Diversity and three-dimensional structures of the alpha Mcr of the methanogenic Archaea from the anoxic region of Tucuruí Lake, in Eastern Brazilian Amazonia

Methanogenic archaeans are organisms of considerable ecological and biotechnological interest that produce methane through a restricted metabolic pathway, which culminates in the reaction catalyzed by the Methyl-coenzyme M reductase (Mcr) enzyme, and results in the release of methane. Using a metagenomic approach, the gene of the α subunit of mcr (mcrα) was isolated from sediment sample from an anoxic zone, rich in decomposing organic material, obtained from the Tucuruí hydroelectric dam reservoir in eastern Brazilian Amazonia. The partial nucleotide sequences obtained were 83 to 95% similar to those available in databases, indicating a low diversity of archaeans in the reservoir. Two orders were identified - the Methanomicrobiales, and a unique Operational Taxonomic Unit (OTU) forming a clade with the Methanosarcinales according to low bootstrap values. Homology modeling was used to determine the three-dimensional (3D) structures, for this the partial nucleotide sequence of the mcrα were isolated and translated on their partial amino acid sequences. The 3D structures of the archaean Mcrα observed in the present study varied little, and presented approximately 70% identity in comparison with the Mcrα of Methanopyrus klanderi. The results demonstrated that the community of methanogenic archaeans of the anoxic C1 region of the Tucurui reservoir is relatively homogeneous.     

Identification of the mcrC gene product in Methanococcus vannielii

Abstract The polypeptide encoded by the mcr C gene has been identified in Methanococcus vannielii by immunoblotting using rabbit antibodies raised against the product of a lac Z- mcr C gene fusion synthesized and purified from Escherichia coli . The mcr C gene product (gp mcr C) was located in both supernatant and pellet fractions after centrifugation of Mc. vannielii cell extracts for 2 h at 100 000 × g . When anaerobic reducing conditions were maintained during purification, gp mcr C co-sedimented through sucrose gradients to the same position as molecules of the methyl coenzyme M reductase holoenzyme (approx. 300 kDa). This co-sedimentation was lost under aerobic, nonreducing conditions.     

Nucleotide sequence of the R.meliloti nitrogenase reductase (nifH) gene.

The nucleotide sequence of the structural gene (nifH) of nitrogenase reductase (Fe protein) from R.meliloti 41 with its flanking ends is reported. The amino acid sequence of nitrogenase reductase was deduced from the DNA sequence. The predicted R.meliloti nitrogenase reductase protein consists of 297 amino acid residues, has a molecular weight of 32,740 daltons and contains 5 cysteine residues. The codon usage in the nifH gene is presented. In the 5' flanking region, sequences resembling to consensus sequences of bacterial control regions were found. Comparison of the R.meliloti nifH nucleotide and amino acid sequences with those from different nitrogen-fixing organisms showed that the amino acid sequences are more conserved than the nucleotide sequences. This structural conservation of nitrogenase reductase may be related to its function and may explain the conservation of the nifH gene during evolution.     

Differential expression of methanogenesis genes of Methanothermobacter thermoautotrophicus (formerly Methanobacterium thermoautotrophicum) in pure culture and in cocultures with fatty acid-oxidizing syntrophs

