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J G Ferry

Publications and source records attributed to J G Ferry.

At least 37 records · Page 2Linked to original sources
PubMed1999

Kinetic and spectroscopic characterization of the gamma-carbonic anhydrase from the methanoarchaeon Methanosarcina thermophila.

The zinc and cobalt forms of the prototypic gamma-carbonic anhydrase from Methanosarcina thermophila were characterized by extended X-ray absorption fine structure (EXAFS) and the kinetics were investigated using steady-state spectrophotometric and (18)O exchange equilibrium assays. EXAFS results indicate that cobalt isomorphously replaces zinc and that the metals coordinate three histidines and two or three water molecules. The efficiency of either Zn-Cam or Co-Cam for CO(2) hydration (k(cat)/K(m)) was severalfold greater than HCO(3-) dehydration at physiological pH values, a result consistent with the proposed physiological function for Cam during growth on acetate. For both Zn- and Co-Cam, the steady-state parameter k(cat) for CO(2) hydration was pH-dependent with a pK(a) of 6.5-6.8, whereas k(cat)/K(m) was dependent on two ionizations with pK(a) values of 6.7-6.9 and 8.2-8.4. The (18)O exchange assay also identified two ionizable groups in the pH profile of k(cat)/K(m) with apparent pK(a) values of 6.0 and 8.1. The steady-state parameter k(cat) (CO(2) hydration) is buffer-dependent in a saturable manner at pH 8. 2, and the kinetic analysis suggested a ping-pong mechanism in which buffer is the second substrate. The calculated rate constant for intermolecular proton transfer is 3 x 10(7) M(-1) s(-1). At saturating buffer concentrations and pH 8.5, k(cat) is 2.6-fold higher in H(2)O than in D(2)O, suggesting that an intramolecular proton transfer step is at least partially rate-determining. At high pH (pH > 8), k(cat)/K(m) is not dependent on buffer and no solvent hydrogen isotope effect was observed, consistent with a zinc hydroxide mechanism. Therefore, at high pH the catalytic mechanism of Cam appears to resemble that of human CAII, despite significant structural differences in the active sites of these two unrelated enzymes.

Apoenzymes↗
PubMed1999

Enzymology of one-carbon metabolism in methanogenic pathways.

Methanoarchaea, the largest and most phylogenetically diverse group in the Archaea domain, have evolved energy-yielding pathways marked by one-carbon biochemistry featuring novel cofactors and enzymes. All of the pathways have in common the two-electron reduction of methyl-coenzyme M to methane catalyzed by methyl-coenzyme M reductase but deviate in the source of the methyl group transferred to coenzyme M. Most of the methane produced in nature derives from acetate in a pathway where the activated substrate is cleaved by CO dehydrogenase/acetyl-CoA synthase and the methyl group is transferred to coenzyme M via methyltetrahydromethanopterin or methyltetrahydrosarcinapterin. Electrons for reductive demethylation of the methyl-coenzyme M originate from oxidation of the carbonyl group of acetate to carbon dioxide by the synthase. In the other major pathway, formate or H2 is oxidized to provide electrons for reduction of carbon dioxide to the methyl level and reduction of methyl-coenzyme to methane. Methane is also produced from the methyl groups of methanol and methylamines. In these pathways specialized methyltransferases transfer the methyl groups to coenzyme M. Electrons for reduction of the methyl-coenzyme M are supplied by oxidation of the methyl groups to carbon dioxide by a reversal of the carbon dioxide reduction pathway. Recent progress on the enzymology of one-carbon reactions in these pathways has raised the level of understanding with regard to the physiology and molecular biology of methanogenesis. These advances have also provided a foundation for future studies on the structure/function of these novel enzymes and exploitation of the recently completed sequences for the genomes from the methanoarchaea Methanobacterium thermoautotrophicum and Methanococcus jannaschii.

Amino Acid Sequence↗
PubMed1999

A plant-type (beta-class) carbonic anhydrase in the thermophilic methanoarchaeon Methanobacterium thermoautotrophicum.

