Zinc−Thiolate Intermediate in Catalysis of Methyl Group Transfer in <i>Methanosarcina barkeri</i>

Gencic, Simonida; Leclerc, Gilles M.; Gorlatova, Natalia; Peariso, Katrina; Penner‐Hahn, James E.; Grahame, David A.

doi:10.1021/bi0112917

Cited by 31 publications

(57 citation statements)

References 24 publications

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“…The gene for the full-length ACDS ␤ subunit (cdhC) and a truncated form of the gene coding for a protein lacking 75 amino acids at the C terminus (cdhC*) were overexpressed using a modified version of the pQE60 (Qiagen) vector from which the His 6 tag was deleted, designated pQE60⌬His. Modifications to remove the His 6 tag were as described previously (14). PCR amplifications of cdhC and cdhC* were performed using genomic DNA as template isolated from M. thermophila strain TM-1 with forward primers incorporating an NcoI site and reverse primers containing a BamHI site.…”

Section: Methodsmentioning

confidence: 99%

Nickel in Subunit β of the Acetyl-CoA Decarbonylase/Synthase Multienzyme Complex in Methanogens

Gencic

Grahame

2003

Journal of Biological Chemistry

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113

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The methanogenic Archaea utilize a unique metabolic pathway for degradation of acetate under anaerobic conditions, and cleavage of acetate thereby accounts for a major proportion of the methane formed in the environment. The central reaction in this pathway is carried out by an unusual multienzyme complex, designated acetyl-CoA decarbonylase/synthase (ACDS), 1 which contains five different polypeptide subunits and accounts for as much as 25% of the soluble protein in species such as Methanosarcina thermophila and Methanosarcina barkeri growing on acetate. The ACDS complex catalyzes cleavage of the acetyl C-C bond using the substrates acetylCoA and tetrahydrosarcinapterin (H 4 SPt), a tetrahydrofolate analog which serves as methyl acceptor, and yields the products CoA, N 5 -methyltetrahydrosarcinapterin, CO 2 , and two reducing equivalents, as given in Reaction 1 (1).This overall reaction is made up of a series of partial reactions catalyzed by different protein subcomponents of the ACDS complex as shown in Scheme 1 (2). Acetyl-CoA binds to the ␤ subunit, and under low redox potential conditions, as required for activity, transfers the acetyl group to a nucleophilic center on the enzyme forming an acetyl-enzyme species and releasing CoA (Scheme 1, acetyl transfer) (2, 3). The acetyl intermediate then undergoes C-C bond cleavage by a reaction that is presumed to involve metal-based decarbonylation and/or methyl group migration (Scheme 1, cleavage). The nascent methyl group is then transferred to a corrinoid cofactor present on the ␥␦ subcomponent, which catalyzes subsequent methyl transfer to the substrate H 4 SPt (Scheme 1, methyl transfer) (2). The carbonyl group is oxidized to CO 2 by a process involving the ␣⑀ CO dehydrogenase subcomponent, with regeneration of the reduced form of the ␤ subunit. Previous studies on the ␤ subunit have focused on a C-terminally truncated form of the protein purified from the native ACDS complex following partial proteolytic digestion (2-4).The genes encoding the five ACDS subunits are arranged together in an operon along with one additional open reading frame in all species of Methanosarcina and in certain other methanogens as well (5-7, 9).2 The operon structure is shown in Scheme 2, with the designated genes and corresponding subunit molecular masses indicated for M. thermophila TM-1. The additional open reading frame encodes an accessory protein thought to be involved in nickel insertion, and nickel is present in both the large CO dehydrogenase subunit ␣ (CdhA) and in the ␤ subunit (CdhC) containing the active site for

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Section: Methodsmentioning

confidence: 99%

Nickel in Subunit β of the Acetyl-CoA Decarbonylase/Synthase Multienzyme Complex in Methanogens

Gencic

Grahame

2003

Journal of Biological Chemistry

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113

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“…S2). The amino terminal domain of each protein contains a corrinoid-binding motif (GDVH-DIGKNLV) with the conserved active site histidine, whereas the C-terminal methyltransferase domain of each has the potential to co-ordinate Zinc by virtue of a conserved HXCX nC motif Gencic et al, 2001;Kruer et al, 2002). This motif is also conserved in the MtaA1, MtaA2 and MtbA methyltransferase 2 enzymes from all three Methanosarcina spp.…”

