The magnetic properties of the nickel cofactor F430 in the enzyme methyl-coenzyme M reductase of Methanobacterium thermoautotrophicum.
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ABSTRACT:
SUBMITTER: Cheesman MR
PROVIDER: S-EPMC1138715 | biostudies-other | 1989 Jun
REPOSITORIES: biostudies-other
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Project description:BACKGROUND: RNA ligases are essential reagents for many methods in molecular biology including NextGen RNA sequencing. To prevent ligation of RNA to itself, ATP independent mutant ligases, defective in self-adenylation, are often used in combination with activated pre-adenylated linkers. It is important that these ligases not have de-adenylation activity, which can result in activation of RNA and formation of background ligation products. An additional useful feature is for the ligase to be active at elevated temperatures. This has the advantage or reducing preferences caused by structures of single-stranded substrates and linkers. RESULTS: To create an RNA ligase with these desirable properties we performed mutational analysis of the archaeal thermophilic RNA ligase from Methanobacterium thermoautotrophicum. We identified amino acids essential for ATP binding and reactivity but dispensable for phosphodiester bond formation with 5' pre-adenylated donor substrate. The motif V lysine mutant (K246A) showed reduced activity in the first two steps of ligation reaction. The mutant has full ligation activity with pre-adenylated substrates but retained the undesirable activity of deadenylation, which is the reverse of step 2 adenylation. A second mutant, an alanine substitution for the catalytic lysine in motif I (K97A) abolished activity in the first two steps of the ligation reaction, but preserved wild type ligation activity in step 3. The activity of the K97A mutant is similar with either pre-adenylated RNA or single-stranded DNA (ssDNA) as donor substrates but we observed two-fold preference for RNA as an acceptor substrate compared to ssDNA with an identical sequence. In contrast, truncated T4 RNA ligase 2, the commercial enzyme used in these applications, is significantly more active using pre-adenylated RNA as a donor compared to pre-adenylated ssDNA. However, the T4 RNA ligases are ineffective in ligating ssDNA acceptors. CONCLUSIONS: Mutational analysis of the heat stable RNA ligase from Methanobacterium thermoautotrophicum resulted in the creation of an ATP independent ligase. The K97A mutant is defective in the first two steps of ligation but retains full activity in ligation of either RNA or ssDNA to a pre-adenylated linker. The ability of the ligase to function at 65°C should reduce the constraints of RNA secondary structure in RNA ligation experiments.
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Project description:The genes encoding the two isoenzymes of methyl coenzyme M reductase (MRI and MRII) in Methanobacterium thermoautotrophicum delta H have been cloned and sequenced. The MRI-encoding mcr operon (mcrBDCGA) has been located immediately upstream from the mtr operon (mtrEDCBA) that encodes N5-methyltetrahydromethanopterin:coenzyme M methyltransferase, the enzyme that catalyzes the step preceding the MR-catalyzed reaction in methanogenesis. The MRII-encoding mrt operon (mrtBDGA) has been located between the operon that encodes the methyl viologen-reducing hydrogenase and an open reading frame (designated pyrC) predicted to encode dihydroorotase. Surprisingly, the mrt operon has been found to contain only four genes (mrtBDGA), lacking the equivalent of the mcrC gene that is present in all mcr operons. A protocol that isolates transcripts intact from M. thermoautotrophicum delta H cells has been developed and used, with primer extension and Northern (RNA) blot procedures, to identify the sites of transcription initiation upstream of the mcr, mrt, and mtr operons and to determine the relative numbers of these transcripts in cells at different growth stages. Transcription of the mrt operon was found to occur only at early times in batch cultures and was then replaced by transcription of the mcr operon. Transcripts of the mtr operon were detectable at all times; however, at early times, all mtr transcripts were initiated at the mtr promoter, whereas at later times, during mcr transcription, approximately 3% of mcr transcripts were extended to generate mcr plus mtr transcripts that constituted approximately 20% of all mtr transcripts present.
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Project description:The ribosomal protein S28E from the archaeon Methanobacterium thermoautotrophicum is a component of the 30S ribosomal subunit. Sequence homologs of S28E are found only in archaea and eukaryotes. Here we report the three-dimensional solution structure of S28E by NMR spectroscopy. S28E contains a globular region and a long C-terminal tail protruding from the core. The globular region consists of four antiparallel beta-strands that are arranged in a Greek-key topology. Unique features of S28E include an extended loop L2-3 that folds back onto the protein and a 12-residue charged C-terminal tail with no regular secondary structure and greater flexibility relative to the rest of the protein. The structural and surface resemblance to OB-fold family of proteins and the presence of highly conserved basic residues suggest that S28E may bind to RNA. A broad positively charged surface extending over one side of the beta-barrel and into the flexible C terminus may present a putative binding site for RNA.
