by orf1. DHODS employs a serine as catalytic base, which is unique for a cytosolic DHOD. To our knowledge, this work represents not only the first study on an archaeal DHOD but the first on a nonmesophilic DHOD as well.     

Two W-containing formate dehydrogenases (CO2-reductases) involved in syntrophic propionate oxidation by Syntrophobacter fumaroxidans.

Two formate dehydrogenases (CO2-reductases) (FDH-1 and FDH-2) were isolated from the syntrophic propionate-oxidizing bacterium Syntrophobacter fumaroxidans. Both enzymes were produced in axenic fumarate-grown cells as well as in cells which were grown syntrophically on propionate with Methanospirillum hungatei as the H2 and formate scavenger. The purified enzymes exhibited extremely high formate-oxidation and CO2-reduction rates, and low Km values for formate. For the enzyme designated FDH-1, a specific formate oxidation rate of 700 U.mg-1 and a Km for formate of 0.04 mm were measured when benzyl viologen was used as an artificial electron acceptor. The enzyme designated FDH-2 oxidized formate with a specific activity of 2700 U.mg-1 and a Km of 0.01 mm for formate with benzyl viologen as electron acceptor. The specific CO2-reduction (to formate) rates measured for FDH-1 and FDH-2, using dithionite-reduced methyl viologen as the electron donor, were 900 U.mg-1 and 89 U.mg-1, respectively. From gel filtration and polyacrylamide gel electrophoresis it was concluded that FDH-1 is composed of three subunits (89 +/- 3, 56 +/- 2 and 19 +/- 1 kDa) and has a native molecular mass of approximately 350 kDa. FDH-2 appeared to be a heterodimer composed of a 92 +/- 3 kDa and a 33 +/- 2 kDa subunit. Both enzymes contained tungsten and selenium, while molybdenum was not detected. EPR spectroscopy suggested that FDH-1 contains at least four [2Fe-2S] clusters per molecule and additionally paramagnetically coupled [4Fe-4S] clusters. FDH-2 contains at least two [4Fe-4S] clusters per molecule. As both enzymes are produced under all growth conditions tested, but with differences in levels, expression may depend on unknown parameters.     

5-Carboxymethyl-2-hydroxymuconic semialdehyde dehydrogenases of Escherichia coli C and Klebsiella pneumoniae M5a1 show very high N-terminal sequence homology

5-Carboxymethyl-2-hydroxymuconic semialdehyde (CHMS) dehydrogenase from Escherichia coli C and Klebsiella pneumoniae M5a1 have been purified and some of their properties studied. The apparent Km values for NAD and CHMS were 11.7 +/- 1.5 microM and 5.2 +/- 1.9 microM, respectively, for the K. pneumoniae enzyme, and 19.5 +/- 2.7 microM and 9.2 +/- 1.4 microM, respectively, for the E. coli enzyme. Both enzymes were optimally active at pH 7.5 in sodium phosphate buffer. They had subunit molecular weights of 52,000 (+/- 1000) and the native enzymes appeared to be dimers of identical subunits. The first 20 residues of their N-terminal amino acid sequences were 90% homologous. A degenerate oligonucleotide probe constructed to a six amino acid sequence common to both enzymes gave strong hybridization with DNA from E. coli strains B and W as well as with E. coli C and K. pneumoniae but little or no hybridization to DNA from E. coli K12 or Pseudomonas putida.     

Purification and characterization of NADP(+)-dependent 5,10-methylenetetrahydrofolate dehydrogenase from Peptostreptococcus productus marburg.

