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.     

Properties of R-citramalyl-coenzyme A lyase and its role in the autotrophic 3-hydroxypropionate cycle of Chloroflexus aurantiacus

The autotrophic CO(2) fixation pathway (3-hydroxypropionate cycle) in Chloroflexus aurantiacus results in the fixation of two molecules of bicarbonate into one molecule of glyoxylate. Glyoxylate conversion to the CO(2) acceptor molecule acetyl-coenzyme A (CoA) requires condensation with propionyl-CoA (derived from one molecule of acetyl-CoA and one molecule of CO(2)) to beta-methylmalyl-CoA, which is converted to citramalyl-CoA. Extracts of autotrophically grown cells contained both S- and R-citramalyl-CoA lyase activities, which formed acetyl-CoA and pyruvate. Pyruvate is taken out of the cycle and used for cellular carbon biosynthesis. Both the S- and R-citramalyl-CoA lyases were up-regulated severalfold during autotrophic growth. S-Citramalyl-CoA lyase activity was found to be due to l-malyl-CoA lyase/beta-methylmalyl-CoA lyase. This promiscuous enzyme is involved in the CO(2) fixation pathway, forms acetyl-CoA and glyoxylate from l-malyl-CoA, and condenses glyoxylate with propionyl-CoA to beta-methylmalyl-CoA. R-Citramalyl-CoA lyase was further studied. Its putative gene was expressed and the recombinant protein was purified. This new enzyme belongs to the 3-hydroxy-3-methylglutaryl-CoA lyase family and is a homodimer with 34-kDa subunits that was 10-fold stimulated by adding Mg(2) or Mn(2+) ions and dithioerythritol. The up-regulation under autotrophic conditions suggests that the enzyme functions in the ultimate step of the acetyl-CoA regeneration route in C. aurantiacus. Genes similar to those involved in CO(2) fixation in C. aurantiacus, including an R-citramalyl-CoA lyase gene, were found in Roseiflexus sp., suggesting the operation of the 3-hydroxypropionate cycle in this bacterium. Incomplete sets of genes were found in aerobic phototrophic bacteria and in the gamma-proteobacterium Congregibacter litoralis. This may indicate that part of the reactions may be involved in a different metabolic process.     

13C-NMR study of autotrophic CO2 fixation pathways in the sulfur-reducing Archaebacterium Thermoproteus neutrophilus and in the phototrophic Eubacterium Chloroflexus aurantiacus.

The unresolved autotrophic CO2 fixation pathways in the sulfur-reducing Archaebacterium Thermoproteus neutrophilus and in the phototrophic Eubacterium Chloroflexus aurantiacus have been investigated. Autotrophically growing cultures were labelled with [1,4-13C1]succinate, and the 13C pattern in cell constituents was determined by 1H- and 13C-NMR spectroscopy of purified amino acids and other cell constituents. In both organisms succinate contributed to less than 10% of cell carbon, the major part of carbon originated from CO2. All cell constituents became 13C-labelled, but different patterns were observed in the two organisms. This proves that two different cyclic CO2 fixation pathways are operating in autotrophic carbon assimilation in both of which succinate is an intermediate. The 13C-labelling pattern in T. neutrophilus is consistent with the operation of a reductive citric acid cycle and rules out any other known autotrophic CO2 fixation pathway. Surprisingly, the proffered [1,4-13C1]succinate was partially converted to double-labelled [3,4-13C2]glutamate, but not to double-labelled aspartate. These findings suggest that the conversion of citrate to 2-oxoglutarate is readily reversible under the growth conditions used, and a reversible citrate cleavage reaction is proposed. The 13C-labelling pattern in C. aurantiacus disagrees with any of the established CO2 fixation pathways; it therefore demands a novel autotrophic CO2 fixation cycle in which 3-hydroxypropionate and succinate are likely intermediates. The bacterium excreted substantial amounts of 3-hydroxypropionate (5 mM) and succinate (0.5 mM) at the end of autotrophic growth. Autotrophically grown Chloroflexus cells contained acetyl-CoA carboxylase and propionyl-CoA carboxylase activity. These enzymes are proposed to be the main CO2-fixing enzymes resulting in malonyl-CoA and methylmalonyl-CoA formation; from these carboxylation products 3-hydroxypropionate and succinate, respectively, can be formed.     

