The acetyl-CoA pathway of autotrophic growth

Abstract The most direct conceivable route for synthesis of multicarbon compounds from CO₂ is to join two molecules of CO₂ together to make a 2-carbon compound and then polymerize the 2-carbon compound or add CO₂ successively to the 2-carbon compound to make multicarbon compounds. Recently, it has been demonstrated that the bacterium, Clostridium thermoaceticum , grows autotrophically by such a process. The mechanism involves the reduction of one molecule of CO₂ to a methyl group and then its combination with a second molecule of CO₂ and CoA to form acetyl-CoA. We have designated this autotrophic pathway the acetyl-CoA pathway [1]. Evidence is accumulating that this pathway is utilized by other bacteria that grow with CO₂ and H₂ as the source of carbon and energy. This group includes bacteria which, like C. thermoaceticum , produce acetate as a major end product and are called acetogens or acetogenic bacteria. It also includes the methane-producing bacteria and sulfate-reducing bacteria.
The purpose of this review is to examine critically the evidence that the acetyl-CoA pathway occurs in other bacteria by a mechanism that is the same or similar to that found in C. thermoaceticum . For this purpose, the mechanism of the acetyl-CoA pathway, as found in C. thermoaceticum , is described and hypothetical mechanisms for other organisms are presented based on the acetyl-CoA pathway of C. thermoaceticum . The available data have been reviewed to determine if the hypothetical schemes are in accord with presently known facts. We conclude that the formation of acetyl-CoA by other acetogens, the methanogens and sulphate-reducing bacteria occurs by a mechanism very similar to that of C. thermoaceticum .     

A pathway of the autotrophic CO2 fixation in Chloroflexus aurantiacus

Autotrophically grown cells of Chloroflexus aurantiacus B-3 were shown to possess activity of ATP-dependent malate lyase (acetylating CoA). ATP: malate lyase is supposed to be the specific enzyme of the cycle of the autotrophic CO₂ fixation, in which pyruvate synthase, pyruvate phosphate dikinase, phosphoenolpyruvate (PEP) carboxylase and malate dehydrogenase are involved as well. The main product of the CO₂ fixation cycle is glyoxylate, which could further be converted into 3-phosphoglyceric acid (3-PGA) in the reactions of either glycerate or serine pathway. The enzymes of both pathways were detected in C. auratiacus B-3. The results of the in vivo studies of glyxoylate and glycine metabolism, as well as the inhibitor analysis using fluoroacetate (FAc), isonicotinic acid hydrazide (INH), and 4-aminopterin (4-AP) confirm the operation of the proposed pathway in Chloroflexus.Abbreviations 3-PGA 3-phosphoglyceric acid - 4-AP 4-aminopterin - FAc fluoroacetate - INH isonicotinic acid hydrazide - MV methyl viologen - PEP phosphoenolpyruvate - THF tetrahydrofolate - TPP thiamine pyrophosphate     

Enzymes and coenzymes of the carbon monoxide dehydrogenase pathway for autotrophic CO2 fixation in Archaeoglobus lithotrophicus and the lack of carbon monoxide dehydrogenase in the heterotrophic A. profundus

Archaeoglobus lithotrophicus is a hyperthermophilic Archaeon that grows on H₂ and sulfate as energy sources and CO₂ as sole carbon source. The autotrophic sulfate reducer was shown to contain all the enzyme activities and coenzymes of the reductive carbon monoxide dehydrogenase pathway for autotrophic CO₂ fixation as operative in methanogenic Archaea. With the exception of carbon monoxide dehydrogenase these enzymes and coenzymes were also found in A. profundus. This organism grows lithotrophically on H₂ and sulfate, but differs from A. lithotrophicus in that it cannot grow autotrophically: A. profundus requires acetate and CO₂ for biosynthesis. The absence of carbon monoxide dehydrogenase in A. profundus is substantiated by the observation that this organism, in contrast to A. lithotrophicus, is not mini-methanogenic and contains only relatively low concentrations of corrinoids.Abbreviations F ₄₂₀ coenzyme F₄₂₀ - MFR methanofuran - CHO-MFR formylmethanofuran - H ₄MPT 5,6,7,8-tetrahydromethanopterin - CHO–H ₄MPT N⁵ formyl-H₄MPT - CHH₄MPT⁺N⁵ methenyl-H₄MPT - CH ₂=H₄MPT N⁵, N¹⁰ methylene-H₄MPT - CH ₃–H₄MPT N⁵ methyl-H₄MPT - H ₄F tetrahydrofolate - I U 1 mol/min - t _d doubling time     