The 5,10-methylenetetrahydrofolate dehydrogenase of heterotrophically grown Peptostreptococcus productus Marburg was purified to apparent homogeneity. The purified enzyme catalyzed the reversible oxidation of methylenetetrahydrofolate with NADP+ as the electron acceptor at a specific activity of 627 U/mg of protein. The Km values for methylenetetrahydrofolate and for NADP+ were 27 and 113 microM, respectively. The enzyme, which lacked 5,10-methenyltetrahydrofolate cyclohydrolase activity, was insensitive to oxygen and was thermolabile at temperatures above 40 degrees C. The apparent molecular mass of the enzyme was estimated by gel filtration to be 66 kDa. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed the presence of a single subunit of 34 kDa, accounting for a dimeric alpha 2 structure of the enzyme. Kinetic studies on the initial reaction velocities with different concentrations of both substrates in the absence and presence of NADPH as the reaction product were interpreted to indicate that the enzyme followed a sequential reaction mechanism. After gentle ultracentrifugation of crude extracts, the enzyme was recovered to greater than 95% in the soluble (supernatant) fraction. Sodium (10 microM to 10 mM) had no effect on enzymatic activity. The data were taken to indicate that the enzyme was similar to the methylenetetrahydrofolate dehydrogenases of other homoacetogenic bacteria and that the enzyme is not involved in energy conservation of P. productus.     

Purification of a novel coenzyme F420-dependent glucose-6-phosphate dehydrogenase from Mycobacterium smegmatis.

A variety of Mycobacterium species contained the 5-deazaflavin coenzyme known as F420. Mycobacterium smegmatis was found to have a glucose-6-phosphate dehydrogenase that was dependent on F420 as an electron acceptor and which did not utilize NAD or NADP. The enzyme was purified by ammonium sulfate fractionation, phenyl-Sepharose column chromatography, F420-ether-linked aminohexyl-Sepharose 4B affinity chromatography, and quaternary aminoethyl-Sephadex column chromatography, and the sequence of the first 26 N-terminal amino acids has been determined. The response of enzyme activity to a range of pHs revealed a two-peak pattern, with maxima at pH 5.5 and 8.0. The apparent Km values for F420 and glucose-6-phosphate were, respectively, 0.004 and 1.6 mM. The apparent native and subunit molecular masses were 78,000 and approximately 40,000 Da, respectively.     

Purification and properties of heterodisulfide reductase from Methanobacterium thermoautotrophicum (strain Marburg)

The reduction of the heterodisulfide of coenzyme M (H-S-CoM) and 7-mercaptoheptanoyl-L-threonine phosphate (H-S-HTP) is a key reaction in the metabolism of methanogenic bacteria. The heterodisulfide reductase catalyzing this step was purified 80-fold to apparent homogeneity from Methanobacterium thermoautotrophicum. The native enzyme showed an apparent molecular mass of 550 kDa. Sodium dodecyl sulfate/polyacrylamide gel electrophoresis revealed the presence of three different subunits of apparent molecular masses 80 kDa, 36 kDa, and 21 kDa. The enzyme, which was brownish yellow, contained per mg protein 7 +/- 1 nmol FAD, 130 +/- 10 nmol non-heme iron and 130 +/- 10 nmol acid-labile sulfur, corresponding to 4 mol FAD and 72 mol FeS/mol native enzyme. The purified heterodisulfide reductase catalyzed the reduction of CoM-S-S-HTP (app. Km = 0.1 mM) with reduced benzylviologen at a specific rate of 30 mumol.min-1.mg protein-1 (kcat = 68 s-1) and the reduction of methylene blue with H-S-CoM (app. Km = 0.2 mM) plus H-S-HTP (app. Km less than 0.05 mM) at a specific rate of 15 mumol.min-1.mg-1. The enzyme was highly specific for CoM-S-S-HTP and H-S-CoM plus H-S-HTP. The physiological electron donor/acceptor remains to be identified.     

Purification and characterization of dihydroorotate dehydrogenase from the rodent malaria parasite Plasmodium berghei.