The expression of genes involved in methanogenesis in a thermophilic hydrogen-utilizing methanogen, Methanothermobacter thermoautotrophicus strain TM, was investigated both in a pure culture sufficiently supplied with H(2) plus CO(2) and in a coculture with an acetate-oxidizing hydrogen-producing bacterium, Thermacetogenium phaeum strain PB, in which hydrogen partial pressure was constantly kept very low (20 to 80 Pa). Northern blot analysis indicated that only the mcr gene, which encodes methyl coenzyme M reductase I (MRI), catalyzing the final step of methanogenesis, was expressed in the coculture, whereas mcr and mrt, which encodes methyl coenzyme M reductase II (MRII), the isofunctional enzyme of MRI, were expressed at the early to late stage of growth in the pure culture. In contrast to these two genes, two isofunctional genes (mtd and mth) for N(5),N(10)-methylene-tetrahydromethanopterin dehydrogenase, which catalyzes the fourth step of methanogenesis, and two hydrogenase genes (frh and mvh) were expressed both in a pure culture and in a coculture at the early and late stages of growth. The same expression pattern was observed for Methanothermobacter thermoautotrophicus strain DeltaH cocultured with a thermophilic butyrate-oxidizing syntroph, Syntrophothermus lipocalidus strain TGB-C1. Two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis of whole proteins of M. thermoautotrophicus strain TM obtained from a pure culture and a coculture with the acetate-oxidizing syntroph and subsequent N-terminal amino acid sequence analysis confirmed that MRI and MRII were produced in the pure culture, while only MRI was produced in the coculture. These results indicate that under syntrophic growth conditions, the methanogen preferentially utilizes MRI but not MRII. Considering that hydrogenotrophic methanogens are strictly dependent for growth on hydrogen-producing fermentative microbes in the natural environment and that the hydrogen supply occurs constantly at very low concentrations compared with the supply in pure cultures in the laboratory, the results suggest that MRI is an enzyme primarily functioning in natural methanogenic ecosystems.     

Phylogenetic analysis of methyl coenzyme-M reductase detected from the bovine rumen

AIMS: The object of the present study is isolation of methyl coenzyme-M reductase (MCR) genes (mcrA) from the bovine rumen fluid and determination of phylogenetical placements of the genes to investigate mechanisms of methanogenesis in the rumen from a point of view of mcrA genes. METHODS: Genes for methanogen-specific MCR were isolated from the bovine rumen by PCR amplification. The deduced amino acid sequences were fitted to the alignments of mcrA gene products from the referred sequences. SIGNIFICANCE AND IMPACT OF THE STUDY: Although the deduced amino acid sequences of mcrA genes, isolated from the bovine rumen in the present study, were close to that of Methanobrevibacter ruminantium, these amino acid sequences did not fall into known clusters of MCR. The findings suggest that methanogenesis in the rumen would be partially carried out by unknown methanogens.     

The sequence of squash NADH:nitrate reductase and its relationship to the sequences of other flavoprotein oxidoreductases. A family of flavoprotein pyridine nucleotide cytochrome reductases.

Nucleotide sequences were determined for cDNA clones for squash NADH:nitrate oxidoreductase (EC 1.6.6.1), which is one of the most completely characterized forms of this higher plant enzyme. An open reading frame of 2754 nucleotides began at the first ATG. The deduced amino acid sequence contains 918 residues, with a predicted Mr = 103,376. The amino acid sequence is very similar to sequences deduced for other higher plant nitrate reductases. The squash sequence has significant similarity to the amino acid sequences of sulfite oxidase, cytochrome b5, and NADH:cytochrome b5 reductase. Alignment of these sequences with that of squash defines domains of nitrate reductase that appear to bind its 3 prosthetic groups (molybdopterin, heme-iron, and FAD). The amino acid sequence of the FAD domain of squash nitrate reductase was aligned with FAD domain sequences of other NADH:nitrate reductases, NADH:cytochrome b5 reductases, NADPH:nitrate reductases, ferredoxin:NADP+ reductases, NADPH:cytochrome P-450 reductases, NADPH:sulfite reductase flavoproteins, and Bacillus megaterium cytochrome P-450BM-3. In this multiple alignment, 14 amino acid residues are invariant, which suggests these proteins are members of a family of flavoenzymes. Secondary structure elements of the structural model of spinach ferredoxin:NADP+ reductase were used to predict the secondary structure of squash nitrate reductase and the other related flavoenzymes in this family. We suggest that this family of flavoenzymes, nearly all of which reduce a hemoprotein, be called "flavoprotein pyridine nucleotide cytochrome reductases."     