A bicyclic autotrophic CO2 fixation pathway in Chloroflexus aurantiacus

Phototrophic CO(2) assimilation by the primitive, green eubacterium Chloroflexus aurantiacus has been shown earlier to proceed in a cyclic mode via 3-hydroxypropionate, propionyl-CoA, succinyl-CoA, and malyl-CoA. The metabolic cycle could be closed by cleavage of malyl-CoA affording glyoxylate (the primary CO(2) fixation product) with regeneration of acetyl-CoA serving as the starter unit of the cycle. The pathway of glyoxylate assimilation to form gluconeogenic precursors has not been elucidated to date. We could now show that the incubation of cell extract with a mixture of glyoxylate and [1,2,3-(13)C(3)]propionyl-CoA afforded erythro-beta-[1,2,2'-(13)C(3)]methylmalate and [1,2,2'-(13)C(3)]citramalate. Similar experiments using a partially purified protein fraction afforded erythro-beta-[1,2,2'-(13)C(3)]methylmalyl-CoA and [1,2,2'-(13)C(3)]mesaconyl-CoA. Cell extracts of C. aurantiacus were also shown to catalyze the conversion of citramalate into pyruvate and acetyl-CoA in a succinyl-CoA-dependent reaction. The data suggest that glyoxylate obtained by the cleavage of malyl-CoA can be utilized by condensation with propionyl-CoA affording erythro-beta-methylmalyl-CoA, which is converted to acetyl-CoA and pyruvate. This reaction sequence regenerates acetyl-CoA, which serves as the precursor of propionyl-CoA in the 3-hydroxypropionate cycle. Autotrophic CO(2) fixation proceeds by combination of the 3-hydroxypropionate cycle with the methylmalyl-CoA cycle. The net product of that bicyclic autotrophic CO(2) fixation pathway is pyruvate serving as an universal building block for anabolic reactions.     

Enzymology of the Acetyl-CoA Pathway of CO2 Fixation

Abstract

We know of three routes that organisms have evolved to synthesize complex organic molecules from CO₂: the Calvin cycle. the reverse tricarboxylic acid cycle, and the reductive acetyl-CoA pathway. This review describes the enzymatic steps involved in the acetyl-CoA pathway, also called the Wood pathway, which is the major mechanism of CO₂ fixation under anaerobic conditions. The acetyl-CoA pathway is also able to form acetyl-CoA from carbon monoxide.

There are two parts to the acetyl-CoA pathway: (1) reduction of CO₂ to methyltetrahydrofolate (methyl-H₄folate) and (2) synthesis of acetyl-CoA from methyl-H, folate, a carboxyl donor such as CO or CO₂, and CoA. This pathway is unique in that the major intermediates are enzyme-bound and are often organometallic complexes. Our current understanding of the pathway is based on radioactive and stable isotope tracer studies, purification of the component enzymes (some extremely oxygen sensitive), and identification of the enzyme-bound intcrmediates by chromatographic, spectroscopic. and electrochemical techniques. This review describes the remarkable series of enzymatic steps involved in acetyl-CoA formation by this pathway that is a key component of the global carbon cycle.     

Identification of ADP-ribosylation site in human glutamate dehydrogenase isozymes

When the influence of ADP-ribosylation on the activities of the purified human glutamate dehydrogenase isozymes (hGDH1 and hGDH2) was measured in the presence of 100 microM NAD+ for 60 min, hGDH isozymes were inhibited by up to 75%. If incubations were performed for longer time periods up to 3 h, the inhibition of hGDH isozymes did not increased further. This phenomenon may be related to the reversibility of ADP-ribosylation in mitochondria. ADP-ribosylated hDGH isozymes were reactivated by Mg2+-dependent mitochondrial ADP-ribosylcysteine hydrolase. The stoichiometry between incorporated ADP-ribose and GDH subunits shows a modification of one subunit per catalytically active homohexamer. Since ADP and GTP had no effects on the extent of modification, it would appear that the ADP-ribosylation is unlikely to occur in allosteric sites. It has been proposed that Cys residue may be involved in the ADP-ribosylation of GDH, although identification of the reactive Cys residue has not been reported. To identify the reactive Cys residue involved in the ADP-ribosylation, we performed cassette mutagenesis at three different positions (Cys59, Cys119, and Cys274) using synthetic genes of hGDH isozymes. Among the Cys residues tested, only Cys119 mutants showed a significant reduction in the ADP-ribosylation. These results suggest a possibility that the Cys119 residue has an important role in the regulation of hGDH isozymes by ADP-ribosylation.     