Carbonic anhydrase, a zinc enzyme catalyzing the interconversion of carbon dioxide and bicarbonate, is nearly ubiquitous in the tissues of highly evolved eukaryotes. Here we report on the first known plant-type (beta-class) carbonic anhydrase in the archaea. The Methanobacterium thermoautotrophicum DeltaH cab gene was hyperexpressed in Escherichia coli, and the heterologously produced protein was purified 13-fold to apparent homogeneity. The enzyme, designated Cab, is thermostable at temperatures up to 75 degrees C. No esterase activity was detected with p-phenylacetate as the substrate. The enzyme is an apparent tetramer containing approximately one zinc per subunit, as determined by plasma emission spectroscopy. Cab has a CO(2) hydration activity with a k(cat) of 1.7 x 10(4) s(-1) and K(m) for CO(2) of 2.9 mM at pH 8.5 and 25 degrees C. Western blot analysis indicates that Cab (beta class) is expressed in M. thermoautotrophicum; moreover, a protein cross-reacting to antiserum raised against the gamma carbonic anhydrase from Methanosarcina thermophila was detected. These results show that beta-class carbonic anhydrases extend not only into the Archaea domain but also into the thermophilic prokaryotes.

Amino Acid Sequence↗
PubMed1998

Electrochemical and spectroscopic properties of the iron-sulfur flavoprotein from Methanosarcina thermophila.

An iron-sulfur flavoprotein (Isf) from the methanoarchaeaon Methanosarcina thermophila, which participates in electron transfer reactions required for the fermentation of acetate to methane, was characterized by electrochemistry and EPR and Mössbauer spectroscopy. The midpoint potential (Em) of the FMN/FMNH2 couple was -0.277 V. No flavin semiquinone was observed during potentiometric titrations; however, low amounts of the radical were observed when Isf was quickly frozen after reaction with CO and the CO dehydrogenase/acetyl-CoA synthase complex from M. thermophila. Isf contained a [4Fe-4S]2+/1+ cluster with g values of 2.06 and 1.93 and an unusual split signal with g values at 1.86 and 1.82. The unusual morphology was attributed to microheterogeneity among Isf molecules. The Em value for the 2+/1+ redox couple of the cluster was -0.394 V. Extracts from H2-CO2-grown Methanobacterium thermoautotrophicum cells catalyzed either the H2- or CO-dependent reduction of M. thermophila Isf. In addition, Isf homologs were found in the genomic sequences of the CO2-reducing methanoarchaea M. thermoautotrophicum and Methanococcus jannaschii. These results support a general role for Isf in electron transfer reactions of both acetate-fermenting and CO2-reducing methanoarchaea. It is suggested that Isf functions to couple electron transfer from ferredoxin to membrane-bound electron carriers, such as methanophenazine and/or b-type cytochromes.

Amino Acid Sequence↗
PubMed1998

Identification of essential glutamates in the acetate kinase from Methanosarcina thermophila.

Acetate kinase catalyzes the reversible phosphorylation of acetate (CH3COO- + ATP<-->CH3CO2PO3(2-) + ADP). A mechanism which involves a covalent phosphoryl-enzyme intermediate has been proposed, and chemical modification studies of the enzyme from Escherichia coli indicate an unspecified glutamate residue is phosphorylated (J. A. Todhunter and D. L. Purich, Biochem. Biophys. Res. Commun. 60:273-280, 1974). Alignment of the amino acid sequences for the acetate kinases from E. coli (Bacteria domain), Methanosarcina thermophila (Archaea domain), and four other phylogenetically divergent microbes revealed high identity which included five glutamates. These glutamates were replaced in the M. thermophila enzyme to determine if any are essential for catalysis. The histidine-tagged altered enzymes were produced in E. coli and purified to electrophoretic homogeneity by metal affinity chromatography. Replacements of E384 resulted in either undetectable or extremely low kinase activity, suggesting E384 is essential for catalysis which supports the proposed mechanism. Replacement of E385 influenced the Km values for acetate and ATP with only moderate decreases in k(cat), which suggests that this residue is involved in substrate binding but not catalysis. The unaltered acetate kinase was not inactivated by N-ethylmaleimide; however, replacement of E385 with cysteine conferred sensitivity to N-ethylmaleimide which was prevented by preincubation with acetate, acetyl phosphate, ATP, or ADP, suggesting that E385 is located near the active site. Replacement of E97 decreased the Km value for acetate but not ATP, suggesting this residue is involved in binding acetate. Replacement of either E32 or E334 had no significant effects on the kinetic constants, which indicates that neither residue is essential for catalysis or significantly influences the binding of acetate or ATP.