Mitochondrial NADH-cytochrome b(5) reductase plays a crucial role in the reduction of D-erythroascorbyl free radical in Saccharomyces cerevisiae.

The relevance of NADH-cytochrome b(5) reductase to the NADH-dependent reduction of D-erythroascorbyl free radical was investigated in Saccharomyces cerevisiae. MCR1, which is known to encode NADH-cytochrome b(5) reductase in S. cerevisiae, was disrupted by the insertion of URA3 gene into the gene of MCR1. In the mcr1 disruptant cells, the activity of NADH-D-erythroascorbyl free radical reductase almost disappeared and the intracellular level of D-erythroascorbic acid was about 11% of that of the congenic wild-type strain. In the transformant cells carrying MCR1 in multicopy plasmid, the intracellular level of D-erythroascorbic acid and the activity of NADH-D-erythroascorbyl free radical reductase increased up to 1.7-fold and 2.1-fold, respectively. Therefore, it indicated that the MCR1 product, mitochondrial NADH-cytochrome b(5) reductase, plays a key role in the NADH-dependent reduction of D-erythroascorbyl free radical in S. cerevisiae. On the other hand, the mcr1 disruptant cells were hypersensitive to hydrogen peroxide and menadione, and overexpression of MCR1 made the cells more resistant against oxidative stress. These results suggested that the mitochondrial NADH-cytochrome b(5) reductase functions as NADH-D-erythroascorbyl free radical reductase and plays an important role in the response to oxidative damage in S. cerevisiae.     

Evidence that the protein components of bovine erythrocyte green heme binding protein and flavin reductase are identical

Bovine erythrocyte green heme binding protein and bovine erythrocyte flavin reductase have been isolated in highly purified forms and subjected to amino acid analysis and N-terminal amino acid sequence analysis. The two proteins possess similar amino acid compositions and identical N-terminal amino acid sequences. Moreover, the two proteins are immunochemically cross-reactive and are indistinguishable when compared by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by double diffusion technique. This study provides evidence that the protein components of bovine erythrocyte green heme binding protein and flavin reductase are identical.     

Amino acid sequences of pyridoxal 5'-phosphate binding sites and fluorescence resonance energy transfer in chicken liver fatty acid synthase

The amino acid sequences associated with pyridoxal 5'-phosphate binding sites in chicken liver fatty acid synthase have been determined: a site whose modification causes selective inhibition of the enoyl reductase activity and a site (site I) that is not associated with enzymatic activity. The amino acid sequences of peptides obtained by trypsin hydrolysis of the pyridoxamine 5'-phosphate labeled enzyme were determined. For the site associated with enoyl reductase activity, the sequence similarities between chicken and goose are extensive and include the sequence Ser-X-X-Lys, a characteristic structural feature of pyridoxamine enzymes. In addition, the spatial relationships between the pyridoxal 5'-phosphate binding sites and reductase site(s) have been studied with fluorescence resonance energy-transfer techniques. The distances between site I and the enoyl reductase and beta-ketoacyl reductase sites are greater than 50 and 41-44 A, respectively. The distance between the two reductase sites is greater than 49 A.     

Sequence analysis of the large and small subunits of human ribonucleotide reductase.

We have isolated and sequenced overlapping cDNA clones from a breast carcinoma cDNA library containing the entire coding region of both the R1 and R2 subunits of the human ribonucleotide reductase gene. The coding region of the human R1 subunit comprises 2376 nucleotides and predicts a polypeptide of 792 amino acids (calculated molecular mass 90,081). The sequence of this subunit is almost identical to the equivalent mouse ribonucleotide reductase subunit with 97.7% homology between the mouse and human R1 subunit amino acid sequences. The coding region of the human R2 subunit of ribonucleotide reductase comprises 1170 nucleotides and predicts a polypeptide of 389 amino acids (calculated molecular mass 44,883), which is one amino acid shorter than the equivalent mouse subunit. The human and mouse R2 subunits display considerable homology in their carboxy-terminal amino acid sequences, with 96.3% homology downstream of amino acid 68 of the human and mouse R2 proteins. However, the amino-terminal portions of these two proteins are more divergent in sequence, with only 69.2% homology in the first 68 amino acids.     