Propionyl-coenzyme A synthase from Chloroflexus aurantiacus, a key enzyme of the 3-hydroxypropionate cycle for autotrophic CO2 fixation

The 3-hydroxypropionate cycle has been proposed as a new autotrophic CO(2) fixation pathway for the phototrophic green non-sulfur eubacterium Chloroflexus aurantiacus and for some chemotrophic archaebacteria. The cycle requires the reductive conversion of the characteristic intermediate 3-hydroxypropionate to propionyl-CoA. The specific activity of the 3-hydroxypropionate-, CoA-, K(+)-, and MgATP-dependent oxidation of NADPH in autotrophically grown cells was 0.09 micromol min(-1) mg(-1) protein, which was 2-fold down-regulated in heterotrophically grown cells. Unexpectedly, a single enzyme catalyzes the entire reaction sequence: 3-hydroxypropionate + MgATP + CoA + NADPH + H(+) --> propionyl-CoA + MgAMP + PP(i) + NADP(+) + H(2)O. The enzyme was purified 30-fold to near homogeneity and has a very large native molecular mass between 500 and 800 kDa, with subunits of about 185 kDa as judged by SDS-PAGE, suggesting a homotrimeric or homotetrameric structure. Upon incubation of this new enzyme, termed propionyl-CoA synthase, with the proteinase trypsin, the NADPH oxidation function of the enzyme was lost, whereas the enzyme still activated 3-hydroxypropionate to its CoA-thioester and dehydrated it to acrylyl-CoA. SDS-PAGE revealed that the subunits of propionyl-CoA synthase had been cleaved once and the N-terminal amino acid sequences of the two trypsin digestion products were determined. Two parts of the gene encoding propionyl-CoA synthase (pcs) were identified on two contigs of an incomplete genome data base of C. aurantiacus, and the sequence of the pcs gene was completed. Propionyl-CoA synthase is a natural fusion protein of 201 kDa consisting of a CoA ligase, an enoyl-CoA hydratase, and an enoyl-CoA reductase, the reductase domain containing the trypsin cleavage site. Similar polyfunctional large enzymes are common in secondary metabolism (e.g. polyketide synthases) but rare in primary metabolism (e.g. eukaryotic type I fatty acid synthase). These results lend strong support to the operation of the proposed pathway in autotrophic CO(2) fixation.     

L-Malyl-coenzyme A lyase/beta-methylmalyl-coenzyme A lyase from Chloroflexus aurantiacus,a bifunctional enzyme involved in autotrophic CO(2) fixation

The 3-hydroxypropionate cycle is a bicyclic autotrophic CO(2) fixation pathway in the phototrophic Chloroflexus aurantiacus (Bacteria), and a similar pathway is operating in autotrophic members of the Sulfolobaceae (Archaea). The proposed pathway involves in a first cycle the conversion of acetyl-coenzyme A (acetyl-CoA) and two bicarbonates to L-malyl-CoA via 3-hydroxypropionate and propionyl-CoA; L-malyl-CoA is cleaved by L-malyl-CoA lyase into acetyl-CoA and glyoxylate. In a second cycle, glyoxylate and another molecule of propionyl-CoA (derived from acetyl-CoA and bicarbonate) are condensed by a putative beta-methylmalyl-CoA lyase to beta-methylmalyl-CoA, which is converted to acetyl-CoA and pyruvate. The putative L-malyl-CoA lyase gene of C. aurantiacus was cloned and expressed in Escherichia coli, and the recombinant enzyme was purified and studied. Beta-methylmalyl-CoA lyase was purified from cell extracts of C. aurantiacus and characterized. We show that these two enzymes are identical and that both enzymatic reactions are catalyzed by one single bifunctional enzyme, L-malyl-CoA lyase/beta-methylmalyl-CoA lyase. Interestingly, this enzyme works with two different substrates in two different directions: in the first cycle of CO(2) fixation, it cleaves L-malyl-CoA into acetyl-CoA and glyoxylate (lyase reaction), and in the second cycle it condenses glyoxylate with propionyl-CoA to beta-methylmalyl-CoA (condensation reaction). The combination of forward and reverse directions of a reversible enzymatic reaction, using two different substrates, is rather uncommon and reduces the number of enzymes required in the pathway. In summary, L-malyl-CoA lyase/beta-methylmalyl-CoA lyase catalyzes the interconversion of L-malyl-CoA plus propionyl-CoA to beta-methylmalyl-CoA plus acetyl-CoA.     

