Respiratory ubiquinone-9 from Hyphomicrobium spec. Strain ZV 580

Ubiquinone-9, an ubiquinone with a side-chain containing 9 prenyl residues, was purified from Hyphomicrobium spec. strain ZV 580, and identified by thin-layer chromatography, UV spectroscopy, and mass spectrometry. The participation of the quinone in the reactions of the respiratory chain was established by observing its increasing reduction in a membrane fraction upon the addition of NADH, the exhaustion of oxygen, and in the presence of NADH plus cyanide. The degrees of reduction in these states matched those of the cytochromes b and c.     

Development of a fed-batch process for the production of a dye-linked formaldehyde dehydrogenase in <Emphasis Type="Italic">Hyphomicrobium zavarzinii ZV 580</Emphasis>

The dye-linked formaldehyde dehydrogenase (dlFalDH) from Hyphomicrobium zavarzinii ZV 580 processes formaldehyde in a highly selective manner and without need for NAD(P). The enzyme thus has considerable potential for technical applications if the difficulties associated with its efficient production can be resolved. In this contribution, a fed-batch bioprocess is developed, which improves both the biomass production of H. zavarzinii ZV 580 (from 0.6 to 2 g l⁻¹ dry mass) and the specific dlFalDH production (from 0.1 to 0.3 units g⁻¹ biomass), resulting in an overall improvement of the productivity by more than an order of magnitude compared to the previously reported process (Klein et al., Biochem J 301:289–295, 1994). In particular, the process uses an automated feeding strategy controlled via the dissolved oxygen concentration. In addition, our results show that the growth of H. zavarzinii ZV 580 is rather sensitive toward increasing salt concentration in the culture medium. Growth is also inhibited by the presence of surfactant-based antifoam reagents. Adjustment of the pH via the addition of methylamine instead of NaOH, on the other hand, leads to an increase in biomass yield.     

Comparative Analysis of Denitrifying Activities of Hyphomicrobium nitrativorans,Hyphomicrobium denitrificans,and Hyphomicrobium zavarzinii

Hyphomicrobium spp. are commonly identified as major players in denitrification systems supplied with methanol as a carbon source. However, denitrifying Hyphomicrobium species are poorly characterized, and very few studies have provided information on the genetic and physiological aspects of denitrification in pure cultures of these bacteria. This is a comparative study of three denitrifying Hyphomicrobium species, H. denitrificans ATCC 51888, H. zavarzinii ZV622, and a newly described species, H. nitrativorans NL23, which was isolated from a denitrification system treating seawater. Whole-genome sequence analyses revealed that although they share numerous orthologous genes, these three species differ greatly in their nitrate reductases, with gene clusters encoding a periplasmic nitrate reductase (Nap) in H. nitrativorans, a membrane-bound nitrate reductase (Nar) in H. denitrificans, and one Nap and two Nar enzymes in H. zavarzinii. Concurrently with these differences observed at the genetic level, important differences in the denitrification capacities of these Hyphomicrobium species were determined. H. nitrativorans grew and denitrified at higher nitrate and NaCl concentrations than did the two other species, without significant nitrite accumulation. Significant increases in the relative gene expression levels of the nitrate (napA) and nitrite (nirK) reductase genes were also noted for H. nitrativorans at higher nitrate and NaCl concentrations. Oxygen was also found to be a strong regulator of denitrification gene expression in both H. nitrativorans and H. zavarzinii, although individual genes responded differently in these two species. Taken together, the results presented in this study highlight the potential of H. nitrativorans as an efficient and adaptable bacterium that is able to perform complete denitrification under various conditions.     

Characterization of a second methylene tetrahydromethanopterin dehydrogenase from Methylobacterium extorquens AM1.

