INTRODUCTION

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Chloroflexus aurantiacus is a phototrophic green nonsulfur bacterium that grows facultatively autotrophically. Energy can be obtained by anoxic photosynthesis, respiration with oxygen, or fermentation (24, 31, 32). The classic Calvin-Benson-Bassham cycle does not appear to operate in autotrophic carbon fixation, although early work reported the presence of ribulose 1,5-bisphosphate carboxylase (37). The key enzymes of this cycle were not detectable by other groups, and the ¹³C-¹²C isotope discrimination was outside the range of values reported for Calvin cycle organisms (14), indicating the possibility of a different pathway. Indeed, evidence has been accumulated for an alternative carbon fixation cycle, which has been termed the 3-hydroxypropionate cycle (12, 14, 15, 16, 39, 40). This proposed pathway also seems to operate in the branch of archaebacteria comprising autotrophic Acidianus, Sulfolobus, and Metallosphaera species (4, 18, 27, 30).

The proposed 3-hydroxypropionate cycle (40) is shown in Fig. 1. Each turn of the cycle results in the net fixation of two molecules of bicarbonate into one molecule of glyoxylate. Acetyl coenzyme A (acetyl-CoA) is carboxylated to malonyl-CoA by conventional ATP-dependent biotin-containing acetyl-CoA carboxylase. The unprecedented reduction of malonyl-CoA to propionyl-CoA requires three NADPH molecules and one MgATP molecule and proceeds via free 3-hydroxypropionate as an intermediate. 3-Hydroxypropionate and succinate are even excreted into the medium by autotrophically grown cultures when factors other than the availability of an electron donor (hydrogen gas) or a carbon source (carbon dioxide) become limiting (15, 16, 39). Propionyl-CoA is carboxylated to methylmalonyl-CoA, followed by the isomerization of methylmalonyl-CoA to succinyl-CoA; these reactions are conventional and are used in many organisms for propionate assimilation. Interestingly, succinyl-CoA appears to be used for malate activation by CoA transfer, forming succinate and malyl-CoA; succinate in turn is oxidized to malate by conventional enzymes. Malyl-CoA is cleaved to acetyl-CoA and glyoxylate. In conclusion, acetyl-CoA is considered the starting molecule of the cycle and is converted to malate. Two CO₂ fixation steps, acetyl-CoA carboxylation and propionyl-CoA carboxylation, are involved in this reaction sequence.

View larger version (28K): [in this window] [in a new window]   FIG. 1.   Proposed 3-hydroxypropionate cycle of autotrophic CO2 fixation in the phototrophic green nonsulfur bacterium C. aurantiacus (12, 39). Enzyme activities: (1) acetyl-CoA carboxylase; (2) malonyl-CoA reductase (NADPH); (3) 3-hydroxypropionate dehydrogenase (NADP+); (4) 3-hydroxypropionyl-CoA synthetase; (5) 3-hydroxypropionyl-CoA dehydratase; (6) acryloyl-CoA reductase (NADPH); (7) propionyl-CoA carboxylase; (8) methylmalonyl-CoA epimerase; (9) methylmalonyl-CoA mutase; (10) succinyl-CoA:L-malate CoA transferase; (11) succinate dehydrogenase; (12) fumarate hydratase; (13) L-malyl-CoA lyase. Note that different enzyme activities do not necessarily mean different enzymes.

A different pathway has been proposed in which acetyl-CoA is reductively carboxylated to pyruvate by ferredoxin-dependent pyruvate synthase (19, 21). Pyruvate then can be converted to phosphoenolpyruvate (PEP) and oxaloacetate via pyruvate phosphate dikinase and PEP carboxylase. Oxaloacetate then can be reduced to malate and malate can be converted to acetyl-CoA and glyoxylate, as described for the 3-hydroxypropionate cycle. Based on this proposal, the labeling pattern of cellular building blocks can be predicted, e.g., when cells are fed with ¹³C-labeled acetate or succinate. However, the results of labeling studies were not consistent with this proposal (12, 39). Furthermore, the postulated CO₂-fixing enzyme pyruvate synthase could not be demonstrated unequivocally (42). In addition, the roles of malonyl-CoA reduction to propionyl-CoA, propionyl-CoA carboxylation, and the excretion of 3-hydroxypropionate could not be explained. According to this hypothetical pathway, malate should be formed solely from oxaloacetate.

Contrary to this proposal, in the 3-hydroxypropionate cycle PEP carboxylation is believed to function as an anaplerotic reaction filling up C₄ compounds which are withdrawn from the cycle for the biosynthesis of building blocks such as amino acids of the aspartate family. Hence, malate can be formed either from succinyl-CoA or from oxaloacetate.

