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1.  Novel Pyrophosphate-Forming Acetate Kinase from the Protist Entamoeba histolytica 
Eukaryotic Cell  2012;11(10):1249-1256.
Acetate kinase (ACK) catalyzes the reversible synthesis of acetyl phosphate by transfer of the γ-phosphate of ATP to acetate. Here we report the first biochemical and kinetic characterization of a eukaryotic ACK, that from the protist Entamoeba histolytica. Our characterization revealed that this protist ACK is the only known member of the ASKHA structural superfamily, which includes acetate kinase, hexokinase, and other sugar kinases, to utilize inorganic pyrophosphate (PPi)/inorganic phosphate (Pi) as the sole phosphoryl donor/acceptor. Detection of ACK activity in E. histolytica cell extracts in the direction of acetate/PPi formation but not in the direction of acetyl phosphate/Pi formation suggests that the physiological direction of the reaction is toward acetate/PPi production. Kinetic parameters determined for each direction of the reaction are consistent with this observation. The E. histolytica PPi-forming ACK follows a sequential mechanism, supporting a direct in-line phosphoryl transfer mechanism as previously reported for the well-characterized Methanosarcina thermophila ATP-dependent ACK. Characterizations of enzyme variants altered in the putative acetate/acetyl phosphate binding pocket suggested that acetyl phosphate binding is not mediated solely through a hydrophobic interaction but also through the phosphoryl group, as for the M. thermophila ACK. However, there are key differences in the roles of certain active site residues between the two enzymes. The absence of known ACK partner enzymes raises the possibility that ACK is part of a novel pathway in Entamoeba.
doi:10.1128/EC.00169-12
PMCID: PMC3485911  PMID: 22903977
2.  Purification and characterization of two reversible and ADP-dependent acetyl coenzyme A synthetases from the hyperthermophilic archaeon Pyrococcus furiosus. 
Journal of Bacteriology  1996;178(20):5897-5903.
Pyrococcus furiosus is a strictly anaerobic archaeon (archaebacterium) that grows at temperatures up to 105 degrees C by fermenting carbohydrates and peptides. Cell extracts have been previously shown to contain an unusual acetyl coenzyme A (acetyl-CoA) synthetase (ACS) which catalyzes the formation of acetate and ATP from acetyl-CoA by using ADP and phosphate rather than AMP and PPi. We show here that P. furiosus contains two distinct isoenzymes of ACS, and both have been purified. One, termed ACS I, uses acetyl-CoA and isobutyryl-CoA but not indoleacetyl-CoA or phenylacetyl-CoA as substrates, while the other, ACS II, utilizes all four CoA derivatives. Succinyl-CoA did not serve as a substrate for either enzyme. ACS I and ACS II have similar molecular masses (approximately 140 kDa), and both appear to be heterotetramers (alpha2beta2) of two different subunits of 45 (alpha) and 23 (beta) kDa. They lack metal ions such as Fe2+, Cu2+, Zn2+, and Mg2+ and are stable to oxygen. At 25 degrees C, both enzymes were virtually inactive and exhibited optimal activities above 90 degrees C (at pH 8.0) and at pH 9.0 (at 80 degrees C). The times required to lose 50% of their activity at 80 degrees C were about 18 h for ACS I and 8 h for ACS II. With both enzymes in the acid formation reactions, ADP and phosphate could be replaced by GDP and phosphate but not by CDP and phosphate or by AMP and PPi. The apparent Km values for ADP, GDP, and phosphate were approximately 150, 132, and 396 microM, respectively, for ACS I (using acetyl-CoA) and 61, 236, and 580 microM, respectively, for ACS II (using indoleacetyl-CoA). With ADP and phosphate as substrates, the apparent Km values for acetyl-CoA and isobutyryl-CoA were 25 and 29 microM, respectively, for ACS I and 26 and 12 microM, respectively, for ACS II. With ACS II, the apparent Km value for phenylacetyl-CoA was 4 microM. Both enzymes also catalyzed the reverse reaction, the ATP-dependent formation of the CoA derivatives of acetate (I and II), isobutyrate (I and II), phenylacetate (II only), and indoleacetate (II only). The N-terminal amino acid sequences of the two subunits of ACS I were similar to those of ACS II and to that of a hypothetical 67-kDa protein from Escherichia coli but showed no similarity to mesophilic ACS-type enzymes. To our knowledge, ACS I and II are the first ATP-utilizing enzymes to be purified from a hyperthermophile, and ACS II is the first enzyme of the ACS type to utilize aromatic CoA derivatives.
PMCID: PMC178444  PMID: 8830684
3.  Characterization of an Archaeal Medium-Chain Acyl Coenzyme A Synthetase from Methanosarcina acetivorans▿  
Journal of Bacteriology  2010;192(22):5982-5990.
Short- and medium-chain acyl coenzyme A (acyl-CoA) synthetases catalyze the formation of acyl-CoA from an acyl substrate, ATP, and CoA. These enzymes catalyze mechanistically similar two-step reactions that proceed through an enzyme-bound acyl-AMP intermediate. Here we describe the characterization of a member of this enzyme family from the methane-producing archaeon Methanosarcina acetivorans. This enzyme, a medium-chain acyl-CoA synthetase designated MacsMa, utilizes 2-methylbutyrate as its preferred substrate for acyl-CoA synthesis but cannot utilize acetate and thus cannot catalyze the first step of acetoclastic methanogenesis in M. acetivorans. When propionate or other less favorable acyl substrates, such as butyrate, 2-methylpropionate, or 2-methylvalerate, were utilized, the acyl-CoA was not produced or was produced at reduced levels. Instead, acyl-AMP and PPi were released in the absence of CoA, whereas in the presence of CoA, the intermediate was broken down into AMP and the acyl substrate, which were released along with PPi. These results suggest that although acyl-CoA synthetases may have the ability to utilize a broad range of substrates for the acyl-adenylate-forming first step of the reaction, the intermediate may not be suitable for the thioester-forming second step. The MacsMa structure has revealed the putative acyl substrate- and CoA-binding pockets. Six residues proposed to form the acyl substrate-binding pocket, Lys256, Cys298, Gly351, Trp259, Trp237, and Trp254, were targeted for alteration. Characterization of the enzyme variants indicates that these six residues are critical in acyl substrate binding and catalysis, and even conservative alterations significantly reduced the catalytic ability of the enzyme.
doi:10.1128/JB.00600-10
PMCID: PMC2976453  PMID: 20851904
4.  Isolation and characterization of acetyl-coenzyme A synthetase from Methanothrix soehngenii. 
Journal of Bacteriology  1989;171(10):5430-5435.