Cell extracts of Methylobacterium extorquens AM1 were recently found to catalyze the dehydrogenation of methylene tetrahydromethanopterin (methylene H4MPT) with NAD+ and NADP+. The purification of a 32-kDa NADP-specific methylene H4MPT dehydrogenase (MtdA) was described already. Here we report on the characterization of a second methylene H4MPT dehydrogenase (MtdB) from this aerobic alpha-proteobacterium. Purified MtdB with an apparent molecular mass of 32 kDa was shown to catalyze the oxidation of methylene H4MPT to methenyl H4MPT with NAD+ and NADP+ via a ternary complex catalytic mechanism. The Km for methylene H4MPT was 50 microM with NAD+ (Vmax = 1100 U x mg(-1) and 100 microM with NADP+ (Vmax = 950 U x mg(-1). The Km value for NAD+ was 200 microM and for NADP+ 20 microM. In contrast to MtdA, MtdB could not catalyze the dehydrogenation of methylene tetrahydrofolate. Via the N-terminal amino-acid sequence, the MtdB encoding gene was identified to be orfX located in a cluster of genes whose translated products show high sequence identities to enzymes previously found only in methanogenic and sulfate reducing archaea. Despite its location, MtdB did not show sequence similarity to archaeal enzymes. The highest similarity was to MtdA, whose encoding gene is located outside of the archaeal island. Mutants defective in MtdB were unable to grow on methanol and showed a pronounced sensitivity towards formaldehyde. On the basis of the mutant phenotype and of the kinetic properties, possible functions of MtdB and MtdA are discussed. We also report that both MtdB and MtdA can be heterologously overproduced in Escherichia coli making these two enzymes readily available for structural analysis.     

Purification and properties of methanol dehydrogenase from Hyphomicrobium x.

(1) A method for the isolation of methanol dehydrogenase (alcohol:(acceptor) oxidoreductase, EC 1.1.99.8) from Hyphomicrobium X is decribed. The purified enzyme was resolved by polyacrylamide gel electrophoresis into one main and two minor active bands. Iron and manganese were the only detected metals in the enzyme preparation. (2) The substrate, methanol, was oxidized to formic acid by a stoichiometric amount of artificial electron acceptor. During the reaction, no free formaldehyde could be detected. Other primary alcohols were oxidized to the corresponding aldehydes were a poor substrate or no substrate at all. (3) Some new and efficient one-electron acceptors were found. With these electron acceptors, the enzyme had a high pH optimum and ammonia was still required in the assay system. (4) ESR spectroscopy showed the presence of an enzyme-bound organic free radical. With X-band ESR the signal had a peak-to-peak linewidth of about 0.7 mT. The signal was further resolved by Q-band ESR and the values gparallel = 2.0024 and gperpendicular = 2.0056 were derived. (5) Under denaturing conditions the ESR signal and enzymatic activity disappeared at the same time as fluorescence appeared. Enzymatic activity is not restored when extracted cofactor and apoenzyme are brought together under normal conditions. Some properties of the unusual prosthetic group are presented in a preliminary form.     

Purification and characterization of dimethylsulfide monooxygenase from Hyphomicrobium sulfonivorans

