Showing papers on "Methanosarcina barkeri published in 2007"

A cysteine-rich CCG domain contains a novel [4Fe-4S] cluster binding motif as deduced from studies with subunit B of heterodisulfide reductase from Methanothermobacter marburgensis.

Abstract: Heterodisulfide reductase (HDR1) (EC 1.8.98.1) is a unique disulfide reductase with a key function in the energy metabolism of methane-producing archaea. The enzyme catalyzes the reversible reduction of the mixed disulfide (CoM-S-S-CoB) of the two methanogenic thiol coenzymes, designated coenzyme M (CoM-SH) and coenzyme B (CoB-SH). This disulfide is generated in the final step of methanogenesis (1, 2). Two types of HDRs from phylogenetically distantly related methanogens, represented by the enzymes from Methanothermobacter marburgensis (3) and from Methanosarcina barkeri and Methanosarcina thermophila (4, 5), have been identified and characterized (1, 6). Neither type of enzyme belongs to the family of pyridine nucleotide disulfide oxidoreductases (7). HDR from M. marburgensis is an iron–sulfur flavoprotein composed of three subunits: HdrA, HdrB, and HdrC. The primary sequence of HdrA indicates that it contains the FAD binding site and four canonical binding motifs for [4Fe-4S] clusters. HdrB contains no sequence motif characteristic for the binding of known cofactors but has two unique cysteine-rich sequence motifs (CX31–39CCX35–36CXXC) of unknown function, designated as the CCG domain in the Pfam protein families database (accession number PF02754) (8). The ferredoxin-like subunit HdrC contains two canonical binding motifs for [4Fe-4S] clusters (9). HDR from Methanosarcina species lacks a homologue of the M. marburgensis HdrA subunit, whereas subunits HdrC and HdrB are conserved in the putative fusion protein HdrD (Figure 1). Subunits HdrC and HdrB are also conserved in subunit TfrB of thiol:fumarate reductase (TFR) (Figure 1), an anabolic enzyme of methanogens that catalyzes the reduction of fumarate to succinate with CoM-SH plus CoB-SH as electron donors (10). HDR with highly conserved HdrB and HdrC subunits is also encoded by the genomes of uncultivated anaerobic methanotrophic archaea in which the enzyme is thought to catalyze formation of CoM-S-S-CoB from CoM-SH and CoB-SH during anaerobic methane oxidation (11). Figure 1 Schematic alignment of heterodisulfide reductase from Methanothermobacter marburgensis (Mt Hdr), heterodisulfide reductase from Methanosarcina barkeri (Mb Hdr), and thiol: fumarate reductase from Methanothermobacter marburgensis (Mt Tfr). Homologous subunits ... Studies focused on elucidation of the catalytic mechanism of HDR performed with both the enzyme from M. marburgensis and M. barkeri led to identification of a mechanistic-based paramagnetic intermediate generated upon half-reaction of the oxidized enzyme with CoM-SH in the absence of CoB-SH (12, 14). The S = 1/2 species, designated as CoM-HDR, is observed at temperatures below 50 K with principal g values of 2.013, 1.991, and 1.938 (M. marburgensis HDR). The resonance is lost on reduction (Em = −185 mV versus NHE at pH 7.6) and on reaction with CoB-SH. Hence, it was attributed to the product of the oxidative half-reaction that occurs in the absence of CoB-SH in which case it is likely to correspond to a trapped intermediate in the catalytic cycle. Signal broadening in the 57Fe-enriched enzyme indicated that the intermediate is iron based. The combination of variable-temperature magnetic circular dichroism (VT-MCD) spectroscopy and EPR spectroscopy with 33S-labeled CoM-SH led to the proposal that the CoM-HDR reaction intermediate is a novel substrate-bound [4Fe-4S]3+ cluster with two thiolate ligands at a unique Fe site (13, 14). 57Fe-pulsed ENDOR at two very different frequencies, 9 and 94 GHz, provided direct evidence for a [4Fe-4S] cluster with unusually large 57Fe isotropic hyperfine coupling values, which reveals the unusual nature of the cluster (15). From these data it was concluded that HDR uses an active-site iron–sulfur cluster to mediate disulfide reduction in two one-electron steps via site-specific cluster chemistry (6, 13). The sequence of HDR is not related to that of ferredoxin: thioredoxin reductase (FTR), the only other known enzyme which uses an active-site [4Fe-4S] cluster to mediate disulfide reduction (16, 17). An assignment of the active-site cluster to a specific subunit has not yet been achieved. It has been suggested that cysteine residues present in the two CCG domains of M. marburgensis HdrB, M. barkeri HdrD and M. marburgensis TfrB (Figure 1), could provide the ligands to this cluster (4, 10). A database search indicates that the CCG domain is also conserved in a large number of proteins from non-methanogens in the archaeal and bacterial domain. This protein family currently has 1871 members. In most of these proteins the CCG domain is present in two copies, but in some proteins the N-terminal CCG domain is degenerated and conserved cysteine residues are replaced by other amino acid residues. The role of a CCG domain has not been defined for any of these proteins (6). In previous studies HdrB was produced in Bacillus subtilis to address the function of its CCG domains (12). M. marburgensis HdrB rather than M. barkeri HdrD was selected because it contains no additional canonical iron–sulfur cluster binding motifs (Figure 1). Heterologous production of HdrB resulted in a protein containing a [2Fe-2S] cluster as deduced by UV/vis absorption, MCD, and resonance Raman spectroscopies. The cluster was labile and irreversibly lost upon reduction. Since a [2Fe-2S] cluster was not observed in native HdrABC it was considered as an artifact of the heterologous expression system (12). Here we reinvestigated the heterologous production of HdrB using E. coli as the expression host. We present evidence that after an in vitro reconstitution step a [4Fe-4S] cluster with spectroscopic properties reminiscent of CoM-HDR was generated. The cluster ligands are located in the C-terminal CCG domain as shown by site-directed mutagenesis. Via X-ray absorption spectroscopy a Zn site was identified in HdrB, proposed to be coordinated by cysteine residues of the N-terminal CCG domain. This work provides the first assignment of a CCG domain as an iron–sulfur cluster binding site and establishes the basis for future studies on the widespread CCG domain proteins.