Identification of a novel gene, aut, involved in autotrophic growth of Alcaligenes eutrophus.

The aerobic facultative chemoautotroph Alcaligenes eutrophus was found to possess a novel gene, designated aut, required for both lithoautotrophic (hydrogen plus carbon dioxide) and organoautotrophic (formate) growth (Aut+ phenotype). Insertional mutagenesis by transposon Tn5-Mob localized the gene on a chromosomal 13-kbp EcoRI fragment. Physiological characterization of various Aut- mutants revealed pleiotropic effects caused by the transposon insertion. Heterotrophic growth of the mutants on substrates catabolized via the glycolytic pathway was slower than that of the parent strains, and the colony morphology of the mutants was altered when grown on nutrient agar. The heterotrophic derepression of the cbb operons encoding Calvin cycle enzymes was abolished, although their expression was still inducible in the presence of formate. Apparently, the mutation did not affect the cbb genes directly but impaired the autotrophic growth in a more general manner. The conjugally transferred wild-type EcoRI fragment allowed phenotypic in trans complementation of the mutants. Further subcloning and sequencing identified a single open reading frame (aut) of 495 bp that was sufficient for complementation. The monocistronic aut gene was constitutively transcribed into a 0.65-kb mRNA. However, its expression appeared to be low. Heterologous expression of aut was achieved in Escherichia coli, resulting in overproduction of an 18-kDa protein. Database searches yielded weak partial sequence similarities of the deduced Aut protein sequence to some cytidylyltransferases, but no indication for the exact function of the aut gene was obtained. Hybridizing DNA sequences that might be similar to the aut gene were detected by Southern hybridization in the genome of two other autotrophic bacteria.     

Acetyl-CoA drives the transcriptional growth program in yeast

Comment on: Cai L, et al. Mol Cell 2011; 42:426-37.     

The role of pyruvate ferredoxin oxidoreductase in pyruvate synthesis during autotrophic growth by the Wood-Ljungdahl pathway

Pyruvate:ferredoxin oxidoreductase (PFOR) catalyzes the oxidative decarboxylation of pyruvate to acetyl-CoA and CO(2). The catalytic proficiency of this enzyme for the reverse reaction, pyruvate synthase, is poorly understood. Conversion of acetyl-CoA to pyruvate links the Wood-Ljungdahl pathway of autotrophic CO(2) fixation to the reductive tricarboxylic acid cycle, which in these autotrophic anaerobes is the stage for biosynthesis of all cellular macromolecules. The results described here demonstrate that the Clostridium thermoaceticum PFOR is a highly efficient pyruvate synthase. The Michaelis-Menten parameters for pyruvate synthesis by PFOR are: V(max) = 1.6 unit/mg (k(cat) = 3.2 s(-1)), K(m)(Acetyl-CoA) = 9 micrometer, and K(m)(CO(2)) = 2 mm. The intracellular concentrations of acetyl-CoA, CoASH, and pyruvate have been measured. The predicted rate of pyruvate synthesis at physiological concentrations of substrates clearly is sufficient to support the role of PFOR as a pyruvate synthase in vivo. Measurements of its k(cat)/K(m) values demonstrate that ferredoxin is a highly efficient electron carrier in both the oxidative and reductive reactions. On the other hand, rubredoxin is a poor substitute in the oxidative direction and is inept in donating electrons for pyruvate synthesis.     

The autotrophic growth of Micrococcus denitrificans on Methanol.

Ribulose bisphosphate carboxylase is present at a high specific activity in extracts of methanol-grown Microccus denitrificans. Enzymic and physiological evidence indicates that, during growth on methanol, the ribulose bisphosphate cycle is the route of carbon assimilation.     