In Methanothrix soehngenii, acetate is activated to acetyl-coenzyme A (acetyl-CoA) by an acetyl-CoA synthetase. Cell extracts contained high activities of adenylate kinase and pyrophosphatase, but no activities of a pyrophosphate:AMP and pyrophosphate:ADP phosphotransferase, indicating that the activation of 1 acetate in Methanothrix requires 2 ATP. Acetyl-CoA synthetase was purified 22-fold in four steps to apparent homogeneity. The native molecular mass of the enzyme from M. soehngenii estimated by gel filtration was 148 kilodaltons (kDa). The enzyme was composed of two subunits with a molecular mass of 73 kDa in an alpha 2 oligomeric structure. The acetyl-CoA synthetase constituted up to 4% of the soluble cell protein. At the optimum pH of 8.5, the Vmax was 55 mumol of acetyl-CoA formed per min per mg of protein. Analysis of enzyme kinetic properties revealed a Km of 0.86 mM for acetate and 48 microM for coenzyme A. With varying amounts of ATP, weak sigmoidal kinetic was observed. The Hill plot gave a slope of 1.58 +/- 0.12, suggesting two interacting substrate sites for the ATP. The kinetic properties of the acetyl-CoA synthetase can explain the high affinity for acetate of Methanothrix soehngenii.
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PMCID: PMC210380  PMID: 2571608
5.  A Colorimetric Assay Method to Measure Acetyl-CoA Synthetase Activity: Application to Woodchuck Model of Hepatitis Virus-induced Hepatocellular Carcinoma 
A new spectrophotometric method for quantitation of acetyl-CoA synthetase (ACAS) activity is developed. It has been applied for ACAS assay in the liver tissues of a woodchuck model of hepatitis virus-induced hepatocellular carcinoma (HCC). The assay is based on the established pyrophosphate (PPi) detection system. ACAS activity is indexed by the amount of PPi, the product of ACAS reaction system of activated form of acetate (acetyl-CoA) with ACAS catalysis. PPi is determined quantitatively as the amount of chromophore formed with molybdate reagent, 1-amino-2-naphthol-4-sulfonic acid in bisulfite and 2-mercaptoethanol. PPi reacts with molybdate reagent to produce phosphomolybdate and PPi-molybdate complexes. 2-mercaptoethanol is responsible for color formation which has the peak absorption at 580nm. This method was sensitive from 1 to 20 nmol of PPi in a 380-μl sample (1-cm cuvette). A ten-fold excess of Pi did not interfere with the determination of PPi. To study the major metabolic pathways of imaging tracer [1-11C]-acetate in tumors for detection of HCC by Positron Emission Tomography (PET), the activity of one of the key enzymes involved in acetate or [1-11C]-acetate metabolism, ACAS was assayed by this newly developed assay in the tissue samples of woodchuck HCCs. An significant increase of ACAS activity was observed in the liver tissues of woodchuck HCCs as compared with neighboring regions surrounding the tumors (P < 0.01). The respective ACAS activities in the subcellular locations were also significantly higher in HCCs than in the surrounding tissues (P < 0.01) (total soluble fraction: 876.61 ± 34.64 vs. 361.62 ± 49.97 mU/g tissue; cytoplasmic fraction: 1122.02 ± 112.39 vs. 732.32 ± 84.44 mU/g tissue; organelle content: 815.79 ± 491.5 vs. 547.91 ± 97.05 mU/ g tissue; sedimentable fragment: 251.92 ± 143.7 vs. 90.94 ± 18.98 mU/ g tissue). The finding suggests an increase in ACAS activity in the liver cancer of woodchuck models of HCC as compared to that in the normal woodchuck liver. The developed assay is rapid, simple and accurate and is suitable for the investigation of ACAS activity under physiologic and pathophysiologic conditions.
doi:10.1016/j.jbbm.2007.02.008
PMCID: PMC1995416  PMID: 17399795
Acetyl-CoA Synthetase; Woodchuck; Woodchuck Hepatitis Virus; Hepatocellular Carcinoma; [1-11C]Acetate; Positron Emission Tomography
6.  Purification and Characterization of Two Extremely Thermostable Enzymes, Phosphate Acetyltransferase and Acetate Kinase, from the Hyperthermophilic Eubacterium Thermotoga maritima 
Journal of Bacteriology  1999;181(6):1861-1867.
Phosphate acetyltransferase (PTA) and acetate kinase (AK) of the hyperthermophilic eubacterium Thermotoga maritima have been purified 1,500- and 250-fold, respectively, to apparent homogeneity. PTA had an apparent molecular mass of 170 kDa and was composed of one subunit with a molecular mass of 34 kDa, suggesting a homotetramer (α4) structure. The N-terminal amino acid sequence showed significant identity to that of phosphate butyryltransferases from Clostridium acetobutylicum rather than to those of known phosphate acetyltransferases. The kinetic constants of the reversible enzyme reaction (acetyl-CoA + Pi ⇌ acetyl phosphate + CoA) were determined at the pH optimum of pH 6.5. The apparent Km values for acetyl-CoA, Pi, acetyl phosphate, and coenzyme A (CoA) were 23, 110, 24, and 30 μM, respectively; the apparent Vmax values (at 55°C) were 260 U/mg (acetyl phosphate formation) and 570 U/mg (acetyl-CoA formation). In addition to acetyl-CoA (100%), the enzyme accepted propionyl-CoA (60%) and butyryl-CoA (30%). The enzyme had a temperature optimum at 90°C and was not inactivated by heat upon incubation at 80°C for more than 2 h. AK had an apparent molecular mass of 90 kDa and consisted of one 44-kDa subunit, indicating a homodimer (α2) structure. The N-terminal amino acid sequence showed significant similarity to those of all known acetate kinases from eubacteria as well that of the archaeon Methanosarcina thermophila. The kinetic constants of the reversible enzyme reaction (acetyl phosphate + ADP ⇌ acetate + ATP) were determined at the pH optimum of pH 7.0. The apparent Km values for acetyl phosphate, ADP, acetate, and ATP were 0.44, 3, 40, and 0.7 mM, respectively; the apparent Vmax values (at 50°C) were 2,600 U/mg (acetate formation) and 1,800 U/mg (acetyl phosphate formation). AK phosphorylated propionate (54%) in addition to acetate (100%) and used GTP (100%), ITP (163%), UTP (56%), and CTP (21%) as phosphoryl donors in addition to ATP (100%). Divalent cations were required for activity, with Mn2+ and Mg2+ being most effective. The enzyme had a temperature optimum at 90°C and was stabilized against heat inactivation by salts. In the presence of (NH4)2SO4 (1 M), which was most effective, the enzyme did not lose activity upon incubation at 100°C for 3 h. The temperature optimum at 90°C and the high thermostability of both PTA and AK are in accordance with their physiological function under hyperthermophilic conditions.
