Methyl viologen hydrogenase II, a new member of the hydrogenase family from Methanobacterium thermoautotrophicum delta H.

Two methyl viologen hydrogenase (MVH) enzymes from Methanobacterium thermoautotrophicum delta H have been separated (resolution, Rs at 1.0) on a Mono Q column after chromatography on DEAE-Sephacel and Superose 6 Prep Grade. The newly discovered MVH (MVH II) was eluted at 0.5 M NaCl with a linear gradient of 0.45 to 0.65 M NaCl (100 ml). The previously described MVH (MVH I) eluted in a NaCl gradient at 0.56 M. The specific activities of MVH I and MVH II were 184.8 and 61.3 U/mg of protein, respectively, when enzyme activity was compared at pH 7.5, the optimal pH for MVH II. Gel electrophoresis in nondenaturing systems indicated that MVH I and MVH II had a similar molecular mass of 145 kDa. Denatured MVH II showed four protein bands (alpha, 50 kDa; beta, 44 kDa; gamma, 36 kDa; delta, 15 kDa), similar to MVH I. The N-terminal amino acid sequences of the alpha, gamma, and delta subunits of MVH II were identical with the sequences of the equivalent subunits of MVH I. However, the N-terminal amino acid sequence of the beta subunit of MVH II was totally different from the sequence of the beta subunit of MVH I. Both MVH I and MVH II had the same optimal temperature of 60 degrees C for maximum activity. The pH optima of MVH I and MVH II were 9.0 and 7.5, respectively. Most of the divalent metal ions tested significantly inhibited MVH I activity, but MVH II activity was only partially inhibited by some divalent cations. Both hydrogenases were shown to be stable for over 8 days at --20 degrees C under anaerobic conditions. When exposed to air, 90% of MVH I activity was lost within 2 min; however, MVH II lost only 50% of its activity in 3 h.     

8-Hydroxy-5-deazaflavin-reducing hydrogenase from Methanobacterium thermoautotrophicum: 1. Purification and characterization

The 8-hydroxy-5-deazaflavin (coenzyme F420) reducing hydrogenase from the obligate anaerobe Methanobacterium thermoautotrophicum delta H has been purified 41-fold to apparent homogeneity. The major active enzyme form is a high molecular weight aggregate of Mr ca. 800,000, composed of three subunits, alpha (Mr 47K), beta (Mr 31K), and gamma (Mr 26K). The hydrogenase is purified aerobically in reversibly inhibited form, and conditions for anaerobic reductive activation with H2, high salt, thiols, and electron acceptors have been defined. The minimal species transferring electrons from H2 to coenzyme F420 appears to be an alpha beta delta (Mr 115K) complex. The tightly associated redox cofactors per 115K species are 0.6-0.7 nickel atom, 0.8-0.9 flavin adenine dinucleotide (FAD), and 13-14 iron atoms in iron-sulfur centers. The subunits have been separated by denaturing gel electrophoresis, which has permitted determination of amino acid composition, subunit N-terminal sequencing, and preparation of subunit-directed antibodies. There is iron associated with the alpha-subunit, but placement of the nickel and FAD has not been established.     

HMt, a histone-related protein from Methanobacterium thermoautotrophicum delta H.

HMt, a histone-related protein, has been isolated and characterized from Methanobacterium thermoautotrophicum delta H. HMt preparations contain two polypeptides designated HMt1 and HMt2, encoded by the hmtA and hmtB genes, respectively, that have been cloned, sequenced, and expressed in Escherichia coli. HMt1 and HMt2 are predicted to contain 68 and 67 amino acid residues, respectively, and have calculated molecular masses of 7,275 and 7,141 Da, respectively. Aligning the amino acid sequences of HMt1 and HMt2 with the sequences of HMf1 and HMf2, the subunit polypeptides of HMf, a histone-related protein from the hyperthermophile Methanothermus fervidus, revealed that 40 amino acid residues (approximately 60%) are conserved in all four polypeptides. In pairwise comparisons, these four polypeptides are 66 to 84% identical. The sequences and locations of the TATA box promoter elements and ribosome binding sites are very similar upstream of the hmtA and hmtB genes in M. thermoautotrophicum and upstream of the hmfA and hmfB genes in M. fervidus. HMt binding compacted linear pUC19 DNA molecules in vitro and therefore increased their electrophoretic mobilities through agarose gels. At protein/DNA mass ratios of < 0.2:1, HMt binding caused an increase in the overall negative superhelicity of relaxed, circular DNA molecules, but at HMt/DNA mass ratios of > 0.2:1, positive supercoils were introduced into these molecules. HMt and HMf are indistinguishable in terms of their abilities to compact and constrain DNA molecules in positive toroidal supercoils in vitro. Histone-related proteins with these properties are therefore not limited to reverse gyrase-containing hyperthermophilic species.     