There are three novel metabolic processes in the proposed 3-hydroxypropionate cycle that need to be clarified. The first one concerns the reformation of acetyl-CoA. Previous work (40) could not provide final evidence as to how malate was converted to the starting molecule acetyl-CoA. Succinyl-CoA- and malate-dependent formation of acetyl-CoA and glyoxylate was shown in cell extracts at relatively low rates. It was assumed that malyl-CoA was an intermediate; however, malyl-CoA was not available to demonstrate such a cleavage reaction.

The second unsolved problem concerns the fate of glyoxylate. So far, no mechanism is known for Chloroflexus sp. by which glyoxylate is converted into a central biosynthetic intermediate, such as acetyl-CoA, pyruvate, or oxaloacetate. Some proposals were made, but experimental evidence was scarce (12, 19, 20, 21, 39, 40). These proposals included the following: (i) condensation of two molecules of glyoxylate to form tartronate semialdehyde and subsequent reduction to glyceratethis proposal suffers from the fact that CO₂ is released, not a useful trait of a CO₂ fixation mechanism; (ii) conversion of glycine to a one-carbon unit, again with the release of CO₂, and reassimilation of the one-carbon unit by condensation with glycine, forming serinethe same objection applies to this proposal; (iii) conversion of one molecule of glyoxylate to glycine and condensation of another molecule of glyoxylate with glycine, forming hydroxyaspartate, or any other condensation reaction of glycine; and (iv) glycine reduction to acetylphosphate.

The third issue is the reduction of malonyl-CoA to propionyl-CoA. This complex reaction sequence, which formally involves five steps (Fig. 1), appears to be brought about by only two enzymes, which have been purified and studied (M. Hügler, B. Alber, and G. Fuchs, unpublished results).

The present work addresses the first two problems. The first aim was to provide evidence for malyl-CoA cleavage to acetyl-CoA and glyoxylate by malyl-CoA lyase and for the activation of malate to malyl-CoA by CoA transfer with succinyl-CoA as a CoA donor. The formation of glyoxylate from malate via these two reactions was unequivocally demonstrated. This result prompted us to further study the second problem, the fate of glyoxylate. A possible route of assimilation would be conversion of glyoxylate to glycine and assimilation of glycine following this conversion. This possibility was tested by long-term labeling of autotrophically grown cells with [1,2-¹³C₂]glycine and determining label incorporation into cellular constituents.

	MATERIALS AND METHODS

Top Abstract Introduction Materials and Methods Results Discussion References

Bacteria and growth conditions. C. aurantiacus strain OK-70-fl (DSM636) was grown in 5- or 12-liter glass fermentors to an optical density of 3.5 to 4 at 55°C and a pH of ~8. Autotrophic growth under anaerobic conditions on a minimal medium supplemented with vitamins and trace elements and gassed with H₂-CO₂ (80:20 [vol/vol]) was described elsewhere (40). Cells were grown under photoheterotrophic anaerobic conditions on modified minimal medium D (5) supplemented with 0.25% Casamino Acids, 0.1% yeast extract, and trace elements. The medium was buffered with 0.05% glycylglycine-Na⁺ buffer.

Cell extracts and membrane fractions. Cell extracts were prepared anaerobically. Cells were suspended in an equal volume of 20 mM Tris-HCl buffer (pH 7.8) containing 1 mg of DNase I per 2 ml of cell suspension. Except for determination of the Mg²⁺ dependence of enzyme activity, the buffer also contained 2 mM MgCl₂. The cell suspension was passed through a French pressure cell at 137 MPa, followed by centrifugation (100,000 × g) at 4°C for 1 h. The supernatant (cell extract) was either used immediately or kept frozen at 70°C. The protein content of the extract was determined by the Bradford method (3) and ranged from 15 to 37 mg of protein ml¹. Membrane protein fractions were obtained aerobically from 1 g of cells resuspended in 2 ml of 20 mM Tris-HCl buffer (pH 7.8) containing 20% (wt/vol) glycerol. Cells were broken in a French pressure cell, the suspension was centrifuged at 20,000 × g (4°C, 1 h), and the supernatant was centrifuged at 100,000 × g (4°C, 1 h). The formed pellet was washed with 2 ml of 20 mM Tris HCl, pH 7.8, containing 20% (wt/vol) glycerol, resuspended in 2 ml of the same buffer containing 0.5% Triton X-100 (vol/vol), and centrifuged at 100,000 × g (4°C, 1 h). The supernatant obtained contained the solubilized membrane fraction (2.3 to 3.0 mg of protein ml¹).