PMCID: PMC93586  PMID: 10074080
7.  3-Hydroxypropionyl-Coenzyme A Synthetase from Metallosphaera sedula, an Enzyme Involved in Autotrophic CO2 Fixation▿  
Journal of Bacteriology  2007;190(4):1383-1389.
A modified 3-hydroxypropionate cycle has been proposed as the autotrophic CO2 fixation pathway for the thermoacidophilic crenarchaeon Metallosphaera sedula. The cycle requires the reductive conversion of 3-hydroxypropionate to propionyl-coenzyme A (propionyl-CoA). The specific activity of the 3-hydroxypropionate-, CoA-, and MgATP-dependent oxidation of NADPH in autotrophically grown cells was 0.023 μmol min−1mg protein−1. The reaction sequence is catalyzed by at least two enzymes. The first enzyme, 3-hydroxypropionyl-CoA synthetase, catalyzes the following reaction: 3-hydroxypropionate + ATP + CoA → 3-hydroxypropionyl-CoA + AMP + PPi. The enzyme was purified 95-fold to a specific activity of 18 μmol min−1 mg protein−1 from autotrophically grown M. sedula cells. An internal peptide sequence was determined and a gene encoding a homologous protein identified in the genome of Sulfolobus tokodaii; similar genes were found in S. solfataricus and S. acidocaldarius. The gene was heterologously expressed in Escherichia coli, and the His-tagged protein was purified. Both the native enzyme from M. sedula and the recombinant enzyme from S. tokodaii not only activated 3-hydroxypropionate to its CoA ester but also activated propionate, acrylate, acetate, and butyrate; however, with the exception of propionate, the affinities for these substrates were reduced. 3-Hydroxypropionyl-CoA synthetase is up-regulated eightfold in autotrophically versus heterotrophically grown M. sedula, supporting its proposed role during CO2 fixation in this archaeon and possibly other members of the Sulfolobaceae family.
doi:10.1128/JB.01593-07
PMCID: PMC2238213  PMID: 18165310
8.  Biochemical and Molecular Characterization of Phenylacetate-Coenzyme A Ligase, an Enzyme Catalyzing the First Step in Aerobic Metabolism of Phenylacetic Acid in Azoarcus evansii 
Journal of Bacteriology  2000;182(2):286-294.
Phenylacetate-coenzyme A ligase (PA-CoA ligase; AMP forming, EC 6.2.1.30), the enzyme catalyzing the first step in the aerobic degradation of phenylacetate (PA) in Azoarcus evansii, has been purified and characterized. The gene (paaK) coding for this enzyme was cloned and sequenced. The enzyme catalyzes the reaction of PA with CoA and MgATP to yield phenylacetyl-CoA (PACoA) plus AMP plus PPi. The enzyme was specifically induced after aerobic growth in a chemically defined medium containing PA or phenylalanine (Phe) as the sole carbon source. Growth with 4-hydroxyphenylacetate, benzoate, adipate, or acetate did not induce the synthesis of this enzyme. This enzymatic activity was detected very early in the exponential phase of growth, and a maximal specific activity of 76 nmol min−1 mg of cell protein−1 was measured. After 117-fold purification to homogeneity, a specific activity of 48 μmol min−1 mg of protein−1 was achieved with a turnover number (catalytic constant) of 40 s−1. The protein is a monomer of 52 kDa and shows high specificity towards PA; other aromatic or aliphatic acids were not used as substrates. The apparent Km values for PA, ATP, and CoA were 14, 60, and 45 μM, respectively. The PA-CoA ligase has an optimum pH of 8 to 8.5 and a pI of 6.3. The enzyme is labile and requires the presence of glycerol for stabilization. The N-terminal amino acid sequence of the purified protein showed no homology with other reported PA-CoA ligases. The gene encoding this enzyme is 1,320 bp long and codes for a protein of 48.75 kDa (440 amino acids) which shows high similarity with other reported PA-CoA ligases. An amino acid consensus for an AMP binding motif (VX2SSGTTGXP) was identified. The biochemical and molecular characteristics of this enzyme are quite different from those of the isoenzyme catalyzing the same reaction under anaerobic conditions in the same bacterium.
PMCID: PMC94275  PMID: 10629172
9.  PPi-dependent phosphofructotransferase (phosphofructokinase) activity in the mollicutes (mycoplasma) Acholeplasma laidlawii. 
Journal of Bacteriology  1986;165(1):53-60.