8-Hydroxy-5-deazaflavin-reducing hydrogenase from Methanobacterium thermoautotrophicum: 2. Kinetic and hydrogen-transfer studies

Steady-state kinetic parameters have been obtained for the pure 8-hydroxy-5-deazaflavin-reducing hydrogenase. With H2 and 8-hydroxy-5-deazariboflavin (F0) as substrates, Km (H2) = 12 microM, Km (F0) = 26 microM, and Kcat = 225 s-1. In the back-direction, F0H2 is reoxidized (anaerobically) at 225 s-1. Initial velocity patterns, product inhibition patterns, dead-end inhibition by carbon monoxide, and transhydrogenation to Procion Red HE-3B suggest a two-site hybrid ping-pong mechanism. A kinetic derivation for the rate equation is provided in the Appendix. Studies with D2 and with D2O reveal that no steps involving D transfer are substantially rate determining. Further, D2 yields F0H2 with no deuterium at C5 while in D2O a 5-monodeuterio F0H2 product is formed, indicating complete exchange of hydrogens from H2 with solvent before final transfer of a hydride ion out from reduced enzyme to C5 of F0.     

The formylmethanofuran:tetrahydromethanopterin formyltransferase from Methanobacterium thermoautotrophicum delta H. Nucleotide sequence and functional expression of the cloned gene

The formylmethanofuran:tetrahydromethanopterin formyltransferase (FTR) from Methanobacterium thermoautotrophicum delta H was cloned and its sequence was determined. The clone was contained on a 4.8-kilobase BamHI fragment of M. thermoautotrophicum DNA ligated into pBR329. When this fragment was subcloned into the phagemid pTZ18R, a functional enzyme was synthesized under control of the lac promoter. Sequence analysis revealed the presence of a ribosome binding site and a possible terminator structure. The absence of an identifiable promoter lends credibility to the open reading frame which is present 5' to ftr. The ftr gene encodes an acidic protein with a calculated molecular weight of 31,401. The sequence of FTR does not appear to be homologous to any other sequenced proteins, including proteins which use pterin substrates.     

Halotolerance of Methanobacterium thermoautotrophicum delta H and Marburg.

Methanobacterium thermoautotrophicum delta H and Marburg were adapted to grow in medium containing up to 0.65 M NaCl. From 0.01 to 0.5 M NaCl, there was a lag before cell growth which increased with increasing external NaCl. The effect of NaCl on methane production was not significant once the cells began to grow. Intracellular solutes were monitored by nuclear magnetic resonance (NMR) spectroscopy as a function of osmotic stress. In the delta H strain, the major intracellular small organic solutes, cyclic-2,3-diphosphoglycerate and glutamate, increased at most twofold between 0.01 and 0.4 M NaCl and decreased when the external NaCl was 0.5 M. M. thermoautotrophicum Marburg similarly showed a decrease in solute (cyclic-2,3-diphosphoglycerate, 1,3,4,6-tetracarboxyhexane, and L-alpha-glutamate) concentrations for cells grown in medium containing > 0.5 M NaCl. At 0.65 M NaCl, a new organic solute, which was visible in only trace amounts at the lower NaCl concentrations, became the dominant solute. Intracellular potassium in the delta H strain, detected by atomic absorption and 39K NMR, was roughly constant between 0.01 and 0.4 M and then decreased as the external NaCl increased further. The high intracellular K+ was balanced by the negative charges of the organic osmolytes. At the higher external salt concentrations, it is suggested that Na+ and possibly Cl- ions are internalized to provide osmotic balance. A striking difference of strain Marburg from strain delta H was that yeast extract facilitated growth in high-NaCl-containing medium. The yeast extract supplied only trace NMR-detectable solutes (e.g., betaine) but had a large effect on endogenous glutamate levels, which were significantly decreased. Exogenous choline and glycine, instead of yeast extract, also aided growth in NaCl-containing media. Both solutes were internalized with the choline converted to betaine; the contribution to osmotic balance of these species was 20 to 25% of the total small-molecule pool. These results indicate that M. thermoautotrophicum shows little changes in its internal solutes over a wide range of external NaCl. Furthermore, they illustrate the considerable differences in physiology in the delta H and Marburg strains of this organism.     