Radiochemicals, isotopes, chemicals, and biochemicals. Radiochemicals were obtained from American Radiolabeled Chemicals Inc./Biotrend Chemikalien GmbH (Köln, Germany), Amersham (Braunschweig, Germany), Sigma-Aldrich (Deisenhofen, Germany), or NEN Life Science (Bad Homburg, Germany). L-[1,4(2,3)-¹⁴C]Malate (1.87 MBq µmol¹), [1,2-¹⁴C₂]oxalate (88.7 kBq µmol¹), [1,2-¹⁴C₂]acetate (1.98 MBq µmol¹), [¹⁴C]Na₂CO₃ (2.02 MBq µmol¹), and [1,2-¹⁴C₂]glycine (4.34 MBq µmol¹) were used. [1,2-¹³C₂]Glycine was obtained from Cambridge Isotope Laboratories (Andover, Mass.). All chemicals except those used for growing cells were analytical grade and were obtained from Fluka (Neu-Ulm, Germany), Sigma-Aldrich, or Roth (Karlsruhe, Germany). Biochemicals were obtained from Roche Diagnostics (Mannheim, Germany), Applichem (Darmstadt, Germany), or Gerbu (Craiberg, Germany).

Syntheses. Various possible intermediates of the 3-hydroxypropionate cycle were not commercially available, notably, ¹⁴C-labeled compounds.

(i) Malonyl-CoA. Monothiophenylmalonate was chemically synthesized as described previously (17, 33) and stored under nitrogen gas at 20°C. CoA (61 µmol) was incubated under anaerobic conditions at room temperature in 40 ml of 0.1 M NaHCO₃ solution (pH 7.5) with 122 µmol of monothiophenylmalonate (dissolved in 300 µl of dioxan and 700 µl of 0.1 M NaHCO₃). After 90 min, the pH was adjusted to pH 3 by the addition of 1 M HCl, and the solution was extracted twice with diethyl ether. The aqueous solution was lyophilized, and the powder was stored at 20°C.

(ii) L-Malyl-CoA L-Malyl-CoA was chemically synthesized as described previously (10), with a slight modification. The synthesis intermediate L-malylcaprylcysteamine (S-[-hydroxysuccinyl]-N-caprylcysteamine) was synthesized by Richard Krieger (Institut für Organische Chemie, Universität Freiburg, Freiburg, Germany) as described previously (10, 28). L-Malyl-CoA was stored as a freeze-dried powder at 20°C. It contained 72% CoA-ester and 28% CoA, as determined by high-pressure liquid chromatography (HPLC) separation and detection at 260 nm.

(iii) Succinyl-CoA, acetyl-CoA, and propionyl-CoA. The CoA-thioesters of succinate, acetate, and propionate were synthesized from their anhydrides by a slightly modified method described previously (36, 38).

(iv) 3-Hydroxypropionate. 3-Hydroxypropionate was obtained by heating an alkaline solution (60 ml; 16 g of NaOH) of 3-hydroxypropionitrile (0.13 mol) until ammonia was completely evaporated. The pH of the solution was adjusted to pH 2, and the free acid formed was extracted in a Kutscher-Steudel apparatus with diethyl ether. The product (9.5 ml) contained ~4.4 M 3-hydroxypropionate and was stored at 20°C.

(v) [1,2-¹⁴C₂]Acetyl-CoA. Radioactively labeled acetyl-CoA was synthesized enzymatically. The reaction mixture (1 ml) contained 0.3 mM CoA, 0.2 mM [1,2-¹⁴C₂]acetate (0.4 MBq), 1 mM ATP, 1 U of acetyl-CoA synthetase (Roche, Basel, Switzerland), and 1 mM MgCl₂ in 50 mM Tris-HCl buffer (pH 8.4). An ATP-regenerating system consisting of 1 mM PEP, 0.5 mM NADH, and the enzymes myokinase (1 U), pyruvate kinase (1.5 U), and L-lactate dehydrogenase (2.8 U) was included. The reaction was carried out at 30°C, and NADH oxidation was monitored spectrophotometrically at 365 nm. The addition of a fivefold amount of ethanol stopped the reaction, and the precipitated protein was removed by centrifugation. Ethanol was evaporated, and the radioactive acetyl-CoA was purified by using an ODS-AQ extraction minicolumn (500 mg; 3 ml; YMC, Schermbeck, Germany) and following the protocols of the supplier. [¹⁴C]Acetyl-CoA was eluted with 6 ml of 80% methanol, and samples containing 16.7 kBq of [¹⁴C]acetyl-CoA each were dried in a Speedvac concentrator and stored at 20°C.

(vi) [1,2-¹⁴C₂]Glyoxylate. [¹⁴C]Glyoxylate was synthesized from [1,2-¹⁴C₂]oxalate by a previously described method (29). It was purified by HPLC and stored at 20°C and pH 7.0.