Dihydroorotate dehydrogenase (DHODase) has been purified 400-fold from the rodent malaria parasite Plasmodium berghei to apparent homogeneity by Triton X-100 solubilization followed by anion-exchange, Cibacron Blue F3GA-agarose affinity, and gel filtration chromatography. The purified enzyme has a molecular mass of 52 +/- 2 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and of 55 +/- 6 kDa by gel filtration chromatography, and it has a pI of 8.2. It is active in monomeric form, contains 2.022 mol of iron and 1.602 acid-labile sulfurs per mole of enzyme, and does not contain a flavin cofactor. The purified DHODase exhibits optimal activity at pH 8.0 in the presence of the ubiquinone coenzyme CoQ6, CoQ7, CoQ9, or CoQ10. The Km values for L-DHO and CoQ6 are 7.9 +/- 2.5 microM and 21.6 +/- 5.5 microM, respectively. The kcat values for both substrates are 11.44 min-1 and 11.70 min-1, respectively. The reaction product orotate and an orotate analogue, 5-fluoroorotate, are competitive inhibitors of the enzyme-catalyzed reaction with Ki values of 30.5 microM and 34.9 microM, respectively. The requirement of the long-chain ubiquinones for activity supports the hypothesis of the linkage of pyrimidine biosynthesis to the electron transport system and oxygen utilization in malaria by DHODase via ubiquinones [Gutteridge, W. E., Dave, D., & Richards, W. H. G. (1979) Biochim. Biophys. Acta 582, 390-401].     

Purification and characterization of a UDP-Gal:beta-D-Gal(1,4)-D-GlcNAc alpha(1,3)-galactosyltransferase from Ehrlich ascites tumor cells

A UDP-Gal:N-acetyllactosaminide alpha (1,3)-galactosyltransferase from Ehrlich ascites tumor cells has been purified over 200,000-fold to apparent electrophoretic homogeneity. The purified enzyme transfers D-galactosyl groups from UDP-Gal to beta-D-Gal-(1,4)-D-GlcNAc in alpha-linkage. The apparent Km values for donor and acceptor substrates are 12.6 microM and 1.15 mM, respectively. The trisaccharides beta-D-Gal(1,4)-beta-D-GlcNAc(1,2)- or (1,6)-D-Man exhibit a Km 5-fold lower than that of N-acetyllactosamine, and an even more pronounced effect is observed with the biantennary pentasaccharide beta-D-Gal(1,4)-beta-D-GlcNAc(1,2)-[beta-D-Gal(1, 4)-beta-D-GlcNAc-(1,6)]-D-Man (Km 0.10 mM). The transferase shows a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions with an apparent subunit molecular weight of 80,000, exhibits a pH optimum at 6.2, and requires Mn2+ ions and detergent for enzymatic activity. Specificity studies using immobilized oligosaccharides show that the minimum acceptor structure for the alpha-galactosyltransferase is N-acetyllactosamine. The narrow specificity of the alpha-galactosyltransferase is indicated by the fact that lactose, beta-D-Gal(1,3)-D-GlcNAc, and beta-D-Gal(1,4)-[alpha-L-Fuc(1,3)]-D-GlcNAc are very poor acceptors. The enzyme differs from the blood-group B-specified galactosyltransferase in that the sequence alpha-L-Fuc(1,2)-beta-D-Gal(1,4)-D-GlcNAc is not an acceptor. Oligosaccharides, glycoproteins, glycolipids, and glycosaminoglycans containing the terminal nonreducing N-acetyllactosamine unit all serve as acceptors for the enzyme.     

Purification and characterization of benzaldehyde dehydrogenase I from Acinetobacter calcoaceticus.