A PPi-dependent phosphofructotransferase (PPi-fructose 6-phosphate 1-phosphotransferase, EC 2.7.1.90) which catalyzes the conversion of fructose 6 phosphate (F-6-P) to fructose 1,6-bisphosphate (F-1, 6-P2) was isolated from a cytoplasmic fraction of Acholeplasma laidlawii B-PG9 and partially purified (430-fold). PPi was required as the phosphate donor. ATP, dATP, CTP, dCTP, GTP, dGTP, UTP, dUTP, ITP, TTP, ADP, or Pi could not substitute for PPi. The PPi-dependent reaction (2.0 mM PPi) was not altered in the presence of any of these nucleotides (2.0 mM) or in the presence of smaller (less than or equal to 300 microM) amounts of fructose 2,6-bisphosphate, (NH4)2SO4, AMP, citrate, GDP, or phosphoenolpyruvate. Mg2+ and a pH of 7.4 were required for maximum activity. The partially purified enzyme in sucrose density gradient experiments had an approximate molecular weight of 74,000 and a sedimentation coefficient of 6.7. A second form of the enzyme (molecular weight, 37,000) was detected, although in relatively smaller amounts, by using Blue Sepharose matrix when performing electrophoresis experiments. The back reaction, F-1, 6-P2 to F-6-P, required Pi; arsenate could substitute for Pi, but not PPi or any other nucleotide tested. The computer-derived kinetic constants (+/- standard deviation) for the reaction in the PPi-driven direction of F-1, 6-P2 were as follows: v, 38.9 +/- 0.48 mM min-1; Ka(PPi), 0.11 +/- 0.04 mM; Kb(F-6-P), 0.65 +/- 0.15 mM; and Kia(PPi), 0.39 +/- 0.11 mM. A. laidlawii B-PG9 required PPi not only for the PPi-phosphofructotransferase reaction which we describe but also for purine nucleoside kinase activity. a dependency unknown in any other organism. In A. laidlawii B-PG9, the PPi requirement may be met by reactions in this organism already known to synthesize PPi (e.g., dUTPase and purine nucleobase phosphoribosyltransferases). In almost all other cells, the conversion of F-6-P to F-1,6-P2 is ATP dependent, and the reaction is generally considered to be the rate-limiting step of glycolysis. The ability of A. laidlawii B-PG9 and one other acholeplasma to use PPi instead of ATP as an energy source may offer these cytochrome-deficient organisms some metabolic advantage and may represent a conserved metabolic remnant of an earlier evolutionary process.
PMCID: PMC214369  PMID: 3001032
10.  Acetate catabolism by Methanosarcina barkeri: evidence for involvement of carbon monoxide dehydrogenase, methyl coenzyme M, and methylreductase. 
Journal of Bacteriology  1985;163(3):1000-1006.
The pathway of acetate catabolism in Methanosarcina barkeri strain MS was studied by using a recently developed assay for methanogenesis from acetate by soluble enzymes in cell extracts. Extracts incubated with [2-14C]acetate, hydrogen, and ATP formed 14CH4 and [14C]methyl coenzyme M as products. The apparent Km for acetate conversion to methane was 5 mM. In the presence of excess acetate, both the rate and duration of methane production was dependent on ATP. Acetyl phosphate replaced the cell extract methanogenic requirement for both acetate and ATP (the Km for ATP was 2 mM). Low concentrations of bromoethanesulfonic acid and cyanide, inhibitors of methylreductase and carbon monoxide dehydrogenase, respectively, greatly reduced the rate of methanogenesis. Precipitation of CO dehydrogenase in cell extracts by antibodies raised to 95% purified enzyme inhibited both CO dehydrogenase and acetate-to-methane conversion activity. The data are consistent with a model of acetate catabolism in which methylreductase, methyl coenzyme M, CO dehydrogenase, and acetate-activating enzymes are components. These results are discussed in relation to acetate uptake and rate-limiting transformation mechanisms in methane formation.
PMCID: PMC219231  PMID: 3928595
11.  Regulation of pyrophosphate levels by H+-PPase is central for proper resumption of early plant development 
Plant Signaling & Behavior  2012;7(1):38-42.
The synthesis of DNA, RNA, and de novo proteins is fundamental for early development of the seedling after germination, but such processes release pyrophosphate (PPi) as a byproduct of ATP hydrolysis. The over-accumulation of the inhibitory metabolite PPi in the cytosol hinders these biosynthetic reactions. All living organisms possess ubiquitous enzymes collectively called inorganic pyrophosphatases (PPases), which catalyze the hydrolysis of PPi into two orthophosphate (Pi) molecules. Defects in PPase activity cause severe developmental defects and/or growth arrest in several organisms. In higher plants, a proton-translocating vacuolar PPase (H+­PPase) uses the energy of PPi hydrolysis to acidify the vacuole. However, the biological implications of PPi hydrolysis are vague due to the widespread belief that the major role of H+­PPase in plants is vacuolar acidification. We have shown that the Arabidopsis fugu5 mutant phenotype, caused by a defect in H+­PPase activity, is rescued by complementation with the yeast cytosolic PPase IPP1. In addition, our analyses have revealed that increased cytosolic PPi levels impair postgerminative development in fugu5 by inhibiting gluconeogenesis. This led us to the conclusion that the role of H+­PPase as a proton-pump is negligible. Here, we present further evidence of the growth-boosting effects of removing PPi in later stages of plant vegetative development, and briefly discuss the biological role of PPases and their potential applications in different disciplines and in various organisms.
doi:10.4161/psb.7.1.18573
PMCID: PMC3357364  PMID: 22301965
fugu5 mutant; compensation; gluconeogenesis; H+-pyrophosphatase; leaf development; oilseeds; sucrose
12.  Studies of the CobA-Type ATP:Co(I)rrinoid Adenosyltransferase Enzyme of Methanosarcina mazei Strain Gö1 
Journal of Bacteriology  2006;188(10):3543-3550.