Section: Mtsd Mtsf and Mtsh Are Fusion Proteins With A Methyltransfementioning

confidence: 99%

Regulation of putative methyl‐sulphide methyltransferases in Methanosarcina acetivorans C2A

Bose

Kulkarni

Metcalf

2009

Molecular Microbiology

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SummaryThe regulation of the Methanosarcina acetivorans mtsD, mtsF and mtsH genes, which encode putative corrinoid/methyltransferase isozymes involved in methylsulphide metabolism, was examined by a variety of methods, suggesting that their expression is regulated at both the transcriptional and posttranscriptional levels. Transcripts of all three genes, measured by quantitative reverse transcription PCR, were shown to be most abundant during growth on methanol with dimethylsulphide (DMS). Transcript levels were also high in media with CO or methylamines, but much lower with methanol. In contrast, translational fusions to mtsD showed high expression levels on CO or methanol with DMS, while the mtsF translational fusion showed highest reporter gene activity on methylamines with much lower expression on CO or methanol with DMS. The activity of mtsD and mtsF fusions was very low when the strains were grown in methanol or acetate. Expression of the mtsH fusion was not detected on any substrate, despite the presence of an mRNA transcript. The transcription start sites of all three genes were determined by 5Ј-RACE revealing large leader sequences for each transcript. Characterization of deletion mutants lacking putative regulatory genes suggests that MA0862 (msrF), MA4383 (msrC) and MA4560 (msrG) act as transcriptional activators of mtsD, mtsF and mtsH respectively.

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“…In the case of methanogenesis, this reaction is carried out by vitamin B 12 -dependent methyltransferases. In methanogenic methyltransferases, zinc plays an integral role in activation of the thiol group of CoM for acceptance of a methyl group from various donors (22,55). Analogously, the zinc atom in EaCoMT functions in the activation of CoM as well, in this instance for attack on the electrophilic epoxide substrate (9).…”

Section: Similarities In Com Utilization During Methanogenesis and Prmentioning

confidence: 99%

Getting a Handle on the Role of Coenzyme M in Alkene Metabolism

Krishnakumar

Sliwa

Endrizzi

et al. 2008

Microbiol Mol Biol Rev

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SUMMARY Coenzyme M (2-mercaptoethanesulfonate; CoM) is one of several atypical cofactors discovered in methanogenic archaea which participate in the biological reduction of CO2 to methane. Elegantly simple, CoM, so named for its role as a methyl carrier in all methanogenic archaea, is the smallest known organic cofactor. It was thought that this cofactor was used exclusively in methanogenesis until it was recently discovered that CoM is a key cofactor in the pathway of propylene metabolism in the gram-negative soil microorganism Xanthobacter autotrophicus Py2. A four-step pathway requiring CoM converts propylene and CO2 to acetoacetate, which feeds into central metabolism. In this process, CoM is used to activate and convert highly electrophilic epoxypropane, formed from propylene epoxidation, into a nucleophilic species that undergoes carboxylation. The unique properties of CoM provide a chemical handle for orienting compounds for site-specific redox chemistry and stereospecific catalysis. The three-dimensional structures of several of the enzymes in the pathway of propylene metabolism in defined states have been determined, providing significant insights into both the enzyme mechanisms and the role of CoM in this pathway. These studies provide the structural basis for understanding the efficacy of CoM as a handle to direct organic substrate transformations at the active sites of enzymes.

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Zinc−Thiolate Intermediate in Catalysis of Methyl Group Transfer in Methanosarcina barkeri

Cited by 31 publications

References 24 publications

Nickel in Subunit β of the Acetyl-CoA Decarbonylase/Synthase Multienzyme Complex in Methanogens

Nickel in Subunit β of the Acetyl-CoA Decarbonylase/Synthase Multienzyme Complex in Methanogens

Regulation of putative methyl‐sulphide methyltransferases in Methanosarcina acetivorans C2A

Getting a Handle on the Role of Coenzyme M in Alkene Metabolism

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