Malonyl-coenzyme A reductase in the modified 3-hydroxypropionate cycle for autotrophic carbon fixation in archaeal Metallosphaera and Sulfolobus spp

Autotrophic members of the Sulfolobales (Crenarchaeota) contain acetyl-coenzyme A (CoA)/propionyl-CoA carboxylase as the CO2 fixation enzyme and use a modified 3-hydroxypropionate cycle to assimilate CO2 into cell material. In this central metabolic pathway malonyl-CoA, the product of acetyl-CoA carboxylation, is further reduced to 3-hydroxypropionate. Extracts of Metallosphaera sedula contained NADPH-specific malonyl-CoA reductase activity that was 10-fold up-regulated under autotrophic growth conditions. Malonyl-CoA reductase was partially purified and studied. Based on N-terminal amino acid sequencing the corresponding gene was identified in the genome of the closely related crenarchaeum Sulfolobus tokodaii. The Sulfolobus gene was cloned and heterologously expressed in Escherichia coli, and the recombinant protein was purified and studied. The enzyme catalyzes the following reaction: malonyl-CoA + NADPH + H+ --> malonate-semialdehyde + CoA + NADP+. In its native state it is associated with small RNA. Its activity was stimulated by Mg2+ and thiols and inactivated by thiol-blocking agents, suggesting the existence of a cysteine adduct in the course of the catalytic cycle. The enzyme was specific for NADPH (Km = 25 microM) and malonyl-CoA (Km = 40 microM). Malonyl-CoA reductase has 38% amino acid sequence identity to aspartate-semialdehyde dehydrogenase, suggesting a common ancestor for both proteins. It does not exhibit any significant similarity with malonyl-CoA reductase from Chloroflexus aurantiacus. This shows that the autotrophic pathway in Chloroflexus and Sulfolobaceae has evolved convergently and that these taxonomic groups have recruited different genes to bring about similar metabolic processes.     

Malonyl-coenzyme A reductase from Chloroflexus aurantiacus, a key enzyme of the 3-hydroxypropionate cycle for autotrophic CO(2) fixation