Although methanogenic archaea use B12 extensively as a methyl carrier for methanogenesis, little is known about B12 metabolism in these prokaryotes or any other archaea. To improve our understanding of how B12 metabolism differs between bacteria and archaea, the gene encoding the ATP:co(I)rrinoid adenosyltransferase in Methanosarcina mazei strain Gö1 (open reading frame MM3138, referred to as cobAMm here) was cloned and used to restore coenzyme B12 synthesis in a Salmonella enterica strain lacking the housekeeping CobA enzyme. cobAMm protein was purified and its initial biochemical analysis performed. In vitro, the activity is enhanced 2.5-fold by the addition of Ca2+ ions, but the activity was not enhanced by Mg2+ and, unlike the S. enterica CobA enzyme, it was >50% inhibited by Mn2+. The CobAMm enzyme had a KmATP of 3 μM and a KmHOCbl of 1 μM. Unlike the S. enterica enzyme, CobAMm used cobalamin (Cbl) as a substrate better than cobinamide (Cbi; a Cbl precursor); the β phosphate of ATP was required for binding to the enzyme. A striking difference between CobASe and CobAMm was the use of ADP as a substrate by CobAMm, suggesting an important role for the γ phosphate of ATP in binding. The results from 31P-nuclear magnetic resonance spectroscopy experiments showed that triphosphate (PPPi) is the reaction by-product; no cleavage of PPPi was observed, and the enzyme was only slightly inhibited by pyrophosphate (PPi). The data suggested substantial variations in ATP binding and probably corrinoid binding between CobASe and CobAMm enzymes.
doi:10.1128/JB.188.10.3543-3550.2006
PMCID: PMC1482872  PMID: 16672609
13.  Crystal Structure of Tryptophanyl-tRNA Synthetase Complexed with Adenosine-5′ Tetraphosphate: Evidence for Distributed Use of Catalytic Binding Energy in Amino Acid Activation by Class I Aminoacyl-tRNA Synthetases 
Journal of molecular biology  2007;369(1):108-128.
Tryptophanyl-tRNA synthetase (TrpRS) is a functionally dimeric ligase, which specifically couples hydrolysis of ATP to AMP and pyrophosphate to the formation of an ester bond between tryptophan and the cognate tRNA. TrpRS from Bacillus stearothermophilus binds the ATP analogue, adenosine-5′ tetraphosphate, AQP, competitively with ATP during pyrophosphate exchange. Estimates of binding affinity from this competitive inhibition and from isothermal titration calorimetry show that AQP binds 200 times more tightly than ATP both under conditions of induced-fit, where binding is coupled to an unfavourable conformational change, and under exchange conditions, where there is no conformational change. These binding data provide an indirect experimental measurement of +3.0 kcal/mole for the conformational free energy change associated with induced-fit assembly of the active site. Thermodynamic parameters derived from the calorimetry reveal very modest enthalpic changes, consistent with binding driven largely by a favorable entropy change. The 2.5 Å structure of the TrpRS:AQP complex, determined de novo by X-ray crystallography, resembles that of the previously described, pre-transition state TrpRS:ATP complexes. The anticodon-binding domain untwists relative to the Rossmann-fold domain by 20% of the way toward the orientation observed for the Products complex. An unexpected tetraphosphate conformation allows the γ̃ and δ̃ phosphate groups to occupy positions equivalent to those occupied by the β̃ and γ̃ phosphates of ATP. The β-phosphate effects a 1.11 Å extension that relocates the α-phosphate toward the tryptophan carboxylate while the PPi mimic moves deeper into the KMSKS loop. This configuration improves interactions between enzyme and nucleotide significantly and uniformly in the adenosine and PPi binding subsites. A new hydrogen bond forms between S194 from the class I KMSKS signature sequence and the PPi mimic. These complementary thermodynamic and structural data are all consistent with the conclusion that the tetraphosphate mimics a transition-state in which the KMSKS loop develops increasingly tight bonds to the PPi leaving group, weakening linkage to the Pα as it is relocated by an energetically favourable domain movement. Consistent with extensive mutational data on Tyrosyl-tRNA synthetase, this aspect of the mechanism develops high transition-state affinity for the adenosine and pyrophosphate moieties, which move significantly, relative to one another, during the catalytic step.
doi:10.1016/j.jmb.2007.01.091
PMCID: PMC2715954  PMID: 17428498
Induced-fit; mechanistic enzymology; phosphoryl-transfer; catalytic use of binding energy; class I aminoacyl-tRNA synthetases
14.  Demonstration of carbon-carbon bond cleavage of acetyl coenzyme A by using isotopic exchange catalyzed by the CO dehydrogenase complex from acetate-grown Methanosarcina thermophila. 
Journal of Bacteriology  1991;173(2):929-932.
The purified nickel-containing CO dehydrogenase complex isolated from methanogenic Methanosarcina thermophila grown on acetate is able to catalyze the exchange of [1-14C] acetyl-coenzyme A (CoA) (carbonyl group) with 12CO as well as the exchange of [3'-32P]CoA with acetyl-CoA. Kinetic parameters for the carbonyl exchange have been determined: Km (acetyl-CoA) = 200 microM, Vmax = 15 min-1. CoA is a potent inhibitor of this exchange (Ki = 25 microM) and is formed under the assay conditions because of a slow but detectable acetyl-CoA hydrolase activity of the enzyme. Kinetic parameters for both exchanges are compared with those previously determined for the acetyl-CoA synthase/CO dehydrogenase from the acetogenic Clostridium thermoaceticum. Collectively, these results provide evidence for the postulated role of CO dehydrogenase as the key enzyme for acetyl-CoA degradation in acetotrophic bacteria.
PMCID: PMC207094  PMID: 1987173
15.  Glucose catabolism by Spirochaeta thermophila RI 19.B1. 
Journal of Bacteriology  1992;174(8):2449-2453.
Spirochaeta thermophila RI 19.B1 (DSM 6192) fermented glucose to lactate, acetate, CO2, and H2 with concomitant formation of cell material. The cell dry mass yield was 20.0 g/mol of glucose. From the fermentation balance data and knowledge of the fermentation pathway, a YATP of 9.22 g of dry mass per mol of ATP was calculated for pH-uncontrolled batch-culture growth on glucose in a mineral medium. Measurement of enzyme activities in glucose-grown cells revealed that glucose was taken up by a permease and then subjected to ATP-dependent phosphorylation by a hexokinase. Glucose-6-phosphate was further metabolized to pyruvate through the Embden-Meyerhof-Parnas pathway. The phosphoryl donor for phosphofructokinase activity was PPi rather than ATP. This was also found for the type strain of S. thermophila, Z-1203 (DSM 6578). PPi was probably formed by pyrophosphoroclastic cleavage of ATP, with recovery of the resultant AMP by the activity of adenylate kinase. All other measured kinase activities utilized ATP as the phosphoryl donor. Pyruvate was further metabolized to acetyl coenzyme A with concomitant production of H2 and CO2 by pyruvate synthase. Lactate was also produced from pyruvate by a fructose-1,6-diphosphate-insensitive lactate dehydrogenase. Evidence was obtained for the transfer of reducing equivalents from the glycolytic pathway to hydrogenase to produce H2. No formate dehydrogenase or significant ethanol-producing enzyme activities were detected.