Dimethylsulfide (DMS) is a volatile organosulfur compound which has been implicated in the biogeochemical cycling of sulfur and in climate control. Microbial degradation is a major sink for DMS. DMS metabolism in some bacteria involves its oxidation by a DMS monooxygenase in the first step of the degradation pathway; however, this enzyme has remained uncharacterized until now. We have purified a DMS monooxygenase from Hyphomicrobium sulfonivorans, which was previously isolated from garden soil. The enzyme is a member of the flavin-linked monooxygenases of the luciferase family and is most closely related to nitrilotriacetate monooxygenases. It consists of two subunits: DmoA, a 53-kDa FMNH₂-dependent monooxygenase, and DmoB, a 19-kDa NAD(P)H-dependent flavin oxidoreductase. Enzyme kinetics were investigated with a range of substrates and inhibitors. The enzyme had a K_m of 17.2 (± 0.48) μM for DMS (k_cat = 5.45 s⁻¹) and a V_max of 1.25 (± 0.01) μmol NADH oxidized min⁻¹ (mg protein⁻¹). It was inhibited by umbelliferone, 8-anilinonaphthalenesulfonate, a range of metal-chelating agents, and Hg²⁺, Cd²⁺, and Pb²⁺ ions. The purified enzyme had no activity with the substrates of related enzymes, including alkanesulfonates, aldehydes, nitrilotriacetate, or dibenzothiophenesulfone. The gene encoding the 53-kDa enzyme subunit has been cloned and matched to the enzyme subunit by mass spectrometry. DMS monooxygenase represents a new class of FMNH₂-dependent monooxygenases, based on its specificity for dimethylsulfide and the molecular phylogeny of its predicted amino acid sequence. The gene encoding the large subunit of DMS monooxygenase is colocated with genes encoding putative flavin reductases, homologues of enzymes of inorganic and organic sulfur compound metabolism, and enzymes involved in riboflavin synthesis.Dimethylsulfide (DMS) is a volatile organosulfur compound, important in the biogeochemical cycling of sulfur and global climate regulation (4, 9). Bacterial metabolism of DMS is an important sink of the compound in nature and is thought to account for degradation of over 80% of the DMS produced in the marine environment. Although bacterial pathways of DMS degradation have been studied previously in Hyphomicrobium spp. and in Thiobacillus spp. (12, 36), they remain poorly characterized, and few enzymes of DMS metabolism have been purified (see reference 32). DMS monooxygenase was first reported from an assay of NADH-dependent oxygen uptake in the presence of DMS by cell extracts of Hyphomicrobium S (12), an activity also demonstrated in cell extracts of other Hyphomicrobium, Thiobacillus, and Arthrobacter isolates (6, 7, 34), with specific activities around 30 nmol NADH oxidized min⁻¹ mg protein⁻¹. The enzyme has not previously been purified or characterized.The aims of this study were to purify and characterize the DMS monooxygenase enzyme from a member of the genus Hyphomicrobium. Since Hyphomicrobium S is no longer available, studies were undertaken using the type strain of H. sulfonivorans. The strain was originally isolated from garden soil and grows on DMS, as well as the related compounds dimethyl sulfoxide (DMSO) and dimethylsulfone (DMSO₂). During growth on DMSO₂, H. sulfonivorans first reduces DMSO₂ to DMSO by a dimethylsulfone reductase, and subsequently a DMSO reductase converts DMSO to DMS, which is further oxidized to methanethiol and formaldehyde by a DMS monooxygenase. Oxidation of methanethiol to formaldehyde by methanethiol oxidase yields another mole of formaldehyde, which is either assimilated into biomass or oxidized to carbon dioxide to provide reducing equivalents (Fig. ​(Fig.1).1). DMS monooxygenase activity is present in the soluble protein fraction during growth on these compounds (6, 7). A 53-kDa polypeptide was previously observed in organisms grown on DMS, DMSO, and DMSO₂ (6, 7), but its significance in the metabolism of these compounds was unknown.Open in a separate windowFIG. 1.Pathway and enzymes of dimethylsulfone degradation in Hyphomicrobium sulfonivorans S1. Reduction of dimethylsulfone [DMSO₂; (CH₃)₂SO₂] to dimethyl sulfoxide [DMSO; (CH₃)₂SO] and further reduction of DMSO to dimethylsulfide provides the substrate for DMS monooxygenase. Formaldehyde is either assimilated (via the serine cycle) or oxidized to CO₂ providing reducing equivalents. Sulfide is oxidized to sulfate; see reference 7 for further details.     