Isolation and Characterization of a Soluble NADPH-Dependent Fe(III) Reductase from Geobacter sulfurreducens

NADPH is an intermediate in the oxidation of organic compounds coupled to Fe(III) reduction in Geobacter species, but Fe(III) reduction with NADPH as the electron donor has not been studied in these organisms. Crude extracts of Geobacter sulfurreducens catalyzed the NADPH-dependent reduction of Fe(III)-nitrilotriacetic acid (NTA). The responsible enzyme, which was recovered in the soluble protein fraction, was purified to apparent homogeneity in a four-step procedure. Its specific activity for Fe(III) reduction was 65 micromol. min(-1). mg(-1). The soluble Fe(III) reductase was specific for NADPH and did not utilize NADH as an electron donor. Although the enzyme reduced several forms of Fe(III), Fe(III)-NTA was the preferred electron acceptor. The protein possessed methyl viologen:NADP(+) oxidoreductase activity and catalyzed the reduction of NADP(+) with reduced methyl viologen as electron donor at a rate of 385 U/mg. The enzyme consisted of two subunits with molecular masses of 87 and 78 kDa and had a native molecular mass of 320 kDa, as determined by gel filtration. The purified enzyme contained 28.9 mol of Fe, 17.4 mol of acid-labile sulfur, and 0.7 mol of flavin adenine dinucleotide per mol of protein. The genes encoding the two subunits were identified in the complete sequence of the G. sulfurreducens genome from the N-terminal amino acid sequences derived from the subunits of the purified protein. The sequences of the two subunits had about 30% amino acid identity to the respective subunits of the formate dehydrogenase from Moorella thermoacetica, but the soluble Fe(III) reductase did not possess formate dehydrogenase activity. This soluble Fe(III) reductase differs significantly from previously characterized dissimilatory and assimilatory Fe(III) reductases in its molecular composition and cofactor content.     

Purification and characterization of homogeneous assimilatory reduced nicotinamide adenine dinucleotide phosphate-nitrate reductase from Neurospora crassa.

Neurospora crassa wild type STA4 NADPH-nitrate reductase (NADPH : nitrate oxidoreductase, EC 1.6.6.3) has been purified 5000-fold with an overall yield of 25--50%. The final purified enzyme contained 4 associated enzymatic activities: NADPH-nitrate reductase, FADH2-nitrate reductase, reduced methyl viologen-nitrate reductase and NADPH-cytochrome c reductase. Polyacrylamide gel electrophoresis yielded 1 major and 1 minor protein band and both bands exhibited NADPH-nitrate and reduced methyl viologen-nitrate reductase activities. SDS gel electrophoresis yielded 2 protein bands corresponding to molecular weights of 115 000 and 130 000. A single N-terminal amino acid (glutamic acid) was found and proteolytic mapping for the two separated subunits appeared similar. Purified NADPH-nitrate reductase contained 1 mol of molybdenum and 2 mol of cytochrome b557 per mol protein. Non-heme iron, zinc and copper were not detectable. It is proposed that the Neurospora assimilatory NADPH-nitrate reductase consists of 2 similar cytochrome b557-containing 4.5-S subunits linked together by one molybdenum cofactor. A revised electron flow scheme is presented. p-Hydroxymercuribenzoate inhibition was reversed by sulfhydryl reagents. Inhibitory pattern of p-hydroxymercuribenzoate and phenylglyoxal revealed accessible sulfhydryl and arginyl residue(s) as functional group(s) in the earlier part of electron transport chain as possibly the binding site of NADPH or FAD.     