Post-translational modifications in the active site region of methyl-coenzyme M reductase from methanogenic and methanotrophic archaea

Abstract: Methyl-coenzyme M reductase (MCR) catalyzes the methane-forming step in methanogenic archaea. Isoenzyme I from Methanothermobacter marburgensiswas shown to contain a thioxo peptide bond and four methylated amino acids in the active site region. We report here that MCRs from all methanogens investigated contain the thioxo peptide bond, but that the enzymes differ in their post-translational methylations. The MS analysis included MCR I and MCR II from Methanothermobacter marburgensis, MCR I from Methanocaldococcus jannaschii and Methanoculleus thermophilus, and MCR from Methanococcus voltae, Methanopyrus kandleri and Methanosarcina barkeri. Two MCRs isolated from Black Sea mats containing mainly methanotrophic archaea of the ANME-1 cluster were also analyzed.

In vivo contextual requirements for UAG translation as pyrrolysine

Abstract: Summary Pyrrolysine and selenocysteine have infiltrated natural genetic codes via the translation of canonical stop codons. UGA translation as selenocysteine is absolutely dependent on message context. Here we describe the first experimental examination of contextual requirements for UAG translation as pyrrolysine. A hexahistidine-tagged Methanosarcina barkeri mtmB1 gene, encoding monomethylamine methyltransferase MtmB1, was introduced into Methanosarcina acetivorans. Host mtmB expression was minimized by growth on methanol and recombinant mtmB1 products monitored by anti-MtmB and anti-hexahistidine immunoblotting. UAG translation was not compromised, as recombinant MtmB1 was 1% of cellular protein with only trace UAG-terminated mtmB1 product detectable. Untranslated regions flanking mtmB1 were not required for UAG translation, but loss of a downstream pyrrolysine insertion sequence (PYLIS) significantly increased the UAG-termination product of mtmB1 and decreased the UAG-translation product, which nonetheless contained pyrrolysine. An in-frame UAG within a bacterial uidA transcript was translated in the methanogen as pyrrolysine with 20% efficiency, suggesting UAG translation in the absence of evolved context. However, predominant UAG-directed termination with enhancement of UAG translation by the PYLIS appears analogous to cis-acting elements for UGA translation as selenocysteine, although different mechanisms may underlie these recoding events.

General Characteristics and Important Model Organisms

Abstract: This chapter provides an overview of the Archaea and some of their morphological, physiological, biochemical, and molecular properties. It introduces model organisms and systems that have been used to study fundamental properties and principles of archaeal biology, in addition to those that have served as models for understanding the biology of more complex eucaryal cells. The chapter describes phylogeny of Archaea and the origin of life, and gives an overview of the characteristic properties of archaeal cells. Rather than one model organism, a broad range of archaea have proven useful for studying morphology, physiology, molecular mechanisms of adaptation, and so forth. These include Methanothermobacter thermautotrophicus, M. marburgensis, and Methanosarcina spp. for methanogenesis; Thermoplasma for proteolysis; Halobacterium for light-driven proton translocation, gene regulation, chemotaxis, and gas vesicles synthesis; Archaeoglobus for sulfate reduction; Pyrococcus and Acidianus ambivalens for inorganic sulfur metabolism; Sulfolobus, A. ambivalens, Pyrococcus, and the methanogens for electron transport chains; Sulfolobus and Pyrococcus for DNA replication and transcription; Halobacterium, Haloarcula, and Sulfolobus for translation. The chapter also describes the properties of major archaeal taxa according to their ecology and molecular similarity. Important characteristics of some of the key organisms are also included in this chapter. Methanosarcina barkeri, M. mazei, and M. acetivorans are the most well studied methanogens and are important model organisms for studies on acetoclastic methanogenesis, transcription, and chaperonins.