Purification,crystallization, and preliminary X-ray studies of a bifunctional 5,10-methenyl/methylene tetrahydrofolate cyclohydrolase/dehydrogenase from Escherichia coli

A bifunctional enzyme that catalyzes the conversion of formyltetrahydrofolate to methylene-tetrahydrofolate (5,10-methenyltetrahydrofolate cyclohydrolase and 5,10-methylene tetrahydrofolate dehydrogenease), has been subcloned from a cDNA library, purified to homogeneity, and crystallized. The crystals belong to space group I222, with unit cell dimensions of a= 64.5 Å b= 84.9 Å c= 146.1 Å. The crystal unit cell and diffraction is consistent with an asymmetric unit consisting of the enzyme monomer, and a specific volume of the unit cell of 3.2 ų/Da. The crystals diffract to at least 2.8 Å resolution after flash-cooling, when using a rotating anode x-ray source and an RAXIS image plate detector. A 2.56 Å resolution native data set has been collected at beamline X12-C at the NSLS. © 1997 Wiley-Liss, Inc.     

Purification and characterization of histidinol dehydrogenase from cabbage

Histidinol dehydrogenase (EC 1.1.1.23) activity was determined in several plant species and in cultured plant cell lines. The enzyme was purified from cabbage (Brassica oleracea) to apparent homogeneity. To render complete purification, a new, specific histidinol-Sepharose 4B affinity chromatography was developed. The apparent molecular mass of the protein is 103 kDa. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the protein migrated as a single band with a molecular mass of 52 kDa, giving evidence for a dimeric quaternary structure. By isoelectric focusing, the enzyme was separated into six protein bands, five of which possessed the dehydrogenase activity when examined by an activity staining method. The Km values for L-histidinol and NAD+ were 15.5 and 42 microM, respectively. Enzyme activity was stimulated by addition of Mn2+, but was inhibited in the presence of Ba2+, Mg2+, Ni2+, Ca2+, Zn2+, or Cu2+. Histidinol dehydrogenase is the first histidine enzyme that has been purified to homogeneity and characterized from plants. This plant enzyme catalyzes the NAD-linked four-electron dehydrogenase reaction leading from histidinol to His. The results indicate a similar pathway of His in plants and show furthermore the last two reaction steps to be identical to those in microorganisms.     

Purification and characterization of phosphoglycerate mutase from methanol-grown Hyphomicrobium X and Pseudomonas AM1.

Phosphoglycerate mutase has been purified from methanol-grown Hyphomicrobium X and Pseudomonas AMI by acid precipitation, heat treatment, ammonium sulphate fractionation, Sephadex G-50 gel filtration and DEAE-cellulose column chromatography. The purification attained using the Hyphomicrobium X extract was 72-fold, and using the Pseudomonas AMI extract, 140-fold. The enzyme purity, as shown by analytical polyacrylamide gel electrophoresis, was 50% from Hyphomicrobium X and 40% from Pseudomonas AMI. The enzyme activity was associated with one band. The purified preparations did not contain detectable amounts of phosphoglycerate kinase, phosphopyruvate hydratase, phosphoglycerate dehydrogenase or glycerate kinase activity. The molecular weight of the enzymic preparation was 32000 +/- 3000. The enzyme from both organisms was stable at low temperatures and, in the presence of 2,3-diphosphoglyceric acid, could withstand exposure to high temperatures. The enzyme from Pseudomonas AMI has a broad pH optimum at 7-0 to 7-6 whilst the enzyme from Hyphomicrobium X has an optimal activity at pH 7-3. The cofactor 2,3-diphosphoglyceric acid was required for maximum enzyme activity and high concentrations of 2-phosphoglyceric acid were inhibitory. The Km values for the Hyphomicrobium X enzyme were: 3-phosphoglyceric acid, 6-0 X 10(-3) M: 2-phosphoglyceric acid, 6-9 X 10(-4) M; 2,3-diphosphoglyceric acid, 8-0 X 10(-6) M; and for the Pseudomonas AMI ENzyme: 3-4 X 10(-3) M, 3-7 X 10(-4) M and 10 X 10(-6) M respectively. The equilibrium constant for the reaction was 11-3 +/- 2-5 in the direction of 2-phosphoglyceric acid to 3-phosphoglyceric acid and 0-09 +/- 0-02 in the reverse direction. The standard free energy for the reaction proceeding from 2-phosphoglyceric acid to 3-phosphoglyceric acid was -5-84 kJ mol(-1) and in the reverse direction +5-81 kJ mol(-1).     