Insights into the autotrophic CO2 fixation pathway of the archaeon Ignicoccus hospitalis: comprehensive analysis of the central carbon metabolism

Ignicoccus hospitalis is an autotrophic hyperthermophilic archaeon that serves as a host for another parasitic/symbiotic archaeon, Nanoarchaeum equitans. In this study, the biosynthetic pathways of I. hospitalis were investigated by in vitro enzymatic analyses, in vivo (13)C-labeling experiments, and genomic analyses. Our results suggest the operation of a so far unknown pathway of autotrophic CO(2) fixation that starts from acetyl-coenzyme A (CoA). The cyclic regeneration of acetyl-CoA, the primary CO(2) acceptor molecule, has not been clarified yet. In essence, acetyl-CoA is converted into pyruvate via reductive carboxylation by pyruvate-ferredoxin oxidoreductase. Pyruvate-water dikinase converts pyruvate into phosphoenolpyruvate (PEP), which is carboxylated to oxaloacetate by PEP carboxylase. An incomplete citric acid cycle is operating: citrate is synthesized from oxaloacetate and acetyl-CoA by a (re)-specific citrate synthase, whereas a 2-oxoglutarate-oxidizing enzyme is lacking. Further investigations revealed that several special biosynthetic pathways that have recently been described for various archaea are operating. Isoleucine is synthesized via the uncommon citramalate pathway and lysine via the alpha-aminoadipate pathway. Gluconeogenesis is achieved via a reverse Embden-Meyerhof pathway using a novel type of fructose 1,6-bisphosphate aldolase. Pentosephosphates are formed from hexosephosphates via the suggested ribulose-monophosphate pathway, whereby formaldehyde is released from C-1 of hexose. The organism may not contain any sugar-metabolizing pathway. This comprehensive analysis of the central carbon metabolism of I. hospitalis revealed further evidence for the unexpected and unexplored diversity of metabolic pathways within the (hyperthermophilic) archaea.     

Identification and dynamics of a heparin-binding site in hepatocyte growth factor.

Hepatocyte growth factor (HGF) is a heparin-binding, multipotent growth factor that transduces a wide range of biological signals, including mitogenesis, motogenesis, and morphogenesis. Heparin or closely related heparan sulfate has profound effects on HGF signaling. A heparin-binding site in the N-terminal (N) domain of HGF was proposed on the basis of the clustering of surface positive charges [Zhou, H., Mazzulla, M. J., Kaufman, J. D., Stahl, S. J., Wingfield, P. T., Rubin, J. S., Bottaro, D. P., and Byrd, R. A. (1998) Structure 6, 109-116]. In the present study, we confirmed this binding site in a heparin titration experiment monitored by nuclear magnetic resonance spectroscopy, and we estimated the apparent dissociation constant (K(d)) of the heparin-protein complex by NMR and fluorescence techniques. The primary heparin-binding site is composed of Lys60, Lys62, and Arg73, with additional contributions from the adjacent Arg76, Lys78, and N-terminal basic residues. The K(d) of binding is in the micromolar range. A heparin disaccharide analogue, sucrose octasulfate, binds with similar affinity to the N domain and to a naturally occurring HGF isoform, NK1, at nearly the same region as in heparin binding. (15)N relaxation data indicate structural flexibility on a microsecond-to-millisecond time scale around the primary binding site in the N domain. This flexibility appears to be dramatically reduced by ligand binding. On the basis of the NK1 crystal structure, we propose a model in which heparin binds to the two primary binding sites and the N-terminal regions of the N domains and stabilizes an NK1 dimer.     

On the mechanism of autotrophic fixation of CO2 bychloroflexus aurantiacus

The activity of two carboxylating enzymes was studied in the green filamentous bacteriumChloroflexus aurantiacus. The carboxylation reaction involving pyruvate synthase was optimized using¹⁴CO₂ and cell extracts. Pyruvate synthase was shown to be absent from cells ofCfl. aurantiacus OK-70 and present (in a quantity sufficient to account for autotrophic growth) in cells ofCfl. aurantiacus B-3. Differences in the levels of acetyl CoA carboxylase activity were revealed between cells of the strains studied grown under different conditions. The data obtained confirm the operation of different mechanisms of autotrophic CO₂ assimilation inCfl. aurantiacus B-3 andCfl. aurantiacus OK-70: in the former organism, it is the reductive cycle of dicarboxylic acids, and in the latter one, it is the 3-hydroxypropionate cycle.     