Acetate Kinase↗
PubMed1998

Biochemical characterization of the 20S proteasome from the methanoarchaeon Methanosarcina thermophila.

The 20S proteasome from the methanoarchaeon Methanosarcina thermophila was produced in Escherichia coli and characterized. The biochemical properties revealed novel features of the archaeal 20S proteasome. A fully active 20S proteasome could be assembled in vitro with purified native alpha ring structures and beta prosubunits independently produced in Escherichia coli, which demonstrated that accessory proteins are not essential for processing of the beta prosubunits or assembly of the 20S proteasome. A protein complex with a molecular mass intermediate to those of the alpha7 ring and the 20S proteasome was detected, suggesting that the 20S proteasome is assembled from precursor complexes. The heterologously produced M. thermophila 20S proteasome predominately catalyzed cleavage of peptide bonds carboxyl to the acidic residue Glu (postglutamyl activity) and the hydrophobic residues Phe and Tyr (chymotrypsinlike activity) in short chromogenic and fluorogenic peptides. Low-level hydrolyzing activities were also detected carboxyl to the acidic residue Asp and the basic residue Arg (trypsinlike activity). Sodium dodecyl sulfate and divalent or monovalent ions stimulated chymotrypsinlike activity and inhibited postglutamyl activity, whereas ATP stimulated postglutamyl activity but had little effect on the chymotrypsinlike activity. The results suggest that the 20S proteasome is a flexible protein which adjusts to binding of substrates. The 20S proteasome also hydrolyzed large proteins. Replacement of the nucleophilic Thr1 residue with an Ala in the beta subunit abolished all activities, which suggests that only one active site is responsible for the multisubstrate activity. Replacement of beta subunit active-site Lys33 with Arg reduced all activities, which further supports the existence of one catalytic site; however, this result also suggests a role for Lys33 in polarization of the Thr1 N, which serves to strip a proton from the active-site Thr1 Ogamma nucleophile. Replacement of Asp51 with Asn had no significant effect on trypsinlike activity, enhanced postglutamyl and trypsinlike activities, and only partially reduced lysozyme-hydrolyzing activity, which suggested that this residue is not essential for multisubstrate activity.

Adenosine Triphosphate↗
PubMed1997

Enzymology of the fermentation of acetate to methane by Methanosarcina thermophila.

Biologically-produced CH4 derives from either the reduction of CO2 or the methyl group of acetate by two separate pathways present in anaerobic mierobes from the Archaea domain. Elucidation of the pathway for CO2 reduction to CH4, the first to be investigated, has yielded several novel enzymes and cofactors. Most of the CH4 produced in nature derives from the methyl group of acetate. Methanosarcina thermophila is a moderate thermophile which ferments acetate by reducing the methyl group to CH4 with electrons derived from oxidation of the carbonyl group to CO2. The pathway in M. thermophila is now understood on a biochemical and genetic level comparable to understanding of the CO2-reducing pathway. Enzymes have been purified and characterized. The genes encoding these enzymes have been cloned, sequenced, transcriptionally mapped, and their regulation defined on a molecular level. This review emphasizes recent developments concerning the enzymes which are unique to the acetate fermentation pathway in M. thermophila.

Acetate Kinase↗
PubMed1997

Crystallization of acetate kinase from Methanosarcina thermophila and prediction of its fold.

The unique biochemical properties of acetate kinase present a classic conundrum in the study of the mechanism of enzyme-catalyzed phosphoryl transfer. Large, single crystals of acetate kinase from Methanosarcina thermophila were grown from a solution of ammonium sulfate in the presence of ATP. The crystals diffract to beyond 1.7 A resolution. Analysis of X-ray data from the crystals is consistent with a space group of C2 and unit cell dimensions a = 181 A, b = 67 A, c = 83 A, beta = 103 degrees. Diffraction data have been collected from the crystals at 110 and 277 K. Data collected at 277 K extend to lower resolution, but are more reproducible. The orientation of a noncrystallographic two-fold axis of symmetry has been determined. Based on an analysis of the predicted amino acid sequences of acetate kinase from several organisms, we hypothesize that acetate kinase is a member of the sugar kinase/actin/hsp70 structural family.