Cloning, expression, and nucleotide sequence of the formate dehydrogenase genes from Methanobacterium formicicum

The genes for the two subunits of the formate dehydrogenase from Methanobacterium formicicum were cloned and their sequences determined. When expressed in Escherichia coli, two proteins were produced which had the appropriate mobility on an SDS gel for the two subunits of formate dehydrogenase and cross-reacted with antibodies raised to purified formate dehydrogenase. The genes for the two formate dehydrogenase subunits overlap by 1 base pair and are preceded by DNA sequences similar to both eubacterial and archaebacterial promoters and ribosome-binding sites. The amino acid sequences deduced from the DNA sequence were analyzed, and the arrangement of putative iron-sulfur centers is discussed.     

Isolation, amino acid analysis and N-terminal sequence determination of the two subunits of the nickel-containing hydrogenase of Desulfovibrio gigas

The two subunits of the nickel-iron hydrogenase from Desulfovibrio gigas have been purified by preparative sodium dodecyl sulfate polyacrylamide gel electrophoresis and their amino acid compositions have been determined. The N-terminal sequences for 15 residues of the large subunit (Mr 62,000) and 25 residues of the small subunit (Mr 26,000), respectively, were established. The occurrence of several cysteine residues in the small subunit is discussed in relation with their possible role in the binding of the redox centers of the enzyme.     

Component A2 of methylcoenzyme M reductase system from Methanobacterium thermoautotrophicum delta H: nucleotide sequence and functional expression by Escherichia coli.

The gene for component A2 of the methylcoenzyme M reductase system from Methanobacterium thermoautotrophicum delta H was cloned, and its nucleotide sequence was determined. The gene for A2, designated atwA, encodes an acidic protein of 59,335 Da. Amino acid sequence analysis revealed partial homology of A2 to a number of eucaryotic and bacterial proteins in the ATP-binding cassette (ABC) family of transport systems. Component A2 possesses two ATP-binding domains. A 2.2-kb XmaI-BamHI fragment containing atwA and the surrounding open reading frames was cloned into pGEM-7Zf(+). A cell extract from this strain replaced purified A2 from M. thermoautotrophicum delta H in an in vitro methylreductase assay.     

Purification and characterization of the reduced-nicotinamide-dependent 2,2''-dithiodiethanesulfonate reductase from Methanobacterium thermoautotrophicum delta H.

A novel reduced nicotinamide-dependent disulfide reductase, the 2,2'-dithiodiethanesulfonate [(S-CoM)2] reductase (CoMDSR) of Methanobacterium thermoautotrophicum was purified 405-fold to electrophoretic homogeneity. Both NADPH and NADH functioned as electron donors, although rates with NADPH were three times higher. Reduced factor F420, the deazaflavin electron carrier characteristic of methanogenic bacteria, was not a substrate for the enzyme. The enzyme was most active with (S-CoM)2 but could also reduce L-cystine at 23% the (S-CoM)2 rate. Results of sodium dodecyl sulfate polyacrylamide gel electrophoresis indicated that the enzyme was monomeric with an Mr of about 64,000; spectral analysis showed that it was a flavoprotein with an estimated composition of one molecule of flavin per polypeptide. Maximal activity occurred at 64 degrees C, and the pH optimum was 8.5. The apparent Km for both NADPH and (S-CoM)2 was 80 microM. The enzyme was completely inactivated by oxygen in crude cell extracts but was oxygen stable in the homogeneous state. The low activity of the CoMDSR in cell extracts as well as its relatively low rate of reducing CoM-S-S-HTP (the heterodisulfide of the two thiol cofactors involved in the last step of methanogenesis) make it unlikely that it plays a role in the methylreductase system. It may be involved in the redox balance of the cell, such as the NADPH-dependent bis-gamma-glutamylcystine reductase with which it shows physical similarity in another archaebacterium, Halobacterium halobium (A. R. Sundquist and R. C. Fahey, J. Bacteriol. 170:3459-3467, 1988). The CoMDSR might also be involved in regenerating the coenzyme M trapped as its homodisulfide, a nonutilizable form of the cofactor.     