Class I and class II lysyl-tRNA synthetase mutants and the genetic encoding of pyrrolysine in Methanosarcina spp.

Abstract: Methanosarcina spp. begin methanogenesis from methylamines with methyltransferases made via the translation of UAG as pyrrolysine. In vitro evidence indicates two possible routes to pyrrolysyl-tRNA(Pyl). PylS ligates pyrrolysine to tRNA(Pyl). Alternatively, class I and class II lysyl-tRNA synthetases (LysRS1 and LysRS2) together form lysyl-tRNA(Pyl), a potential intermediate to pyrrolysyl-tRNA(Pyl). The unusual possession of both LysRS1 and LysRS2 by Methanosarcina spp. may also reflect differences in catalytic properties. Here we assessed the in vivo relevance of these hypotheses. The lysK and mtmB transcripts, encoding LysRS1 and monomethylamine methyltransferase, were detectable in Methanosarcina barkeri during early log growth on trimethylamine, but not methanol. In contrast, lysS transcript encoding LysRS2 was detectable during log phase with either substrate. Methanosarcina acetivorans strains bearing deletions of lysK or lysS grew normally on methanol and methylamines with wild-type levels of monomethylamine methyltransferase and aminoacyl-tRNA(Pyl). The lysK and lysS genes could not replace pylS in a recombinant system employing tRNA(Pyl) for UAG suppression. The results support an association of LysRS1 with growth on methylamine, but not an essential role for LysRS1/LysRS2 in the genetic encoding of pyrrolysine. However, decreased lysyl-tRNA(Lys) in the lysS mutant provides a possible rationale for stable transfer of the bacterial lysS gene to methanoarchaea.

Acid resistance of methanogenic bacteria in a two-stage anaerobic process treating high concentration methanol wastewater

Abstract: In this study, the two-stage upflow anaerobic sludge blanket (UASB) system and batch experiments were employed to evaluate the performance of anaerobic digestion for the treatment of high concentration methanol wastewater. The acid resistance of granular sludge and methanogenic bacteria and their metabolizing activity were investigated. The results show that the pH of the first UASB changed from 4.9 to 5.8 and 5.5 to 6.2 for the second reactor. Apparently, these were not the advisable pH levels that common methanogenic bacteria could accept. The methanogenic bacteria of the system, viz. Methanosarcina barkeri, had some acid resistance and could still degrade methanol at pH 5.0. If the methanogenic bacteria were trained further, their acid resistance would be improved somewhat. Granular sludge of the system could protect the methanogenic bacteria within its body against the impact of the acidic environment and make them degrade methanol at pH 4.5. The performance of granular sludge was attributed to its structure, bacteria species, and the distribution of bacterium inside the granule.

Structure and Function of Enzymes Involved in Tetrapyrrole Biosynthesis

Abstract: This thesis approached four open questions regarding the pathway for the biosynthesis of heme and involved enzymes in bacteria, archaea and plants 1 The novel antibiotic alaremycin was shown to inhibit porphobilinogen synthase from various bacterial, archaeal and eukaryotic sources A co-crystal structure of alaremycin with Pseudomonas aeruginosa porphobilinogen synthase was obtained Thus, the molecular basis of alaremycin function was solved at the atomic level 2 Bioinformatic approaches failed to detect the uroporphyrinogen III synthase gene in plants Our cooperation partner Alison Smith at Cambridge University, UK, had genetically isolated a potential open reading frame from Arabidopsis thaliana In this thesis the corresponding protein was recombinantly produced and uroporphyrinogen III synthase activity was demonstrated, providing final proof for the identity of the gene 3 The recently solved crystal structure of tobacco protoporphyrinogen IX oxidase was used as basis for the functional definition of the corresponding substrate binding site Furthermore, the structural constellation causing the human disease variegate porphyria was functionally explored For this purpose 14 enzyme variants were generated, produced, purified and tested for their kinetics and FAD contents A detailed picture of the various chemical reactions occurring during substrate-enzyme interactions was obtained The chemical basis for the enzymatic defect responsible for variegate porphyria was elucidated 4 An alternative pathway for the biosynthesis of hemes in archaea was discovered and initially characterized It was shown that in the archaeon Methanosarcina barkeri heme is synthesized via precorrin-2 Therefore, the last four to five steps of archaeal heme biosynthesis are completely different to their bacterial and eukaryotic counterparts