Acetate Kinase↗
PubMed1997

Identification of cysteine and arginine residues essential for the phosphotransacetylase from Methanosarcina thermophila.

Phosphotransacetylase catalyzes the following reaction: CoASH + CH3CO2PO3(2-) <==> CH3COSCoA + HPO4(2-) (where CoA is coenzyme A). Based on biochemical characterization of the enzyme from the obligate anaerobe Clostridium kluyveri, a ternary mechanism was proposed in which an unspecified cysteine abstracts a proton from CoASH forming a nucleophilic thiolate anion which attacks acetyl phosphate (J. Henkin and R. H. Abeles, Biochemistry 15:3472-3479, 1976). Heterologous production in Escherichia coli of the phosphotransacetylase from Methanosarcina thermophila, an obligately anaerobic methanoarchaeon, allowed site-specific replacements to identify essential residues. All four cysteines present in the sequence were individually replaced with alanine, and the kinetic constants of the altered enzymes were determined. The results indicated that only C159 is essential for activity; however, replacement with serine resulted in a fully active enzyme. Activity of the unaltered phosphotransacetylase was sensitive to N-ethylmaleimide. Inhibition kinetics of altered enzymes indicated that this sensitivity resulted from modification of C312, which is at the active site but itself is nonessential for catalysis. Five arginines were individually replaced with glutamine. Kinetic analysis of the altered enzymes identified R310 as essential for activity. Of the four nonessential for activity, R87 and R133 appear to be involved in binding CoA.

Amino Acid Sequence↗
PubMed1996

Characterization of an iron-sulfur flavoprotein from Methanosarcina thermophila.

A gene (isf) encoding an iron-sulfur flavoprotein (Isf) from Methanosarcina thermophila was cloned and sequenced. The gene was located directly upstream of the genes (pta and ack) encoding phosphotransacetylase and acetate kinase and is transcribed in the opposite direction. The amino acid sequence deduced from isf contained a cluster of cysteine residues reminiscent of proteins that accommodate either a [4Fe-4S] or [3Fe-4S] center. The protein was heterologously produced in Escherichia coli and purified to apparent homogeneity. The 29-kDa subunit molecular mass of heterologously produced Isf (determined by SDS-polyacrylamide gel electrophoresis) corresponded to the molecular mass of 30,451 Da calculated from the amino acid composition deduced from isf. Gel filtration estimated a molecular mass of 65 kDa for the native Isf indicating an alpha2 homodimer. The UV-visible absorption spectrum was characteristic of iron-sulfur flavoproteins with maxima at 484, 452, 430, 378, and 280 nm. Analyses identified 2 FMN, 7-8 non-heme iron atoms, and 6-7 acid-labile sulfur atoms per alpha2 homodimer. Comparisons of the deduced Isf sequence with sequences in available protein data bases suggested Isf is a novel iron-sulfur flavoprotein. Western blot analysis indicated the presence of Isf in extracts of acetate-grown M. thermophila. Ferredoxin stimulated the CO-dependant reduction of Isf by the CO dehydrogenase middle dotacetyl-CoA synthase complex that suggested ferredoxin is a physiological electron donor to Isf.

Archaeal Proteins↗
PubMed1996

A left-hand beta-helix revealed by the crystal structure of a carbonic anhydrase from the archaeon Methanosarcina thermophila.

A carbonic anhydrase from the thermophilic archaeon Methanosarcina thermophila that exhibits no significant sequence similarity to known carbonic anhydrases has recently been characterized. Here we present the structure of this enzyme, which adopts a left-handed parallel beta-helix fold. This fold is of particular interest since it contains only left-handed crossover connections between the parallel beta-strands, which so far have been observed very infrequently. The active form of the enzyme is a trimer with three zinc-containing active sites, each located at the interface between two monomers. While the arrangement of active site groups differs between this enzyme and the carbonic anhydrases from higher vertebrates, there are structural similarities in the zinc coordination environment, suggestive of convergent evolution dictated by the chemical requirements for catalysis of the same reaction. Based on sequence similarities, the structure of this enzyme is the prototype of a new class of carbonic anhydrases with representatives in all three phylogenetic domains of life.