Isolation and sequence studies of cysteinyl peptides from Spirulina glutathione reductase: comparison of active site cysteine peptides with those of other flavoprotein disulfide oxidoreductases

The amino acid sequences of the cysteinyl peptides of Spirulina sp. glutathione reductase were determined. Spirulina glutathione reductase was covalently bound to Thiopropyl-Sepharose 6B in the presence of 8M urea through thiol-disulfide exchange. After tryptic digestion, 4 distinct cysteinyl peptides were finally isolated from NADPH-reduced glutathione reductase and 2 from oxidized glutathione reductase. The amino acid sequences of the two cysteinyl peptides which could not be isolated from the oxidized glutathione reductase were very similar to those around the active site disulfide of the other flavoprotein disulfide oxidoreductases and a unique replacement of asparagine and valine by isoleucine and arginine between the two cysteine residues was found. The other two peptides isolated from both oxidized and reduced glutathione reductase also show considerable homology to the corresponding parts of human and Escherichia coli glutathione reductases.     

Thioredoxin-thioredoxin reductase system of Streptomyces clavuligerus: sequences, expression, and organization of the genes.

The genes that encode thioredoxin and thioredoxin reductase of Streptomyces clavuligerus were cloned, and their DNA sequences were determined. Previously, we showed that S. clavuligerus possesses a disulfide reductase with broad substrate specificity that biochemically resembles the thioredoxin oxidoreductase system and may play a role in the biosynthesis of beta-lactam antibiotics. It consists consists of two components, a 70-kDa NADPH-dependent flavoprotein disulfide reductase with two identical subunits and a 12-kDa heat-stable protein general disulfide reductant. In this study, we found, by comparative analysis of their predicted amino acid sequences, that the 35-kDa protein is in fact thioredoxin reductase; it shares 48.7% amino acid sequence identity with Escherichia coli thioredoxin reductase, the 12-kDa protein is thioredoxin, and it shares 28 to 56% amino acid sequence identity with other thioredoxins. The streptomycete thioredoxin reductase has the identical cysteine redox-active region--Cys-Ala-Thr-Cys--and essentially the same flavin adenine dinucleotide- and NADPH dinucleotide-binding sites as E. coli thioredoxin reductase and is partially able to accept E. coli thioredoxin as a substrate. The streptomycete thioredoxin has the same cysteine redox-active segment--Trp-Cys-Gly-Pro-Cys--that is present in virtually all eucaryotic and procaryotic thioredoxins. However, in vivo it is unable to donate electrons to E. coli methionine sulfoxide reductase and does not serve as a substrate in vitro for E. coli thioredoxin reductase. The S. clavuligerus thioredoxin (trxA) and thioredoxin reductase (trxB) genes are organized in a cluster. They are transcribed in the same direction and separated by 33 nucleotides. In contrast, the trxA and trxB genes of E. coli, the only other organism in which both genes have been characterized, are physically widely separated.     

cDNA sequence of adrenodoxin reductase. Identification of NADP-binding sites in oxidoreductases

Adrenodoxin reductase is an NADP dependent flavoenzyme which functions as the reductase of mitochondrial P 450 systems. We sequenced two adrenodoxin reductase cDNAs isolated from a bovine adrenal cortex cDNA library. The deduced amino acid sequence shows no similarity to the sequence of the microsomal P 450 systems or other known protein sequences. Nonetheless, by sequence analysis and c comparisons with known sequences of dinucleotide-binding folds of two NADP-binding flavoenzymes, two regions of adrenodoxin reductase sequence were identified as the FAD- and NADP-binding sites. These analyses revealed a consensus sequence for the NADP-binding dinucleotide fold (GXGXXAXXXAXXXXXXG, in one-letter amino acid code) that differs from FAD and NAD-binding dinucleotide-fold sequences. In the data base of protein sequences, the NADP-binding-site sequence appears solely in NADP-dependent enzymes, the binding sites of which were not known to date. Thus, this sequence may be used for identification of a certain type of NADP-binding site of enzymes that show no significant sequence similarity.