Purification and characterization of hydroxypyruvate reductase from a serine-producing methylotroph, Hyphomicrobium methylovorum GM2

Hydroxypyruvate reductase of a serine-producing methylotroph, Hyphomicrobium methylovorum GM2, was purified to complete homogeneity, crystallized and characterized, the first time for an enzyme from a methylotroph. The enzyme was found to be a dimer composed of identical subunits (38 kDa), the molecular mass of the enzyme being about 70 kDa. The enzyme was stable against heating at 25 degrees C for 10 min at pH values between 5 and 9. Optimal activity was observed at pH 6.8 and around 45 degrees C. The enzyme catalyzed the reduction of hydroxypyruvate with the oxidation of only NADH. Other than hydroxypyruvate, only glyoxylate served as a substrate. The Km values were found to be 0.175 mM for hydroxypyruvate and 10.8 mM for glyoxylate. Taking advantage of the high substrate specificity of this enzyme, a means of enzymatic determination of hydroxypyruvate was established.     

Purification and characterization of the malate dehydrogenase from Streptomyces aureofaciens

The malate dehydrogenase (MDH) from Streptomyces aureofaciens was purified to homogeneity and its physical and biochemical properties were studied. Its amino-terminal sequence perfectly matched the amino-terminal sequence of the MDH from Streptomyces atratus whose biochemical characteristics have never been determined. The molecular mass of the native enzyme, estimated by size-exclusion chromatography, was 70 kDa. The protein was a homodimer, with a 38-kDa subunit molecular mass. It showed a strong specificity for NADH and was much more efficient for the reduction of oxaloacetate than for the oxidation of malate, with a pH optimum of 8. Unlike MDHs from other sources, it was not inhibited by excess oxaloacetate. This first complete functional characterization of an MDH from Streptomyces shows that the enzyme is very similar in many respects to other bacterial MDHs with the notable exception of a lack of inhibition by excess substrate.     

Purification and characterization of the d-lactate dehydrogenase from Leuconostoc lactis

• 1.1. The d-lactate dehydrogenase from Leuconostoc lactis has been purified in high yield.
• 2.2.The enzyme is a dimer of subunits of M_r = 39,000 and each subunit contains a single thiol group. The N-terminal residue is methionine.
• 3.3. The amino acid composition has been determined and is typical of that of a soluble globular protein.

     

Purification and characterization of aldehyde dehydrogenase from human brain

Aldehyde dehydrogenase (EC 1.2.1.3) has been purified from human brain; this constitutes the first purification to homogeneity from the brain of any mammalian species. Of the three isozymes purified two are mitochondrial in origin (Peak I and Peak II) and one is cytoplasmic (Peak III). By comparison of properties, the cytoplasmic Peak III enzyme could be identified as the same as the liver cytoplasmic E1 isozyme (N.J. Greenfield and R. Pietruszko (1977) Biochim. Biophys. Acta 483, 35-45). The Peak I and Peak II enzymes resemble the liver mitochondrial E2 isozyme, but both have properties that differ from those of the liver enzyme. The Peak I enzyme is extremely sensitive to disulfiram while the Peak II enzyme is totally insensitive; liver mitochondrial E2 isozyme is partially sensitive to disulfiram. The specific activity is 0.3 mumol/mg/min for the Peak I and 3.0 mumol/mg/min for the Peak II enzyme; the specific activity of the liver mitochondrial E2 isozyme is 1.6 mumol/min/mg under the same conditions. The Peak I enzyme is also inhibited by acetaldehyde at low concentrations, while the Peak II enzyme and the liver mitochondrial E2 isozyme are not inhibited under the same conditions. The precise relationship of brain Peak I and II enzymes to the liver E2 isozyme is not clear but it cannot be excluded at the present time that the two brain mitochondrial enzymes are brain specific.     