The 3-hydroxypropionate cycle is a new autotrophic CO(2) fixation pathway in Chloroflexus aurantiacus and some archaebacteria. The initial step is acetyl-coenzyme A (CoA) carboxylation to malonyl-CoA by acetyl-CoA carboxylase, followed by NADPH-dependent reduction of malonyl-CoA to 3-hydroxypropionate. This reduction step was studied in Chloroflexus aurantiacus. A new enzyme was purified, malonyl-CoA reductase, which catalyzed the two-step reduction malonyl-CoA + NADPH + H(+) --> malonate semialdehyde + NADP(+) + CoA and malonate semialdehyde + NADPH + H(+) --> 3-hydroxypropionate + NADP(+). The bifunctional enzyme (aldehyde dehydrogenase and alcohol dehydrogenase) had a native molecular mass of 300 kDa and consisted of a single large subunit of 145 kDa, suggesting an alpha(2) composition. The N-terminal amino acid sequence was determined, and the incomplete gene was identified in the genome database. Obviously, the enzyme consists of an N-terminal short-chain alcohol dehydrogenase domain and a C-terminal aldehyde dehydrogenase domain. No indication of the presence of a prosthetic group was obtained; Mg(2+) and Fe(2+) stimulated and EDTA inhibited activity. The enzyme was highly specific for its substrates, with apparent K(m) values of 30 microM malonyl-CoA and 25 microM NADPH and a turnover number of 25 s(-1) subunit(-1). The specific activity in autotrophically grown cells was 0.08 micromol of malonyl-CoA reduced min(-1) (mg of protein)(-1), compared to 0.03 micromol min(-1) (mg of protein)(-1) in heterotrophically grown cells, indicating downregulation under heterotrophic conditions. Malonyl-CoA reductase is not required in any other known pathway and therefore can be taken as a characteristic enzyme of the 3-hydroxypropionate cycle. Furthermore, the enzyme may be useful for production of 3-hydroxypropionate and for a coupled spectrophotometric assay for activity screening of acetyl-CoA carboxylase, a target enzyme of potent herbicides.     

Autotrophic CO2 Fixation by Chloroflexus aurantiacus: Study of Glyoxylate Formation and Assimilation via the 3-Hydroxypropionate Cycle

In the facultative autotrophic organism Chloroflexus aurantiacus, a phototrophic green nonsulfur bacterium, the Calvin cycle does not appear to be operative in autotrophic carbon assimilation. An alternative cyclic pathway, the 3-hydroxypropionate cycle, has been proposed. In this pathway, acetyl coenzyme A (acetyl-CoA) is assumed to be converted to malate, and two CO(2) molecules are thereby fixed. Malyl-CoA is supposed to be cleaved to acetyl-CoA, the starting molecule, and glyoxylate, the carbon fixation product. Malyl-CoA cleavage is shown here to be catalyzed by malyl-CoA lyase; this enzyme activity is induced severalfold in autotrophically grown cells. Malate is converted to malyl-CoA via an inducible CoA transferase with succinyl-CoA as a CoA donor. Some enzyme activities involved in the conversion of malonyl-CoA via 3-hydroxypropionate to propionyl-CoA are also induced under autotrophic growth conditions. So far, no clue as to the first step in glyoxylate assimilation has been obtained. One possibility for the assimilation of glyoxylate involves the conversion of glyoxylate to glycine and the subsequent assimilation of glycine. However, such a pathway does not occur, as shown by labeling of whole cells with [1,2-(13)C(2)]glycine. Glycine carbon was incorporated only into glycine, serine, and compounds that contained C(1) units derived therefrom and not into other cell compounds.     

FTIR spectroscopy of the reaction center of Chloroflexus aurantiacus: photoreduction of the bacteriopheophytin electron acceptor

Mid-infrared spectral changes associated with the photoreduction of the bacteriopheophytin electron acceptor H(A) in reaction centers (RCs) of the filamentous anoxygenic phototrophic bacterium Chloroflexus (Cfl.) aurantiacus are examined by light-induced Fourier transform infrared (FTIR) spectroscopy. The light-induced H(A)(-)/H(A) FTIR (1800-1200cm(-1)) difference spectrum of Cfl. aurantiacus RCs is compared to that of the previously well characterized purple bacterium Rhodobacter (Rba.) sphaeroides RCs. The most notable feature is that the large negative IR band at 1674cm(-1) in Rba. sphaeroides R-26, attributable to the loss of the absorption of the 13(1)-keto carbonyl of H(A) upon the radical anion H(A)(-) formation, exhibits only a very minor upshift to 1675cm(-1) in Cfl. aurantiacus. In contrast, the absorption band of the 131-keto C=O of H(A)(-) is strongly upshifted in the spectrum of Cfl. aurantiacus compared to that of Rba. sphaeroides (from 1588 to 1623cm(-1)). The data are discussed in terms of: (i) replacing the glutamic acid at L104 in Rba. sphaeroides R-26 RCs by a weaker hydrogen bond donor, a glutamine, at the equivalent position L143 in Cfl. aurantiacus RCs; (ii) a strengthening of the hydrogen-bonding interaction of the 131-keto C=O of H(A) with Glu L104 and Gln L143 upon H(A)(-) formation and (iii) a possible influence of the protein dielectric environment on the 131-keto C=O stretching frequency of neutral H(A). A conformational heterogeneity of the 133-ester C=O group of H(A) is detected for Cfl. aurantiacus RCs similar to what has been previously described for purple bacterial RCs.     