Amino Acid Sequence↗
PubMed1996

Characterization of heterologously produced carbonic anhydrase from Methanosarcina thermophila.

The gene encoding carbonic anhydrase from Methanosarcina thermophila was hyperexpressed in Escherichia coli, and the heterologously produced enzyme was purified 14-fold to apparent homogeneity. The enzyme purified from E. coli has properties (specific activity, inhibitor sensitivity, and thermostability) similar to those of the authentic enzyme isolated from M. thermophila; however, a discrepancy in molecular mass suggests that the carbonic anhydrase is posttranslationally modified in either E. coli or M. thermophila. Both the authentic and heterologously produced enzymes were stable to heating at 55 degrees C for 15 min but were inactivated at higher temperatures. No esterase activity was detected with p-nitrophenylacetate as the substrate. Plasma emission spectroscopy revealed approximately 0.6 Zn per subunit. As judged from the estimated native molecular mass, the enzyme is either a trimer or a tetramer. Western blot (immunoblot) analysis of cell extract proteins from M. thermophila indicates that the levels of carbonic anhydrase are regulated in response to the growth substrate, with protein levels higher in acetate than in methanol- or trimethylamine-grown cells.

Amino Acid Sequence↗
PubMed1996

Characterization of the cdhD and cdhE genes encoding subunits of the corrinoid/iron-sulfur enzyme of the CO dehydrogenase complex from Methanosarcina thermophila.

The CO dehydrogenase enzyme complex from Methanosarcina thermophila contains a corrinoid/iron-sulfur enzyme composed of two subunits (delta and gamma). The cdhD and cdhE genes, which encode the delta and gamma subunits, respectively, were cloned and sequenced. The cdhD gene is upstream of and separated by 3 bp from cdhE. Both genes are preceded by apparent ribosome-binding sites. Northern (RNA) blot and primer extension analyses indicated that cdhD and cdhE are cotranscribed from a promoter located several kilobases upstream of cdhD. The putative CdhD and CdhE sequences are 37% identical to the sequences deduced from the genes encoding the beta and alpha subunits of the corrinoid/iron-sulfur enzyme from Clostridium thermoaceticum. The CdhE sequence had a four-cysteine motif with the potential to bind a 4Fe-4S cluster previously identified in the corrinoid/iron-sulfur enzyme by electron paramagnetic resonance spectroscopy. A T7 RNA polymerase/promoter system was used to produce CdhD and CdhE independently in Escherichia coli. The purified CdhD protein was reconstituted with hydroxocobalamin in the base-on configuration. The purified CdhE protein exhibited an Fe-S center and base-off cobalamin binding in which the benzimidazole base nitrogen atom was no longer a lower axial ligand to the cobalt atom.

Aldehyde Oxidoreductases↗
PubMed1996

Analysis of the CO dehydrogenase/acetyl-coenzyme A synthase operon of Methanosarcina thermophila.

The cdhABC genes encoding the respective alpha, epsilon, and beta subunits of the five-subunit (alpha, beta, gamma, delta, and epsilon) CO dehydrogenase/acetyl-coenzyme synthase (CODH/ACS) complex from Methanosarcina thermophila were cloned and sequenced. Northern (RNA) blot analyses indicated that the cdh genes encoding the five subunits and an open reading frame (ORF1) with unknown function are cotranscribed during growth on acetate. Northern blot and primer extension analyses suggested that mRNA processing and multiple promoters may be involved in cdh transcript synthesis. The putative CdhA (alpha subunit) and CdhB (epsilon subunit) proteins each have 40% identity to CdhA and CdhB of the CODH/ACS complex from Methanosaeta soehngenii. The cdhC gene encodes the beta subunit (CdhC) of the CODH/ACS complex from M. thermophila. The N-terminal 397 amino acids of CdhC are 42% identical to the C-terminal half of the alpha subunit of CODH/ACS from the acetogenic anaerobe Clostridium thermoaceticum. Sequence analysis suggested potential structures and functions for the previously uncharacterized beta subunit from M. thermophila. The deduced protein sequence of ORF1, located between the cdhC and cdhD genes, has 29% identity to NifH2 from Methanobacterium ivanovii.

