A5048493695- Porphyrin Metabolism and Disorders
- Enzyme Structure and Function
- Metalloenzymes and iron-sulfur proteins
- Microbial Metabolic Engineering and Bioproduction
- Metal-Catalyzed Oxygenation Mechanisms
- Metabolism and Genetic Disorders
- Photosynthetic Processes and Mechanisms
- Enzyme Production and Characterization
- Microbial Metabolites in Food Biotechnology
- Neurological diseases and metabolism
- Anaerobic Digestion and Biogas Production
- Microbial metabolism and enzyme function
- Phytase and its Applications
- Biofuel production and bioconversion
- Folate and B Vitamins Research
- Enzyme Catalysis and Immobilization
- Hemoglobin structure and function
- Heme Oxygenase-1 and Carbon Monoxide
- Polyamine Metabolism and Applications
- Bacteriophages and microbial interactions
- Amino Acid Enzymes and Metabolism
- Microbial bioremediation and biosurfactants
- bioluminescence and chemiluminescence research
- Aldose Reductase and Taurine
- Cannabis and Cannabinoid Research
Uniformed Services University of the Health Sciences
2002-2024
Lawrence Berkeley National Laboratory
2003
University of California, Davis
2003
Lawrence Livermore National Laboratory
2003
University of Michigan
2001
University of Kentucky
1998
The Ohio State University
1982-1996
National Heart Lung and Blood Institute
1987-1996
National Institutes of Health
1987-1996
Albert B. Chandler Hospital
1996
Methanogenesis, the biological production of methane, plays a pivotal role in global carbon cycle and contributes significantly to warming. The majority methane nature is derived from acetate. Here we report complete genome sequence an acetate-utilizing methanogen, Methanosarcina acetivorans C2A. Methanosarcineae are most metabolically diverse methanogens, thrive broad range environments, unique among Archaea forming complex multicellular structures. This diversity reflected M. . At...
Dismutation of superoxide has been shown previously to be catalyzed by stable nitroxide compounds. In the present study, mechanism (.O2-) dismutation various five-membered ring and six-membered nitroxides was studied electron paramagnetic resonance spectrometry, UV-visible spectrophotometry, cyclic voltammetry, bulk electrolysis. Electron signals from carbocyclic derivatives (piperidinyl, pyrrolidinyl, pyrrolinyl) were unchanged when exposed enzymatically generated .O2-, whereas, in presence...
The formate-hydrogen lyase complex of Escherichia coli decomposes formic acid to hydrogen and carbon dioxide under anaerobic conditions in the absence exogenous electron acceptors. consists two separable enzymatic activities: a formate dehydrogenase hydrogenase. component (FDHH) was purified near homogeneity column chromatographic steps. enzyme composed single polypeptide molecular weight 80,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Metal analysis showed...
Methyl transfer from dimethylamine to coenzyme M was reconstituted <i>in vitro</i> for the first time using only highly purified proteins. These proteins isolated from<i>Methanosarcina barkeri</i> included previously unidentified corrinoid protein MtbC, which copurified with MtbA, methylcorrinoid:Coenzyme methyltransferase specific methanogenesis methylamines. MtbC binds 1.0 mol of cofactor/mol 24-kDa polypeptide and stimulated dimethylamine:coenzyme methyl 3.4-fold in a cell extract....
An enzyme complex containing carbon monoxide dehydrogenase and a corrinoid protein has been isolated from Methanosarcina barkeri. Sodium dodecyl sulfate-gel electrophoresis revealed five polypeptides of molecular masses alpha = 19,700, beta 84,500, gamma 63,200, delta 53,000, epsilon 51,400 Da in equimolar amounts. One mol cobamide cofactor was found per minimal unit. The mass the native 1,600,000 by high pressure liquid chromatography (HPLC) gel filtration, which suggested an 6 oligomeric...
Clostridium difficile is the leading cause of hospital-acquired antibiotic-associated diarrhea and only widespread human pathogen that contains a complete set genes encoding Wood-Ljungdahl pathway (WLP). In acetogenic bacteria, synthesis acetate from 2 CO2 molecules by WLP functions as terminal electron accepting pathway; however, C. various other reductive pathways, including heavy reliance on Stickland reactions, which questions role in this bacterium. rich medium containing high levels...
Approximately two-thirds of the estimated one-billion metric tons methane produced annually by methanogens is derived from cleavage acetate. Acetate broken down a Ni-Fe-S-containing A-cluster within enzyme acetyl-CoA synthase (ACS) to carbon monoxide (CO) and methyl group (CH
The acetyl-CoA decarbonylase/synthase (ACDS) complex catalyzes the central reaction of acetyl C–C bond cleavage in methanogens growing on acetate and is also responsible for synthesis units during growth C-1 substrates. ACDS β subunit contains nickel an Fe/S center reacts with forming acetyl-enzyme intermediate presumably directly involved activation. To investigate role this process two forms Methanosarcina thermophila were overexpressed anaerobically grown Escherichia coli. Both contained...
Carbon monoxide dehydrogenase from acetategrown cells of Methanosarcina burkeri exists in a high molecular weight form (-3 X lo6) under conditions ionic strength but is converted to much smaller by dialysis.The enzyme was purified procedure which exploits isolation the aggregated gel filtration and subsequent dissociation.Following this, within 92% homogeneity chromatography on phenyl-Sepharose finally hydroxylapatite.Due extreme oxygen lability enzyme, entire carried out anaerobic...