PMCID: PMC205880  PMID: 1556064
16.  Identification of Essential Glutamates in the Acetate Kinase from Methanosarcina thermophila 
Journal of Bacteriology  1998;180(5):1129-1134.
Acetate kinase catalyzes the reversible phosphorylation of acetate (CH3COO− + ATP⇄CH3CO2PO32− + 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 kcat, 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.
PMCID: PMC106999  PMID: 9495750
17.  A Continuous Kinetic Assay for Adenylation Enzyme Activity and Inhibition 
Analytical biochemistry  2010;404(1):56-63.
Adenylation/adenylate-forming enzymes catalyze the activation of a carboxylic acid at the expense of ATP to form an acyl-adenylate intermediate and pyrophosphate (PPi). In a second half-reaction, adenylation enzymes catalyze the transfer of the acyl moiety of the acyl-adenylate onto an acceptor molecule, which can be either a protein or a small molecule. We describe the design, development, and validation of a coupled continuous spectrophotometric assay for adenylation enzymes that employs hydroxylamine as a surrogate acceptor molecule leading to the formation of a hydroxamate. The released pyrophosphate from the first half-reaction is measured using the pyrophosphatase–purine nucleoside phosphorylase coupling system with the chromogenic substrate 7-methylthioguanosine (MesG). The coupled hydroxamate–MesG assay is especially useful for characterizing the activity and inhibition of adenylation enzymes that acylate a protein substrate and/or fail to undergo rapid ATP-PPi exchange.
doi:10.1016/j.ab.2010.04.033
PMCID: PMC2900519  PMID: 20450872
adenylation; adenylate-forming; hydroxamate; MesG; enzyme assay
18.  O-Acetylserine Sulfhydrylase from Methanosarcina thermophila 
Journal of Bacteriology  2000;182(1):45-50.
Cysteine is the major source of fixed sulfur for the synthesis of sulfur-containing compounds in organisms of the Bacteria and Eucarya domains. Though pathways for cysteine biosynthesis have been established for both of these domains, it is unknown how the Archaea fix sulfur or synthesize cysteine. None of the four archaeal genomes sequenced to date contain open reading frames with identities to either O-acetyl-l-serine sulfhydrylase (OASS) or homocysteine synthase, the only sulfur-fixing enzymes known in nature. We report the purification and characterization of OASS from acetate-grown Methanosarcina thermophila, a moderately thermophilic methanoarchaeon. The purified OASS contained pyridoxal 5′-phosphate and catalyzed the formation of l-cysteine and acetate from O-acetyl-l-serine and sulfide. The N-terminal amino acid sequence has high sequence similarity with other known OASS enzymes from the Eucarya and Bacteria domains. The purified OASS had a specific activity of 129 μmol of cysteine/min/mg, with a Km of 500 ± 80 μM for sulfide, and exhibited positive cooperativity and substrate inhibition with O-acetyl-l-serine. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a single band at 36 kDa, and native gel filtration chromatography indicated a molecular mass of 93 kDa, suggesting that the purified OASS is either a homodimer or a homotrimer. The optimum temperature for activity was between 40 and 60°C, consistent with the optimum growth temperature for M. thermophila. The results of this study provide the first evidence for a sulfur-fixing enzyme in the Archaea domain. The results also provide the first biochemical evidence for an enzyme with the potential for involvement in cysteine biosynthesis in the Archaea.
PMCID: PMC94238  PMID: 10613861
19.  Physiological and Transcriptomic Analyses of the Thermophilic, Aceticlastic Methanogen Methanosaeta thermophila Responding to Ammonia Stress 
Microbes and Environments  2014;29(2):162-167.
The inhibitory effects of ammonia on two different degradation pathways of methanogenic acetate were evaluated using a pure culture (Methanosaeta thermophila strain PT) and defined co-culture (Methanothermobacter thermautotrophicus strain TM and Thermacetogenium phaeum strain PB), which represented aceticlastic and syntrophic methanogenesis, respectively. Growth experiments with high concentrations of ammonia clearly demonstrated that sensitivity to ammonia stress was markedly higher in M. thermophila PT than in the syntrophic co-culture. M. thermophila PT also exhibited higher sensitivity to high pH stress, which indicated that an inability to maintain pH homeostasis is an underlying cause of ammonia inhibition. Methanogenesis was inhibited in the resting cells of M. thermophila PT with moderate concentrations of ammonia, suggesting that the inhibition of enzymes involved in methanogenesis may be one of the major factors responsible for ammonia toxicity. Transcriptomic analysis revealed a broad range of disturbances in M. thermophila PT cells under ammonia stress conditions, including protein denaturation, oxidative stress, and intracellular cation imbalances. The results of the present study clearly demonstrated that syntrophic acetate degradation dominated over aceticlastic methanogenesis under ammonia stress conditions, which is consistent with the findings of previous studies on complex microbial community systems. Our results also imply that the co-existence of multiple metabolic pathways and their different sensitivities to stress factors confer resiliency on methanogenic processes.
doi:10.1264/jsme2.ME14021
PMCID: PMC4103522  PMID: 24920170
ammonia stress; methanogenesis; Methanosaeta thermophila; syntrophic acetate oxidation; transcriptome
20.  Electron transport in acetate-grown Methanosarcina acetivorans 
BMC Microbiology  2011;11:165.