Purification and characterization of NADH dehydrogenase from Bacillus subtilis

NADH dehydrogenase from Bacillus subtilis W23 has been isolated from membrane vesicles solubilized with 0.1% Triton X-100 by hydrophobic interaction chromatography on an octyl-Sepharose CL-4B column. A 70-fold purification is achieved. No other components could be detected with sodium dodecyl sulphate polyacrylamide gel electrophoresis. Ferguson plots of the purified protein indicated no anomalous binding of sodium dodecyl sulphate and an accurate molecular weight of 63 000 could be determined. From the amino acid composition a polarity of 43.8% was calculated indicating that the protein is not very hydrophobic. Optical absorption spectra and acid extraction of the enzyme chromophore followed by thin-layer chromatography showed that the enzyme contains 1 molecule FAD/molecule. The enzyme was found to be specific for NADH. NADPH is oxidized at a rate which is less than 6% of the rate of NADH oxidation. The activity of the enzyme as determined by NADH:3-(4'-5'-dimethyl-thiazol-2-yl)2,4-diphenyltetrazolium bromide oxidoreduction is optimal at 37 C and pH 7.5-8.0. The purified enzyme has a Kapp for NADH of 60 microM and a V of 23.5 mumol NADH/min X mg protein. These parameters are not influenced by phospholipids. The enzyme activity is hardly or not at all affected by NADH-related compounds such as ATP, ADP, AMP, adenosine, deoxyadenosine, adenine and nicotinic amide indicating the high binding specificity of the enzyme for NADH.     

Purification and characterization of threonine dehydrogenase from Clostridium sticklandii

Threonine dehydrogenase from Clostridium sticklandii has been purified 76-fold from cells grown in a defined medium to a homogeneous preparation of 234 units · mg^-1 protein. Purification was obtained by chromatography on Q-Sepharose fast flow and Reactive green 19-Agarose. The native enzyme had a molecular mass of 67 kDa and consisted of two identical subunits (33 kDa each). The optimum pH for catalytic activity was 9.0. Only l-threo-threo-nine, dl--hydroxynorvaline and acetoin were substrates; only NAD was used as the natural electron acceptor. The apparent K _m values for l-threonine and NAD were 18 mM and 0.1 mM, respectively. Zn²⁺, Co²⁺ and Cu²⁺ ions (0.9 mM) inhibited enzyme activity. The N-terminal amino acid sequence revealed similarities to the class of non-metal short-chain alcohol dehydrogenases, whereas the threonine dehydrogenase from Escherichia coli belongs to the class of medium chain, zinc-containing alcohol dehydrogenases.Abbreviations PMSF phenylmethylsulfonyl fluoride - Dea diethanolamine - Tris tris-(hydroxy-methyl)-aminomethane - Nbs ₂ 5,5-dithiobis-(2-nitrobenzoic acid) - ApADN 3-acetylpyridine adenine diucleotide - thio-NAD thionicotinamide adenine dinucleotide - NBT nitro blue tetrazolium chloride     

Purification and characterization of lipoamide dehydrogenase from Trypanosoma cruzi