Kinetic parameters of the selenium-containing, formate dehydrogenase component Escherichia coli formate-hydrogenlyase complex have been determined with purified enzyme.A ping-pong Bi kinetic mechanism was observed.The K,,, for is 26 mM, and electron-accepting dye, benzyl viologen, in range 1-5 mM.The maximal turnover rate formate-dependent catalysis viologen reduction calculated to be 1.7 % 10' min".Isotope exchange analysis showed that enzyme catalyzes carbon between dioxide absence other...
Enzymological studies on the multienzyme acetyl-CoA decarbonylase synthase (ACDS) complex from Methanosarcina barkeri have been conducted in order to identify and characterize physiologically relevant substrates reactions synthesis decomposition methanogens. Whereas previous investigations employed carbon monoxide as substrate reducing agent for synthesis, we discovered that bicarbonate (or CO2) acts a highly efficient carbonyl group precursor presence of either hydrogen or Ti3+.EDTA agent....
An immunochemical approach was employed as a direct test for functional activities of isozymes methylcobamide:coenzyme M methyltransferase (MT2-M and MT2-A) in the metabolic pathways methane formation from: methanol, acetate, monomethylamine, dimethylamine, trimethylamine.Specific removal MT2 from buffer soluble cell extracts Methanosarcina barkeri accomplished by use immobilized, affinity-purified, ovine polyclonal antibodies.Extracts methanol-grown cells depleted MT2-M lost entirely...
The selenocysteine-containing formate dehydrogenase H (FDH) is an 80-kDa component of the Escherichia coli formate-hydrogen lyase complex. molybdenum-coordinated selenocysteine essential for catalytic activity native enzyme. FDH in dilute solutions (30 μg/ml) was rapidly inactivated at basic pH or presence under anaerobic conditions, but higher enzyme concentrations (≥3 mg/ml) relatively stable. formate-reduced extremely sensitive to air inactivation all conditions examined. Active...
In methanogens, the acetyl-CoA decarbonylase synthase (ACDS) complex, which has five different subunits, catalyzes synthesis and cleavage of according to reaction: CO2+ 2H++ 2e-+ CH3-H4SPt + CoA ⇌ H4SPt H2O, where are tetrahydrosarcinapterin N5-methyl-tetrahydrosarcinapterin, respectively. We have dissociated ACDS complex into three protein components by limited proteolytic digestion. Catalysis was lost in parallel with loss intact β subunit; however, no decrease activity detected any...
Two forms of methylcobalamin:2-mercaptoethanesulfonate methyltransferase were observed in Methanosarcina barkeri. Resolution the enzymes was accomplished by chromatography on hydroxylapatite. The exhibited different electrophoretic mobilities under nondenaturing conditions, and separated based upon differences net charge. Both isozymes similar size, having molecular weights approximately 34,000. Antibody binding experiments demonstrated that when M. barkeri grown methanol, one enzyme...
The 5-subunit-containing acetyl-CoA decarbonylase/synthase (ACDS) complex plays an important role in methanogenic Archaea that convert acetate to methane, by catalyzing the central reaction of C-C bond cleavage which serves as acetyl donor substrate reacting at ACDS beta subunit active site. properties Ni site A-cluster from Methanosarcina thermophila were investigated. A recombinant, C-terminally truncated form was employed, mimics native previously isolated complex, and contains composed...
Direct synthesis and cleavage of acetyl-CoA are carried out by the bifunctional CO dehydrogenase/acetyl-CoA synthase enzyme in anaerobic bacteria decarbonylase/synthase (ACDS) multienzyme complex Archaea. In both systems, a nickel- Fe/S-containing active site metal center, A cluster, catalyzes acetyl C-C bond formation/breakdown. Carbonyl group exchange [1-(14)C]acetyl-CoA with unlabeled CO, hallmark CODH/ACS, is weakly ACDS, CO(2) was up to 350 times faster, indicating tight coupling...
Acetyl-CoA decarbonylase/synthase (ACDS) is a multienzyme complex that plays central role in energy metabolism Methanosarcina barkeri grown on acetate. The ACDS carries out an unusual reaction involving net cleavage of the acetyl C−C and thioester bonds acetyl-CoA. overall composed several partial reactions, one which involves catalysis group transfer. To gain insight into reaction, study was carried kinetics mechanism acetyltransferase reaction. Analysis by HPLC used to quantify rates...
Acetyl-CoA decarbonylase/synthase (ACDS) is a multienzyme complex found in methanogens and certain other Archaea that carries out the overall synthesis cleavage of acetyl C−C C−S bonds acetyl-CoA. The reaction involved both autotrophic fixation carbon process methanogenesis from acetate, takes place at unique active site metal center known as A cluster, located on beta subunit ACDS composed binuclear Ni−Ni bridged by cysteine thiolate to an Fe4S4 center. In this work, high rate acetyl-CoA...
The acetyl-CoA decarbonylase/synthase (ACDS) complex catalyzes the cleavage of in methanogens that metabolize acetate to CO2 and CH4, also carries out synthesis during growth on one-carbon substrates. ACDS contains five subunits, among which β possesses an Ni−Fe−S active-site metal cluster, A-cluster, at reaction with takes place, generating acetyl-enzyme species poised for C−C bond cleavage. We have used Ni Fe K fluorescence XANES EXAFS analyses characterize these metals subunit, expressed...
Carbon monoxide dehydrogenase was purified to homogeneity from Methanococcus vannielii grown with formate as the sole carbon source. The enzyme is composed of subunits molecular weights 89,000 and 21,000 in an alpha 2 beta oligomeric structure. native weight dehydrogenase, determined by gel electrophoresis, 220,000. M. contains g-atoms nickel per mol enzyme. Except for its relatively high pH optimum 10.5 slightly greater net positive charge, closely resembles isolated previously...