Properties of succinyl-coenzyme A:L-malate coenzyme A transferase and its role in the autotrophic 3-hydroxypropionate cycle of Chloroflexus aurantiacus

The 3-hydroxypropionate cycle has been proposed to operate as the autotrophic CO2 fixation pathway in the phototrophic bacterium Chloroflexus aurantiacus. In this pathway, acetyl coenzyme A (acetyl-CoA) and two bicarbonate molecules are converted to malate. Acetyl-CoA is regenerated from malyl-CoA by L-malyl-CoA lyase. The enzyme forming malyl-CoA, succinyl-CoA:L-malate coenzyme A transferase, was purified. Based on the N-terminal amino acid sequence of its two subunits, the corresponding genes were identified on a gene cluster which also contains the gene for L-malyl-CoA lyase, the subsequent enzyme in the pathway. Both enzymes were severalfold up-regulated under autotrophic conditions, which is in line with their proposed function in CO2 fixation. The two CoA transferase genes were cloned and heterologously expressed in Escherichia coli, and the recombinant enzyme was purified and studied. Succinyl-CoA:L-malate CoA transferase forms a large (alphabeta)n complex consisting of 46- and 44-kDa subunits and catalyzes the reversible reaction succinyl-CoA + L-malate --> succinate + L-malyl-CoA. It is specific for succinyl-CoA as the CoA donor but accepts L-citramalate instead of L-malate as the CoA acceptor; the corresponding d-stereoisomers are not accepted. The enzyme is a member of the class III of the CoA transferase family. The demonstration of the missing CoA transferase closes the last gap in the proposed 3-hydroxypropionate cycle.     

Carbon metabolism of filamentous anoxygenic phototrophic bacteria of the family Oscillochloridaceae

The carbon metabolism of representatives of the family Oscillochloridaceae (Oscillochloris trichoides DG6 and the recent isolates Oscillochloris sp. R, KR, and BM) has been studied. Based on data from an inhibitory analysis of autotrophic CO2 assimilation and measurements of the activities of the enzymes involved in this process, it is concluded that, in all Oscillochloris strains, CO2 fixation occurs via the operation of the Calvin cycle. Phosphoenolpyruvate (PEP), which is formed in this cycle, can be involved in the metabolism via the following reaction sequence: PEP (+ CO2) --> oxalacetate --> malate --> fumarate --> succinate --> succinyl-CoA (+ CO2) --> 2-oxoglutarate (+ CO2) --> isocitrate. Acetate, utilized as and additional carbon source, can be carboxylated to pyruvate by pyruvate synthase and further involved in the metabolism via the above reaction sequence. Propionyl-CoA synthase and malonyl-CoA reductase, the key enzymes of the 3-hydroxypropionate cycle, have not been detected in Oscillochloris representatives.     

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.     