Aldehyde Oxidoreductases↗
PubMed1995

A proteasome from the methanogenic archaeon Methanosarcina thermophila.

A 645-kDa proteasome was purified from Methanosarcina thermophila which had chymotrypsin-like and peptidylglutamyl-peptide hydrolase activities and contained alpha (24-kDa) and beta (22-kDa) subunits. Processing of both subunits was suggested by comparison of N-terminal sequences with the sequences deduced from the alpha- and beta-encoding genes (psmA and psmB). Alignment of deduced sequences for the alpha and beta subunits revealed high similarity; however, the N-terminal sequence of the alpha subunit contained an additional 24 amino acids that were not present in the beta subunit. The alpha and beta subunits had high sequence identity with alpha- and beta-type subunits of proteasomes from eucaryotic organisms and the distantly related archaeon Thermoplasma acidophilum. The psmB gene was transcribed in vivo as a monocistronic message from a consensus archaeal promoter. The results suggest that proteasomes are more widespread in the Archaea than previously proposed. Southern blotting experiments suggested the presence of ubiquitin-like sequences in M. thermophila.

Amino Acid Sequence↗
PubMed1995

Transcriptional regulation of the phosphotransacetylase-encoding and acetate kinase-encoding genes (pta and ack) from Methanosarcina thermophila.

Phosphotransacetylase and acetate kinase catalyze the activation of acetate to acetyl coenzyme A in the first step of methanogenesis from acetate in Methanosarcina thermophila. The genes encoding these enzymes (pta and ack) have been cloned and sequenced. They are arranged on the chromosome with pta upstream of ack (M.T. Latimer, and J. G. Ferry, J. Bacteriol. 175:6822-6829, 1993). The activities of phosphotransacetylase and acetate kinase are at least 8- to 11-fold higher in acetate-grown cells than in cells grown on methanol, monomethylamine, dimethylamine, or trimethylamine. Northern blot (RNA) analyses demonstrated that pta and ack are transcribed as an approximately 2.4-kb polycistronic message and that the regulation of enzyme synthesis occurs at the mRNA level. Primer extension analyses revealed a transcriptional start site located 27 bp upstream from the translational start of the pta gene and 24 bp downstream from a consensus archaeal boxA promoter sequence. S1 nuclease protection assays detected transcripts with four different 3' ends, each of which mapped to the beginning of four consecutive direct repeats. Northern blot analysis using an ack-specific probe detected both the 2.4-kb polycistronic transcript and a smaller 1.4-kb transcript which is the estimated size of monocistronic ack mRNA. A primer extension product was detected with an ack-specific primer; the 5' end of the product was in the intergenic region between the pta and ack genes but did not follow a consensus archaeal boxA sequence. This result, as well as detection of an additional 1.4-kb mRNA species, suggests processing of the polycistronic 2.4-kb transcript.

Acetate Kinase↗
PubMed1995

CO dehydrogenase.

Structurally and functionally diverse CO dehydrogenases are key components of various energy-yielding pathways in aerobic and anaerobic microbes from the Bacteria and Archaea domains. Aerobic microbes utilize Mo-Fe-flavin CO dehydrogenases to oxidize CO in respiratory pathways. Phototrophic anaerobes grow by converting CO to H2, a process initiating with a CO dehydrogenase that contains nickel and iron-sulfur centers. Acetate-producing anaerobes employ a nickel/iron-sulfur CO dehydrogenase to synthesize acetyl-CoA from a methyl group, CO, and CoA. A similar enzyme is responsible for the cleavage of acetyl-CoA by anaerobic Archaea that obtain energy by fermenting acetate to CH4 and CO2. Acetotrophic sulfate reducers from the Bacteria and Archaea also utilize CO dehydrogenase to cleave acetyl-CoA yielding methyl and carbonyl groups. These microbes obtain energy for growth via a respiratory pathway in which the methyl and carbonyl groups are oxidized to CO2, and sulfate is reduced to sulfide.

Aldehyde Oxidoreductases↗