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Project description:Mini-chromosome maintenance (MCM) proteins form a conserved family found in all eukaryotes and are essential for DNA replication. They exist as heteromultimeric complexes containing as many as six different proteins. These complexes are believed to be the replicative helicases, functioning as hexameric rings at replication forks. In most archaea a single MCM protein exists. The protein from Methanobacterium thermoautotrophicum (mtMCM) has been reported to assemble into a large complex consistent with a dodecamer. We show that mtMCM can assemble into a heptameric ring. This ring contains a C-terminal helicase domain that can be fit with crystal structures of ring helicases and an N-terminal domain of unknown function. While the structure of the ring is very similar to that of hexameric replicative helicases such as bacteriophage T7 gp4, our results show that such ring structures may not be constrained to have only six subunits.
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Project description:As part of our structural proteomics initiative, we have determined the crystal structure of MTH1491, a previously uncharacterized hypothetical protein from Methanobacterium thermoautotrophicum. MTH1491 is one of numerous structural genomics targets selected in a genome-wide survey of uncharacterized proteins. It belongs to a family of proteins whose biological function is not known. The crystal structure of MTH1491, the first structure for this family of proteins, consists of an overall five-stranded parallel beta-sheet with strand order 51234 and flanking helices. The oligomeric form of this molecule is a trimer as seen from both crystal contacts and gel filtration studies. Analysis revealed that the structure of MTH1491 is similar to that of dehydrogenases, amidohydrolases, and oxidoreductases. Using a combination of sequence and structural analyses, we showed that MTH1491 does not belong to either the dehydrogenase or the amidohydrolase superfamilies of proteins.
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Project description:Methyl-coenzyme M reductase (MCR) from methanogenic archaea catalyzes the final step in the biological synthesis of methane. Using coenzyme B (CoBSH) as the two-electron donor, MCR reduces methyl-coenzyme M (methyl-SCoM) to methane and the mixed disulfide, CoB-S-S-CoM. MCR contains coenzyme F430, an essential redox-active nickel tetrahydrocorphin, at its active site. The active form of MCR (MCRred1) contains Ni(I)-F430. When 3-bromopropane sulfonate (BPS) is incubated with MCRred1, an alkyl-Ni(III) species is formed that elicits the MCRPS EPR signal. Here we used EPR and UV-visible spectroscopy and transient kinetics to study the reaction between MCR from Methanothermobacter marburgensis and a series of brominated carboxylic acids, with carbon chain lengths of 4-16. All of these compounds give rise to an alkyl-Ni intermediate with an EPR signal similar to that of the MCRPS species. Reaction of the alkyl-Ni(III) adduct, formed from brominated acids with eight or fewer total carbons, with HSCoM as nucleophile at pH 10.0 results in the formation of a thioether coupled to regeneration of the active MCRred1 state. When reacted with 4-bromobutyrate, MCRred1 forms the alkyl-Ni(III) MCRXA state and then, surprisingly, undergoes "self-reactivation" to regenerate the Ni(I) MCRred1 state and a bromocarboxy ester. The results demonstrate an unexpected reactivity and flexibility of the MCR active site in accommodating a broad range of substrates, which act as molecular rulers for the substrate channel in MCR.
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Project description:Methanobacterium thermoautotrophicum when grown on ordinary culture medium has a tough cell wall which is lysozyme-resistant and difficult to disrupt by physical means. The cell wall, however, can be weakened by the addition of D-sorbitol to the growth medium and the organisms form protoplasts after lysozyme addition. This technique allowed the isolation of two types of intracellular small vesicles: (a) isolated by disruption of the total cell population (lysozyme-sensitive and lysozyme-resistant cells) by ultrafrequency sound and (b) isolated by osmotic lysis of protoplasts. For the first time, a small vesicle fraction isolated as in (a) was capable of synthesizing methane from CO2 and H2 without cytoplasm. There was, however, an absolute requirement for a small, heat-stable, oxygen-sensitive cofactor which was isolated from the cytoplasm. Methane synthesis with this vesicle fraction was inhibited by the detergent deoxycholate, and by the protonophores 2,4-dinitrophenol and carbonyl cyanide m-chlorophenylhydrazone. Mg2+-ATPase appeared to be located on the outer or cytoplasmic surface of the small vesicle fraction isolated as in (b). The results were consistent with a previously made suggestion [Sauer, Erfle & Mahadevan (1981) J. Biol. Chem. 256, 9843-9848] that the interior of the small intracellular vesicles becomes acid during methane synthesis.
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