Identification of an alternative nucleoside triphosphate: 5'-deoxyadenosylcobinamide phosphate nucleotidyltransferase in Methanobacterium thermoautotrophicum delta H

Computer analysis of the archaeal genome databases failed to identify orthologues of all of the bacterial cobamide biosynthetic enzymes. Of particular interest was the lack of an orthologue of the bifunctional nucleoside triphosphate (NTP):5'-deoxyadenosylcobinamide kinase/GTP:adenosylcobinamide-phosphate guanylyltransferase enzyme (CobU in Salmonella enterica). This paper reports the identification of an archaeal gene encoding a new nucleotidyltransferase, which is proposed to be the nonorthologous replacement of the S. enterica cobU gene. The gene encoding this nucleotidyltransferase was identified using comparative genome analysis of the sequenced archaeal genomes. Orthologues of the gene encoding this activity are limited at present to members of the domain Archaea. The corresponding ORF open reading frame from Methanobacterium thermoautotrophicum Delta H (MTH1152; referred to as cobY) was amplified and cloned, and the CobY protein was expressed and purified from Escherichia coli as a hexahistidine-tagged fusion protein. This enzyme had GTP:adenosylcobinamide-phosphate guanylyltransferase activity but did not have the NTP:AdoCbi kinase activity associated with the CobU enzyme of S. enterica. NTP:adenosylcobinamide kinase activity was not detected in M. thermoautotrophicum Delta H cell extract, suggesting that this organism may not have this activity. The cobY gene complemented a cobU mutant of S. enterica grown under anaerobic conditions where growth of the cell depended on de novo adenosylcobalamin biosynthesis. cobY, however, failed to restore adenosylcobalamin biosynthesis in cobU mutants grown under aerobic conditions where de novo synthesis of this coenzyme was blocked, and growth of the cell depended on the assimilation of exogenous cobinamide. These data strongly support the proposal that the relevant cobinamide intermediates during de novo adenosylcobalamin biosynthesis are adenosylcobinamide-phosphate and adenosylcobinamide-GDP, not adenosylcobinamide. Therefore, NTP:adenosylcobinamide kinase activity is not required for de novo cobamide biosynthesis.     

Identification of the gltX gene encoding glutamyl-tRNA synthetase from Methanobacterium thermoautotrophicum

The gltX gene encoding glutamyl-tRNA synthetase from Methanobacterium thermoautotrophicum has been cloned, sequenced, and identified. The gene is located immediately downstream of idsA in an operon containing at least three additional ORFs. The deduced protein sequence from gltX contains conserved regions (HIGH and KMSKS) indicative of a class I aminoacyl-tRNA synthetase.     

F390 synthetase and F390 hydrolase from Methanobacterium thermoautotrophicum (strain delta H).

Factor F390 is the 8-OH adenylated form of the deazaflavin coenzyme F420, which is a central electron carrier in methanogenic bacteria. The enzymes catalysing the formation of F390 from ATP and F420 (F390 synthetase) and its hydrolysis into AMP and F420 (F390 hydrolase) were isolated and partially purified from Methanobacterium thermoautotrophicum. Both enzymes were oxygen-stable. The F390 synthetase tended to coelute with coenzyme F420 reducing hydrogenase during all purification steps. The 30-fold purified enzyme was still contaminated with the hydrogenase. The F390 hydrolase was purified 135-fold to a specific activity of 8.6 mumol/min/mg protein. The colourless enzyme consisted of one polypeptide of approximately 27,000 kd.     

Characterization of an 8-hydroxy-5-deazaflavin:NADPH oxidoreductase from Streptomyces griseus

An 8-hydroxy-5-deazaflavin-dependent oxidoreductase was isolated from the actinomycete Streptomyces griseus and purified 590-fold with 72% overall yield. The enzyme catalyzes electron transfer between 8-hydroxy-5-deazaflavins and NADPH. It seems to be more specific than methanogenic oxidoreductase as it has an absolute requirement for both the 5-deazaflavin structure and the presence of an 8-hydroxy group in the substrate. A molecular weight of 42,000 was found with gel permeation chromatography, while SDS gel electrophoresis indicated the presence of two identical subunits. Maximal enzymatic activity was found at 0.32 M NaCl and pH 5.9 for reduction of 8-hydroxy-5-deazaflavin and pH 7.9 for the reverse reaction. From the kinetic constants it was estimated that the main function of this oxidoreductase is probably to provide cells with reduced 8-hydroxy-5-deazaflavin to be used in specific reduction reactions. These results indicate the occurrence of 8-hydroxy-5-deazaflavin-dependent electron transfer in microorganisms not belonging to the archaebacteria.     

Reductive activation of the methyl coenzyme M methylreductase system of Methanobacterium thermoautotrophicum delta H.