Citric acid cycle in the hyperthermophilic archaeon Pyrobaculum islandicum grown autotrophically, heterotrophically, and mixotrophically with acetate

The hyperthermophilic archaeon Pyrobaculum islandicum uses the citric acid cycle in the oxidative and reductive directions for heterotrophic and autotrophic growth, respectively, but the control of carbon flow is poorly understood. P. islandicum was grown at 95 degrees C autotrophically, heterotrophically, and mixotrophically with acetate, H2, and small amounts of yeast extract and with thiosulfate as the terminal electron acceptor. The autotrophic growth rates and maximum concentrations of cells were significantly lower than those in other media. The growth rates on H2 and 0.001% yeast extract with and without 0.05% acetate were the same, but the maximum concentration of cells was fourfold higher with acetate. There was no growth with acetate if 0.001% yeast extract was not present, and addition of H2 to acetate-containing medium greatly increased the growth rates and maximum concentrations of cells. P. islandicum cultures assimilated 14C-labeled acetate in the presence of H2 and yeast extract with an efficiency of 55%. The activities of 11 of 19 enzymes involved in the central metabolism of P. islandicum were regulated under the three different growth conditions. Pyruvate synthase and acetate:coenzyme A (CoA) ligase (ADP-forming) activities were detected only in heterotrophically grown cultures. Citrate synthase activity decreased in autotrophic and acetate-containing cultures compared to the activity in heterotrophic cultures. Acetylated citrate lyase, acetate:CoA ligase (AMP forming), and phosphoenolpyruvate carboxylase activities increased in autotrophic and acetate-containing cultures. Citrate lyase activity was higher than ATP citrate synthase activity in autotrophic cultures. These data suggest that citrate lyase and AMP-forming acetate:CoA ligase, but not ATP citrate synthase, work opposite citrate synthase to control the direction of carbon flow in the citric acid cycle.     

Autotrophic CO<Subscript>2</Subscript> fixation pathways in archaea (Crenarchaeota)

Representative autotrophic and thermophilic archaeal species of different families of Crenarchaeota were examined for key enzymes of the known autotrophic CO(2) fixation pathways. Pyrobaculum islandicum ( Thermoproteaceae) contained key enzymes of the reductive citric acid cycle. This finding is consistent with the operation of this pathway in the related Thermoproteus neutrophilus. Pyrodictium abyssi and Pyrodictium occultum ( Pyrodictiaceae) contained ribulose 1,5-bisphosphate carboxylase, which was active in boiling water. Yet, phosphoribulokinase activity was not detectable. Operation of the Calvin cycle remains to be demonstrated. Ignicoccus islandicus and Ignicoccus pacificus ( Desulfurococcaceae) contained pyruvate oxidoreductase as potential carboxylating enzyme, but apparently lacked key enzymes of known pathways; their mode of autotrophic CO(2) fixation is at issue. Metallosphaera sedula, Acidianus ambivalens and Sulfolobus sp. strain VE6 ( Sulfolobaceae) contained key enzymes of a 3-hydroxypropionate cycle. This finding is in line with the demonstration of acetyl-coenzyme A (CoA) and propionyl-CoA carboxylase activities in the related Acidianus brierleyi and Sulfolobus metallicus. Enzymes of central carbon metabolism in Metallosphaera sedula were studied in more detail. Enzyme activities of the 3-hydroxypropionate cycle were strongly up-regulated during autotrophic growth, supporting their role in CO(2) fixation. However, formation of acetyl-CoA from succinyl-CoA could not be demonstrated, suggesting a modified pathway of acetyl-CoA regeneration. We conclude that Crenarchaeota exhibit a mosaic of three or possibly four autotrophic pathways. The distribution of the pathways so far correlates with the 16S-rRNA-based taxa of the Crenarchaeota.     