Benzaldehyde dehydrogenase I was purified from Acinetobacter calcoaceticus by DEAE-Sephacel, phenyl-Sepharose and f.p.l.c. gel-filtration chromatography. The enzyme was homogeneous and completely free from the isofunctional enzyme benzaldehyde dehydrogenase II, as judged by denaturing and non-denaturing polyacrylamide-gel electrophoresis. The subunit Mr value was 56,000 (determined by SDS/polyacrylamide-gel electrophoresis). Estimations of the native Mr value by gel-filtration chromatography gave values of 141,000 with a f.p.l.c. Superose 6 column, but 219,000 with Sephacryl S300. Chemical cross-linking of the enzyme subunits indicated that the enzyme is tetrameric. Benzaldehyde dehydrogenase I was activated more than 100-fold by K+, Rb+ and NH4+, and the apparent Km for K+ was 11.2 mM. The pH optimum in the presence of K+ was 9.5 and the pI of the enzyme was 5.55. The apparent Km values for benzaldehyde and NAD+ were 0.69 microM and 96 microM respectively, and the maximum velocity was approx. 110 mumol/min per mg of protein. Various substituted benzaldehydes were oxidized at significant rates, and NADP+ was also used as cofactor, although much less effectively than NAD+. Benzaldehyde dehydrogenase I had an NAD+-activated esterase activity with 4-nitrophenol acetate as substrate, and the dehydrogenase activity was inhibited by a range of thiol-blocking reagents. The absorption spectrum indicated that there was no bound cofactor or prosthetic group. Some of the properties of the enzyme are compared with those of other aldehyde dehydrogenases, specifically the very similar isofunctional enzyme benzaldehyde dehydrogenase II from the same organism.     

Purification, characterization, and properties of an aryl aldehyde oxidoreductase from Nocardia sp. strain NRRL 5646.

An aryl aldehyde oxidoreductase from Nocardia sp. strain NRRL 5646 was purified 196-fold by a combination of Mono-Q, Reactive Green 19 agarose affinity, and hydroxyapatite chromatographies. The purified enzyme runs as a single band of 140 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The molecular mass was estimated to be 163 +/- 3.8 kDa by gel filtration, indicating that this enzyme is a monomeric protein. The binding of the enzyme to Reactive Green 19 agarose was Mg2+ dependent. The binding capacity was estimated to be about 0.2 mg of Reactive Green agarose per ml in the presence of 10 mM MgCl2. This enzyme can catalyze the reduction of a wide range of aryl carboxylic acids, including substituted benzoic acids, phenyl-substituted aliphatic acids, heterocyclic carboxylic acids, and polyaromatic ring carboxylic acids, to produce the corresponding aldehydes. The Km values for benzoate, ATP, and NADPH were determined to be 645 +/- 75, 29.3 +/- 3.1, and 57.3 +/- 12.5 microM, respectively. The Vmax was determined to be 0.902 +/- 0.04 micromol/min/mg of protein. Km values for (S)-(+)-alpha-methyl-4-(2-methylpropyl)-benzeneacetic acid (ibuprofen) and its (R)-(-) isomer were determined to be 155 +/- 18 and 34.5 +/- 2.5 microM, respectively. The Vmax for the (S)-(+) and (R)-(-) isomers were 1.33 and 0.15 micromol/min/mg of protein, respectively. Anthranilic acid is a competitive inhibitor with benzoic acid as a substrate, with a Ki of 261 +/- 30 microM. The N-terminal and internal amino acid sequences of a 76-kDa peptide from limited alpha-chymotrypsin digestion were determined.     

Purification and characterization of membrane-bound hydrogenase from Hydrogenobacter thermophilus strain TK-6, an obligately autotrophic, thermophilic, hydrogen-oxidizing bacterium

A membrane-bound hydrogenase was purified to electrophoretic homogeneity from the cells of Hydrogenobacter thermophilus strain TK-6, an obligately autotrophic, thermophilic, hydrogen-oxidizing bacterium. Solubilization and purification were done aerobically in the presence of Triton X-100. Three chromatography steps were done for purification; Butyl-Sepharose, Mono-Q, and Superose 6, in this order. Purification was completed with 6.73% yield of total activity and with 21.4-fold increase of specific activity when compared with the values for the membrane fraction. The purified hydrogenase was shown to be a tetramer with alpha2beta2 structure, with a molecular mass of 60,000 Da for the large subunit and 38,000 Da for the small subunit. The purified hydrogenase directly reduced methionaquinone with an apparent Km of around 300 microM and with a turnover number around 2900 (min(-1)). Metal analysis and EPR properties of the hydrogenase have shown that the enzyme is one of the [NiFe]-hydrogenases. Also, optimum pH and temperature for reaction, thermal stability, and electron acceptor specificity were reported. Finally, a model is presented for energy and central metabolism of H. thermophilus strain TK-6.