When titanium(III) citrate was used as electron donor for the reduction of methyl coenzyme M by the methyl coenzyme M methylreductase system of Methanobacterium thermoautotrophicum delta H, component A1 was no longer required. The simpler system thus obtained required components A2, A3, and C as well as catalytic amounts of ATP, vitamin B12, and the disulfide of 7-mercaptoheptanoylthreonine phosphate in addition to titanium(III) citrate. This three component enzyme system also could produce CH4 when stoichiometric amounts of 7-mercaptoheptanoylthreonine phosphate were used as a source of electrons under an H2 atmosphere. When 7-mercaptoheptanoylthreonine phosphate or H2 was used alone no CH4 was produced, indicating a dual requirement for reducing equivalents: one to activate the methylreductase system and the other to reduce methyl coenzyme M. This is the first evidence that the activation of methyl coenzyme M methylreductase is a reductive process.     

Purification and properties of 5,10-methylenetetrahydromethanopterin reductase, a coenzyme F420-dependent enzyme, from Methanobacterium thermoautotrophicum strain delta H

5,10-Methylenetetrahydromethanopterin reductase was purified 22-fold to apparent homogeneity from the methanogenic bacterium Methanobacterium thermoautotrophicum. The enzyme catalyzes the reduction of 5,10-methylene- to 5-methyltetrahydromethanopterin. The electron carrier coenzyme F420 is specifically used as the cosubstrate. The reductase reaction may proceed in both directions, methylene reduction is, however, thermodynamically favored. In addition, the velocity of the reaction in this direction exceeds the reverse reaction by a factor of 26. The reductase is composed of a single subunit with an estimated Mr = 35,000. The active enzyme does not contain a flavin prosthetic group or iron-sulfur clusters, in contrast to 5,10-methylenetetrahydrofolate reductases purified from eukaryotic and eubacterial sources, which catalyze an analogous reaction as the methanogenic reductase.     

Cloning of genes, purification and properties investigation of recombinant DNA ligases from the thermophilic archaeon Pyrococcus abyssi and Methanobacterium thermoautotrophicum

The genes encoding of DNA ligases from the thermophilic archaeon Pyrococcus abyssi (PabDNA ligase) and Methanobacterium thermoautotrophicum (MthDNA ligase) were cloned and expressed in Escherichia coli. The activity of purified enzymes was studied by ligation of two oligonucleotides, one of which had preformed hairpin structure. In the used system the maximal output of reaction products for both DNA ligases was observed near 70 degrees C that is explained by substrate thermostability. At stoichiometric ratio of enzymes and substrate the output of a product reaches of plateau at 70-75% of theoretical ones. Investigated DNA ligases showed different thermostability. The half-time life of PabDNA ligase was about 60 min at 90 degrees C. MthDNA ligase was completely inactivated at this temperature during 10 min. Recombinant DNA ligases from P. abyssi and M. thermoautotrophicum possessed high stability during a storage at 4 degrees C.     

Cloning and sequencing of the genes encoding the large and the small subunits of the H2 uptake hydrogenase (hup) of Rhodobacter capsulatus

Summary The structural genes (hup) of the H₂ uptake hydrogenase of Rhodobacter capsulatus were isolated from a cosmid gene library of R. capsulatus DNA by hybridization with the structural genes of the H₂ uptake hydrogenase of Bradyrhizobium japonicum. The R. capsulatus genes were localized on a 3.5 kb HindIII fragment. The fragment, cloned onto plasmid pAC76, restored hydrogenase activity and autotrophic growth of the R. capsulatus mutant JP91, deficient in hydrogenase activity (Hup^-). The nucleotide sequence, determined by the dideoxy chain termination method, revealed the presence of two open reading frames. The gene encoding the large subunit of hydrogenase (hupL) was identified from the size of its protein product (68108 dalton) and by alignment with the NH₂ amino acid protein sequence determined by Edman degradation. Upstream and separated from the large subunit by only three nucleotides was a gene encoding a 34 256 dalton polypeptide. Its amino acid sequence showed 80% identity with the small subunit of the hydrogenase of B. japonicum. The gene was identified as the structural gene of the small subunit of R. capsulatus hydrogenase (hupS). The R. capsulatus hydrogenase also showed homology, but to a lesser extent, with the hydrogenase of Desulfovibrio baculatus and D. gigas. In the R. capsulatus hydrogenase the Cys residues, (13 in the small subunit and 12 in the large subunit) were not arranged in the typical configuration found in [4Fe–4S] ferredoxins.