The enzyme of carbon metabolism in the thermotolerant sulfobacillus strain K1

To determine enzymatic activities in the thermotolerant strain K1 (formerly "Sulfobacillus thermosulfidooxidans subsp. thermotolerans"), it was grown in a mineral medium with (1) thiosulfate and Fe2+ or pyrite (autotrophic conditions), (2) Fe2+, thiosulfate, and yeast extract or glucose (mixotrophic conditions), and (3) yeast extract (heterotrophic conditions). Cells grown mixo-, hetero-, and autotrophically were found to contain enzymes of the tricarboxylic acid (TCA) cycle, as well as malate synthase, an enzyme of the glyoxylate cycle. Cells grown organotrophically in a medium with yeast extract exhibited the activity of the key enzymes of the Embden-Meyerhof-Parnas and Entner-Doudoroff pathways. An increased content of carbon dioxide (up to 5 vol%) in the auto- and mixotrophic media enhanced the activity of the enzymes involved in the terminal reactions of the TCA cycle and the enzymes of the pentose phosphate pathway. Carbon dioxide was fixed in the Calvin cycle. The highest activity of ribulose bisphosphate carboxylase was detected in cells grown autotrophically at the atmospheric content of CO2 in the air used for aeration of the growth medium. The activities of pyruvate carboxylase, phosphoenolpyruvate carboxylase, phosphoenolpyruvate carboxykinase, and phosphoenolpyruvate carboxytransphosphorylase decreased with the increasing content of CO2 in the medium.     

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     

Autotrophic acetyl coenzyme A biosynthesis in Methanococcus maripaludis.

To detect autotrophic CO2 assimilation in cell extracts of Methanococcus maripaludis, lactate dehydrogenase and NADH were added to convert pyruvate formed from autotrophically synthesized acetyl coenzyme A to lactate. The lactate produced was determined spectrophotometrically. When CO2 fixation was pulled in the direction of lactate synthesis, CO2 reduction to methane was inhibited. Bromoethanesulfonate (BES), a potent inhibitor of methanogenesis, enhanced lactate synthesis, and methyl coenzyme M inhibited it in the absence of BES. Lactate synthesis was dependent on CO2 and H2, but H2 + CO2-independent synthesis was also observed. In cell extracts, the rate of lactate synthesis was about 1.2 nmol min-1 mg of protein-1. When BES was added, the rate of lactate synthesis increased to 2.3 nmol min-1 mg of protein-1. Because acetyl coenzyme A did not stimulate lactate synthesis, pyruvate synthase may have been the limiting activity in these assays. Radiolabel from 14CO2 was incorporated into lactate. The percentages of radiolabel in the C-1, C-2, and C-3 positions of lactate were 73, 33, and 11%, respectively. Both carbon monoxide and formaldehyde stimulated lactate synthesis. 14CH2O was specifically incorporated into the C-3 of lactate, and 14CO was incorporated into the C-1 and C-2 positions. Low concentrations of cyanide also inhibited autotrophic growth, CO dehydrogenase activity, and autotrophic lactate synthesis. These observations are in agreement with the acetogenic pathway of autotrophic CO2 assimilation.     

Characterization of macrophage cell line A640-BB-2: A640-BB-2 resembles peritoneal exudate macrophages in cell morphology,tumor cell recognition,responsiveness to immunomodulator OK-432 and lysosomal enzyme activity

Characteristics of mouse macrophage (MP) cell lines A640-BB-2, J774.1 and P388D1 and mouse peritoneal exudate MPs were studied and compared in cell morphology, ability to recognize tumor cells in the presence and absence of OK-432 known to activate MPs, and in lysosomal enzyme activity. In A640-BB-2 cells and exudate MPs, cell surfaces showed a few ridge-like processes and microvilli; spontaneous cytotoxicity was moderate against tumor target L929, and little or absent against targets SV3T3, B-16 and U937; and lysosomal enzyme activity of nonspecific esterase, acid phosphatase, and -glucuronidase was high. After culture in the presence of OK-432, A640-BB-2 cells and exudate MPs showed more extensive spreading with larger surface areas and with increased numbers of ridge-like processes and microvilli, and their cytotoxicity against target L929 became more extensive. The stable soluble factor did not participate in the mechanism of cytotoxicity against target L929 mediated by A640-BB-2 cells and exudate MPs. J774.1 and P388D1 cells were different from exudate MPs in cell morphology and ability to recognize tumor cells when cultured either with or without OK-432, and in lysosomal enzyme activity. A640-BB-2 cells seem to be useful in studying MP-tumor cell interaction and MP activation, and in detecting the trace biological activating factor of MPs.Abbreviations DEM Dulbecco's modified Eagle's medium - MP macrophage - PBS phosphate-buffered saline - SEM scanning electron microscopy     