Background
Acetate is the major source of methane in nature. The majority of investigations have focused on acetotrophic methanogens for which energy-conserving electron transport is dependent on the production and consumption of H2 as an intermediate, although the great majority of acetotrophs are unable to metabolize H2. The presence of cytochrome c and a complex (Ma-Rnf) homologous to the Rnf (Rhodobacter nitrogen fixation) complexes distributed in the domain Bacteria distinguishes non-H2-utilizing Methanosarcina acetivorans from H2-utilizing species suggesting fundamentally different electron transport pathways. Thus, the membrane-bound electron transport chain of acetate-grown M. acetivorans was investigated to advance a more complete understanding of acetotrophic methanogens.
Results
A component of the CO dehydrogenase/acetyl-CoA synthase (CdhAE) was partially purified and shown to reduce a ferredoxin purified using an assay coupling reduction of the ferredoxin to oxidation of CdhAE. Mass spectrometry analysis of the ferredoxin identified the encoding gene among annotations for nine ferredoxins encoded in the genome. Reduction of purified membranes from acetate-grown cells with ferredoxin lead to reduction of membrane-associated multi-heme cytochrome c that was re-oxidized by the addition of either the heterodisulfide of coenzyme M and coenzyme B (CoM-S-S-CoB) or 2-hydoxyphenazine, the soluble analog of methanophenazine (MP). Reduced 2-hydoxyphenazine was re-oxidized by membranes that was dependent on addition of CoM-S-S-CoB. A genomic analysis of Methanosarcina thermophila, a non-H2-utilizing acetotrophic methanogen, identified genes homologous to cytochrome c and the Ma-Rnf complex of M. acetivorans.
Conclusions
The results support roles for ferredoxin, cytochrome c and MP in the energy-conserving electron transport pathway of non-H2-utilizing acetotrophic methanogens. This is the first report of involvement of a cytochrome c in acetotrophic methanogenesis. The results suggest that diverse acetotrophic Methanosarcina species have evolved diverse membrane-bound electron transport pathways leading from ferredoxin and culminating with MP donating electrons to the heterodisulfide reductase (HdrDE) for reduction of CoM-S-S-CoB.
doi:10.1186/1471-2180-11-165
PMCID: PMC3160891  PMID: 21781343
21.  Carbon Monoxide, Hydrogen, and Formate Metabolism during Methanogenesis from Acetate by Thermophilic Cultures of Methanosarcina and Methanothrix Strains 
Applied and Environmental Microbiology  1992;58(10):3323-3329.
CO and H2 have been implicated in methanogenesis from acetate, but it is unclear whether they are directly involved in methanogenesis or electron transfer in acetotrophic methanogens. We compared metabolism of H2, CO, and formate by cultures of the thermophilic acetotrophic methanogens Methanosarcina thermophila TM-1 and Methanothrix sp. strain CALS-1. M. thermophila accumulated H2 to partial pressures of 40 to 70 Pa (1 Pa = 0.987 × 10-5 atm), as has been previously reported for this and other Methanosarcina cultures. In contrast, Methanothrix sp. strain CALS-1 accumulated H2 to maximum partial pressures near 1 Pa. Growing cultures of Methanothrix sp. strain CALS-1 initially accumulated CO, which reached partial pressures near 0.6 Pa (some CO came from the rubber stopper) during the middle of methanogenesis; this was followed by a decrease in CO partial pressures to less than 0.01 Pa by the end of methanogenesis. Accumulation or consumption of CO by cultures of M. thermophila growing on acetate was not detected. Late-exponential-phase cultures of Methanothrix sp. strain CALS-1, in which the CO partial pressure was decreased by flushing with N2-CO2, accumulated CO to 0.16 Pa, whereas cultures to which ca. 0.5 Pa of CO was added consumed CO until it reached this partial pressure. Cyanide (1 mM) blocked CO consumption but not production. High partial pressures of H2 (40 kPa) inhibited methanogenesis from acetate by M. thermophila but not by Methanothrix sp. strain CALS-1, and 2 kPa of CO was not inhibitory to M. thermophila but was inhibitory to Methanothrix sp. strain CALS-1. Levels of CO dehydrogenase, hydrogenase, and formate dehydrogenase in Methanothrix sp. strain CALS-1 were 9.1, 0.045, and 5.8 μmol of viologen reduced min-1 mg of protein-1. These results suggest that CO plays a role in Methanothrix sp. strain CALS-1 similar to that of H2 in M. thermophila and are consistent with the conclusion that CO is an intermediate in a catabolic or anabolic pathway in Methanothrix sp. strain CALS-1; however, they could also be explained by passive equilibration of CO with a metabolic intermediate.
PMCID: PMC183098  PMID: 16348788
22.  Synthesis of acetyl coenzyme A by carbon monoxide dehydrogenase complex from acetate-grown Methanosarcina thermophila. 
Journal of Bacteriology  1990;172(12):7145-7150.
The carbon monoxide dehydrogenase (CODH) complex from Methanosarcina thermophila catalyzed the synthesis of acetyl coenzyme A (acetyl-CoA) from CH3I, CO, and coenzyme A (CoA) at a rate of 65 nmol/min/mg at 55 degrees C. The reaction ended after 5 min with the synthesis of 52 nmol of acetyl-CoA per nmol of CODH complex. The optimum temperature for acetyl-CoA synthesis in the assay was between 55 and 60 degrees C; the rate of synthesis at 55 degrees C was not significantly different between pHs 5.5 and 8.0. The rate of acetyl-CoA synthesis was independent of CoA concentrations between 20 microM and 1 mM; however, activity was inhibited 50% with 5 mM CoA. Methylcobalamin did not substitute for CH3I in acetyl-CoA synthesis; no acetyl-CoA or propionyl coenzyme A was detected when sodium acetate or CH3CH2I replaced CH3I in the assay mixture. CO could be replaced with CO2 and titanium(III) citrate. When CO2 and 14CO were present in the assay, the specific activity of the acetyl-CoA synthesized was 87% of the specific activity of 14CO, indicating that CO was preferentially incorporated into acetyl-CoA without prior oxidation to free CO2. Greater than 100 microM potassium cyanide was required to significantly inhibit acetyl-CoA synthesis, and 500 microM was required for 50% inhibition; in contrast, oxidation of CO by the CODH complex was inhibited 50% by approximately 10 microM potassium cyanide.