Identification of the cysteine residue in the active site of horse liver mitochondrial aldehyde dehydrogenase

Aldehyde dehydrogenase catalyzes the oxidation of aldehydes to acids through the formation of a covalent intermediate. It has been postulated that a cysteine residue could be acting as the active site nucleophilic group. Although N-ethylmaleimide was found to react with many cysteines it was possible by doing the reaction in the presence of chloral hydrate, a substrate analog which functions as a competitive inhibitor, to label cysteine at position 49 in the horse liver mitochondrial enzyme. The dehydrogenase activity was lost as the residue was modified, consistent with the possibility that the residue was an integral component of the active site of the enzyme. Cysteines at positions 162 and 369 also could be modified. It is suggested that cysteine 162 may function as part of a site capable of hydrolyzing nitrophenyl acetate. Details of the second site will appear in the accompanying paper (Tu, G. C., and Weiner, H. (1988) J. Biol. Chem. 263, 1218-1222). It appeared that the substrate-binding domain was in the N-terminal portion of the enzyme while the coenzyme binding domain was in the C-terminal portion. During this investigation 133 of the 500 residues of the horse liver enzyme were sequenced. These showed about 95% sequence identity with those of the human enzyme. Inasmuch as both beef and rat liver enzymes also share 95% identity with the human enzyme it can be expected that the results found with the horse liver enzyme can be applicable to all mammalian aldehyde dehydrogenase.     

Ecological aspects of the distribution of different autotrophic CO2 fixation pathways

Autotrophic CO(2) fixation represents the most important biosynthetic process in biology. Besides the well-known Calvin-Benson cycle, five other totally different autotrophic mechanisms are known today. This minireview discusses the factors determining their distribution. As will be made clear, the observed diversity reflects the variety of the organisms and the ecological niches existing in nature.     

Lactate dehydrogenase from autotrophic and heterotrophic cells of the marine diatom Cylindrotheca fusiformis Reimann & Lewin.

Cultures of Cylindrotheca furisormis grown either autotrohpically or heterotrophically on lactate contained significant amounts of NAD-dependent L(+)-lactate dehydrogenase (EC 1.1.1.27). Polyacylamide gel electrophoresis of crude enzyme extracts revealed a single band which was indistinguishable between autotrohpic and heterotrohpic cells. The Km for lactate of partially purified preparations was lower under heterotrophic conditions. The specific activity in crude extracts was higher under autotrophic than heterotrophic conditions; it dropped precipitously when autotrophic cells were transferred to the dark, increasing again only in the presence of lactate. These and related observations suggest that this enzyme has at most only a minor role in the assimilation of lactate during heterotrophic growth on lactate.     

Identification by mutagenesis of arginines in the substrate binding site of the porcine NADP-dependent isocitrate dehydrogenase

Pig heart mitochondrial NADP-dependent isocitrate dehydrogenase is the most extensively studied among the mammalian isocitrate dehydrogenases. The crystal structure of Escherichia coli isocitrate dehydrogenase and sequence alignment of porcine with E. coli isocitrate dehydrogenase suggests that the porcine Arg(101), Arg(110), Arg(120), and Arg(133) are candidates for roles in substrate binding. The four arginines were separately mutated to glutamine using a polymerase chain reaction method. Wild type and mutant enzymes were each expressed in E. coli, isolated as maltose binding fusion proteins, then cleaved with thrombin, and purified to yield homogeneous porcine isocitrate dehydrogenase. The R120Q mutant has a specific activity, as well as K(m) values for isocitrate, Mn(2+), and NADP(+) similar to wild type enzyme, indicating that Arg(120) is not needed for function. The specific activities of R101Q, R110Q, and R133Q are 1.73, 1.30, and 19.7 micromols/min/mg, respectively, as compared with 39.6 units/mg for wild type enzyme. The R110Q and R133Q enzymes exhibit K(m) values for isocitrate that are increased more than 400- and 165-fold, respectively, as compared with wild type. The K(m) values for Mn(2+), but not for NADP(+), are also elevated indicating that binding of the metal-isocitrate complex is impaired in these mutants. It is proposed that the positive charges of Arg(110) and Arg(133) normally strengthen the binding of the negatively charged isocitrate by electrostatic attraction. The R101Q mutant shows smaller, but significant increases in the K(m) values for isocitrate and Mn(2+); however, the marked decrease in k(cat) suggests a role for Arg(101) in catalysis. The V(max) of wild type enzyme depends on the ionized form of an enzymic group of pK 5.5, and this pK(aes) is similar for the R101Q and R120Q enzymes. In contrast, the pK(aes) for R110Q and R133Q enzymes increases to 6.4 and 7.4, respectively, indicating that the positive charges of Arg(110) and Arg(133) normally lower the pK of the nearby catalytic base to facilitate its ionization. These results may be understood in terms of the structure of the porcine NADP-specific isocitrate dehydrogenase generated by the Insight II Modeler Program, based on the x-ray coordinates of the E. coli enzyme.