Properties of succinyl-coenzyme A:D-citramalate coenzyme A transferase and its role in the autotrophic 3-hydroxypropionate cycle of Chloroflexus aurantiacus

The phototrophic bacterium Chloroflexus aurantiacus uses the 3-hydroxypropionate cycle for autotrophic CO(2) fixation. This cycle starts with acetyl-coenzyme A (CoA) and produces glyoxylate. Glyoxylate is an unconventional cell carbon precursor that needs special enzymes for assimilation. Glyoxylate is combined with propionyl-CoA to beta-methylmalyl-CoA, which is converted to citramalate. Cell extracts catalyzed the succinyl-CoA-dependent conversion of citramalate to acetyl-CoA and pyruvate, the central cell carbon precursor. This reaction is due to the combined action of enzymes that were upregulated during autotrophic growth, a coenzyme A transferase with the use of succinyl-CoA as the CoA donor and a lyase cleaving citramalyl-CoA to acetyl-CoA and pyruvate. Genomic analysis identified a gene coding for a putative coenzyme A transferase. The gene was heterologously expressed in Escherichia coli and shown to code for succinyl-CoA:d-citramalate coenzyme A transferase. This enzyme, which catalyzes the reaction d-citramalate + succinyl-CoA --> d-citramalyl-CoA + succinate, was purified and studied. It belongs to class III of the coenzyme A transferase enzyme family, with an aspartate residue in the active site. The homodimeric enzyme composed of 44-kDa subunits was specific for succinyl-CoA as a CoA donor but also accepted d-malate and itaconate instead of d-citramalate. The CoA transferase gene is part of a cluster of genes which are cotranscribed, including the gene for d-citramalyl-CoA lyase. It is proposed that the CoA transferase and the lyase catalyze the last two steps in the glyoxylate assimilation route.     

Mesaconyl-coenzyme A hydratase, a new enzyme of two central carbon metabolic pathways in bacteria

The coenzyme A (CoA)-activated C5-dicarboxylic acids mesaconyl-CoA and beta-methylmalyl-CoA play roles in two as yet not completely resolved central carbon metabolic pathways in bacteria. First, these compounds are intermediates in the 3-hydroxypropionate cycle for autotrophic CO2 fixation in Chloroflexus aurantiacus, a phototrophic green nonsulfur bacterium. Second, mesaconyl-CoA and beta-methylmalyl-CoA are intermediates in the ethylmalonyl-CoA pathway for acetate assimilation in various bacteria, e.g., in Rhodobacter sphaeroides, Methylobacterium extorquens, and Streptomyces species. In both cases, mesaconyl-CoA hydratase was postulated to catalyze the interconversion of mesaconyl-CoA and beta-methylmalyl-CoA. The putative genes coding for this enzyme in C. aurantiacus and R. sphaeroides were cloned and heterologously expressed in Escherichia coli, and the proteins were purified and studied. The recombinant homodimeric 80-kDa proteins catalyzed the reversible dehydration of erythro-beta-methylmalyl-CoA to mesaconyl-CoA with rates of 1,300 micromol min(-1) mg protein(-1). Genes coding for similar enzymes with two (R)-enoyl-CoA hydratase domains are present in the genomes of Roseiflexus, Methylobacterium, Hyphomonas, Rhodospirillum, Xanthobacter, Caulobacter, Magnetospirillum, Jannaschia, Sagittula, Parvibaculum, Stappia, Oceanicola, Loktanella, Silicibacter, Roseobacter, Roseovarius, Dinoroseobacter, Sulfitobacter, Paracoccus, and Ralstonia species. A similar yet distinct class of enzymes containing only one hydratase domain was found in various other bacteria, such as Streptomyces species. The role of this widely distributed new enzyme is discussed.