PMCID: PMC210839  PMID: 2123865
23.  Engineering Acetyl Coenzyme A Supply: Functional Expression of a Bacterial Pyruvate Dehydrogenase Complex in the Cytosol of Saccharomyces cerevisiae 
mBio  2014;5(5):e01696-14.
ABSTRACT
The energetic (ATP) cost of biochemical pathways critically determines the maximum yield of metabolites of vital or commercial relevance. Cytosolic acetyl coenzyme A (acetyl-CoA) is a key precursor for biosynthesis in eukaryotes and for many industrially relevant product pathways that have been introduced into Saccharomyces cerevisiae, such as isoprenoids or lipids. In this yeast, synthesis of cytosolic acetyl-CoA via acetyl-CoA synthetase (ACS) involves hydrolysis of ATP to AMP and pyrophosphate. Here, we demonstrate that expression and assembly in the yeast cytosol of an ATP-independent pyruvate dehydrogenase complex (PDH) from Enterococcus faecalis can fully replace the ACS-dependent pathway for cytosolic acetyl-CoA synthesis. In vivo activity of E. faecalis PDH required simultaneous expression of E. faecalis genes encoding its E1α, E1β, E2, and E3 subunits, as well as genes involved in lipoylation of E2, and addition of lipoate to growth media. A strain lacking ACS that expressed these E. faecalis genes grew at near-wild-type rates on glucose synthetic medium supplemented with lipoate, under aerobic and anaerobic conditions. A physiological comparison of the engineered strain and an isogenic Acs+ reference strain showed small differences in biomass yields and metabolic fluxes. Cellular fractionation and gel filtration studies revealed that the E. faecalis PDH subunits were assembled in the yeast cytosol, with a subunit ratio and enzyme activity similar to values reported for PDH purified from E. faecalis. This study indicates that cytosolic expression and assembly of PDH in eukaryotic industrial microorganisms is a promising option for minimizing the energy costs of precursor supply in acetyl-CoA-dependent product pathways.
IMPORTANCE
Genetically engineered microorganisms are intensively investigated and applied for production of biofuels and chemicals from renewable sugars. To make such processes economically and environmentally sustainable, the energy (ATP) costs for product formation from sugar must be minimized. Here, we focus on an important ATP-requiring process in baker’s yeast (Saccharomyces cerevisiae): synthesis of cytosolic acetyl coenzyme A, a key precursor for many industrially important products, ranging from biofuels to fragrances. We demonstrate that pyruvate dehydrogenase from the bacterium Enterococcus faecalis, a huge enzyme complex with a size similar to that of a ribosome, can be functionally expressed and assembled in the cytosol of baker’s yeast. Moreover, we show that this ATP-independent mechanism for cytosolic acetyl-CoA synthesis can entirely replace the ATP-costly native yeast pathway. This work provides metabolic engineers with a new option to optimize the performance of baker’s yeast as a “cell factory” for sustainable production of fuels and chemicals.
doi:10.1128/mBio.01696-14
PMCID: PMC4212835  PMID: 25336454
24.  Low Levels of Pyrophosphate in Transgenic Potato Plants Expressing E. coli Pyrophosphatase Lead to Decreased Vitality Under Oxygen Deficiency 
Annals of Botany  2005;96(4):717-726.
• Background and Aims The aim of this study was to investigate the importance of pyrophosphate (PPi) for plant metabolism and survival under low oxygen stress. Responses of roots of wild-type potato plants were compared with roots of transgenic plants containing decreased amounts of PPi as a result of the constitutive expression of Escherichia coli pyrophosphatase in the cytosol.
• Methods For the experiments, roots of young wild-type and transgenic potato plants growing in nutrient solution were flushed for 4 d with nitrogen, and subsequently metabolite contents as well as enzyme activities of the glycolytic pathway were determined.
• Key Results and Conclusions In roots of transgenic plants containing 40 % less PPi, UDPglucose accumulated while the concentrations of hexose-6-phosphate, other glycolytic intermediates and ATP were decreased, leading to a growth retardation in aerated conditions. Apart from metabolic alterations, the activity of sucrose synthase was increased to a lower extent in the transgenic line than in wild type during hypoxia. These data suggest that sucrose cleavage was inhibited due to PPi deficiency already under aerated conditions, which has severe consequences for plant vitality under low oxygen. This is indicated by a reduction in the glycolytic activity, lower ATP levels and an impaired ability to resume growth after 4 d of hypoxia. Interestingly, the phosphorylation of fructose-6-phosphate via PPi-dependent phosphofructokinase was not altered in roots of transgenic plants. Nevertheless, our data provide some evidence for the importance of PPi to maintain plant growth and metabolism under oxygen deprivation.
doi:10.1093/aob/mci223
PMCID: PMC4247038  PMID: 16027130
Fermentation; glycolysis; hypoxia; pyrophosphate; Solanum tuberosum; sucrose synthase
25.  Utilization of PPi as an Energy Source by a Clostridium sp 
The growth of an anaerobic, spore-forming rod we have isolated from the cockroach gut after enrichment on media containing PPi was stimulated by the presence of PPi. The doubling time decreased and cell yield increased proportionately to PPi concentrations of up to 0.35%. A similar stimulation of the growth of Desulfotomaculum sp. by PPi has been reported. The PPi-stimulated Clostridium sp. fermented a number of sugars with the production of hydrogen, acetate, and butyrate, with smaller amounts of ethanol and butanol being produced from some substrates. The fermentation products were not qualitatively changed by the presence of PPi, but significantly more hydrogen was produced. The organism contained several of the enzymes previously reported from Entamoeba sp. and Propionibacterium sp., in which PPi serves as a source of a high-energy bond in place of ATP. These include significant amounts of pyruvate-phosphate dikinase and phosphoenolpyruvate carboxytransphosphorylase. The activities of many of the catabolic enzymes of the organism, as well as of its phosphatases and pyrophosphatase, were similar whether it was grown in the presence or absence of PPi. The organism did not accumulate intracellular polyphosphate granules but stored large amounts of glycogen.
Images
PMCID: PMC239582  PMID: 16346447

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