From Trypanosoma cruzi, the causative agent of Chagas' disease, a lipoamide dehydrogenase was isolated. The enzyme, an FAD-cystine oxidoreductase, shares many physical and chemical properties with T. cruzi trypanothione reductase, the key enzyme of the parasite's thiol metabolism. 1. From 60 g epimastigotic T. cruzi cells, 2.7 mg lipoamide dehydrogenase was extracted. The flavoenzyme was purified 3000-fold to homogeneity with an overall yield of 26%. 2. The enzyme is a dimer with a subunit Mr of 55,000. With 1 mM lipoamide (Km approximately 5 mM) and 100 microM NADH (Km = 23 microM), the specific activity at pH 7.0 is 297 U/mg. 3. With excess NADH, the enzyme is reduced to the EH2.NADH complex and, by addition of lipoamide, it is reoxidized, indicating that it can cycle between the oxidized state E and the two-electron-reduced state, EH2. 4. As shown by N-terminal sequencing of the enzyme, 21 out of 30 positions are identical with those of pig heart and human liver lipoamide dehydrogenase. The sequenced section comprises the GGGPGG stretch, which represents the binding site for the pyrophosphate moiety of FAD. 5. After reduction of Eox to the two-electron-reduced state, the enzyme is specifically inhibited by the nitrosourea drug 1,3-bis(2-chloroethyl)-1-nitrosourea (Carmustine), presumably by carbamoylation at one of the nascent active-site thiols. 6. Polyclonal rabbit antibodies raised against T. cruzi lipoamide dehydrogenase and trypanothione reductase are specific for the respective enzyme, as shown by immunoblots of the pure proteins and of cell extracts.     

Purification and characterization of tetrahydrofolate.protein complex in bovine liver

Gel filtration of bovine liver extract on a Sephadex G-200 column resolved three macromolecular fractions with dihydropteridine reductase-dependent cytochrome c reducing activity. One of the active fractions was purified from the extract through the steps of solvent fractionation, chromatography on DEAE-Sephadex, and gel filtration. Biochemical and microbiological analyses showed that the purified complex consists of a Mr = 70,000 protein and tetrahydropteroyldiglutamate. In contrast to the extreme lability of free tetrahydropteridines the complex was quite stable against autooxidation under aerobic conditions.     

Purification and characterization of dihydropyrimidine dehydrogenase from pig liver

Dihydropyrimidine dehydrogenase was isolated from cytosolic pig liver extracts and purified 3100-fold to apparent homogeneity. Purification made use of ammonium sulfate fractionation, precipitation with acetic acid and chromatography on DEAE-cellulose and 2',5'-ADP-Sepharose with 28% recovery of total activity. The native enzyme has a molecular mass of 206 kDa and is apparently composed of two similar, if not identical, subunits. Proteolytic cleavage reveals two fragments with apparent molecular masses of 92 kDa and 12 kDa. The C-terminal 12-kDa fragment seems to be extremely hydrophobic. The enzyme contains tightly associated compounds including four flavin nucleotide molecules and 32 iron atoms/206-kDa molecule. The iron atoms are probably present in iron-sulfur centers. The flavins released from the enzyme were identified as FAD and FMN in equal amounts. An isoelectric point of 4.65 was determined for the dehydrogenase. Apparent kinetic parameters were obtained for the substrates thymine, uracil, 5-aminouracil, 5-fluorouracil and NADPH.     

Hydrogen-forming and coenzyme-F420-reducing methylene tetrahydromethanopterin dehydrogenase are genetically distinct enzymes in Methanobacterium thermoautotrophicum (Marburg).

A coenzyme-F420-reducing and an H2-forming methylenetetrahydromethanopterin dehydrogenase have been isolated from Methanobacterium thermoautotrophicum (Marburg). Indirect evidence suggested that the former enzyme (32 kDa) might be derived from the latter enzyme (42 kDa) by proteolysis. To test this hypothesis the gene sequence of the H2-forming dehydrogenase was determined and compared with the N-terminal amino acid sequence of the F420-reducing dehydrogenase. No corresponding sequences were found indicating that the two dehydrogenases are genetically distinct enzymes. With purified enzyme preparations it is shown that the activity of the F420-reducing dehydrogenase is inhibited in the presence of the H2-forming enzyme. This finding is discussed in terms of substrate competition.