Enzyme assays. Unless otherwise indicated, tests were carried out at 55°C under aerobic conditions with extracts of autotrophically grown cells; insensitivity of enzyme activity toward oxygen was checked in each case. For comparison, enzyme assays were also performed with extracts of heterotrophically grown cells.

(i) L-Malyl-CoA lyase. Enzyme activity was monitored spectrophotometrically at 324 nm as described previously (13), with some modifications (E₃₂₄ of glyoxylate-phenylhydrazone, 17,000 M¹ cm¹). The assay mixture (0.5 ml) contained 200 mM morpholinopropanesulfonic acid (MOPS)-K⁺ buffer (pH 7.7), 10 mM MgCl₂, 3.5 mM phenylhydrazinium chloride, 0.2 mM L-malyl-CoA, and 0.1 to 0.3 mg of protein. L-Malyl-CoA routinely started the reaction. When the apparent K_m was determined, the value for L-malyl-CoA varied between 0.5 and 0.005 mM. The stoichiometry of the reaction was investigated with 0.01, 0.02, and 0.05 mM L-malyl-CoA.

(ii) L-Malyl-CoA synthetase. Enzyme activity was measured using endogenous L-malyl-CoA lyase activity, which produces glyoxylate; glyoxylate-phenylhydrazone formation was monitored spectrophotometrically at 324 nm. The assay mixture (0.5 ml) contained 200 mM MOPS-K⁺ buffer (pH 7.7), 10 mM MgCl₂, 3.5 mM phenylhydrazinium chloride, 0.3 mM CoA, 3 mM ATP or GTP, 5 mM L-malate, and 0.1 to 1.7 mg of protein. L-Malate started the reaction. The specific activity of endogenous L-malyl-CoA lyase was 52 to 220 nmol min¹ mg of protein¹, depending on the extract.

(iii) Succinyl-CoA:L-malate CoA transferase. The succinyl-CoA- and L-malate-dependent formation of glyoxylate due to L-malyl-CoA cleavage by endogenous L-malyl-CoA lyase was monitored spectrophotometrically at 324 nm to determine enzyme activity (40). The assay mixture (0.5 ml) contained 200 mM MOPS-K⁺ buffer (pH 6.5), 10 mM MgCl₂, 3.5 mM phenylhydrazinium chloride, 1 mM succinyl-CoA, 5 mM L-malate, and 0.3 to 1.1 mg of protein. Either substrate could be used to start the reaction. The apparent K_ms were determined at saturating concentration of the second substrate using 0.1 to 1.3 mM succinyl-CoA and 0.1 to 5.0 mM L-malate. The pH optima of the reactions were determined with MOPS-K⁺ buffer at various pHs (6.0 to 8.9).

(iv) Apparent malate synthase reaction (reverse reaction of malyl-CoA lyase). The formation of free CoA was monitored spectrophotometrically at 412 nm with 5,5'-dithiobis(2-nitrobenzoate) (DTNB; Ellman's reagent) (E₄₁₂, 13,600 M¹ cm¹) (8). The assay mixture (0.5 ml) contained 100 mM potassium phosphate buffer (pH 7.0), 0.25 mM DTNB, 10 mM MgCl₂, 0.3 mM acetyl-CoA, 5 mM glyoxylate, and 0.1 to 0.3 mg of protein. The addition of either substrate started the reaction. The apparent K_ms were determined at saturating concentrations of the second substrate using 0.01 to 0.2 mM acetyl-CoA and 0.1 to 5 mM glyoxylate.

(v) Acetyl-CoA and L-malyl-CoA hydrolysis. The formation of free CoA was monitored spectrophotometrically at 412 nm with 5,5'-dithiobis(2-nitrobenzoate). The assay mixture (0.5 ml) contained 100 mM potassium phosphate buffer (pH 7.0), 0.25 mM DTNB, 10 mM MgCl₂, 0.2 mM L-malyl-CoA or 0.3 mM acetyl-CoA, and 0.1 to 0.3 mg of protein. Either acetyl-CoA or L-malyl-CoA started the reaction. Cell extract was omitted when chemical CoA-ester stability under these conditions was determined.

(vi) Glyoxylate reduction. The reduction of glyoxylate was monitored spectrophotometrically by measuring the oxidation of NADH or NADPH at 365 nm (E₃₆₅ of NADH, 3,390 M¹ cm¹; E₃₆₅ of NADPH, 3,490 M¹ cm¹) (8) and 45°C. The assay mixture (0.5 ml) contained 100 mM potassium phosphate buffer (pH 7.8), 10 mM KCl, 2 mM MgCl₂, 1 mM dithioerythritol (DTE), 0.3 mM thiamine diphosphate, 0.3 mM NADH or NADPH, 10 mM glyoxylate, and 0.5 to 1.0 mg of protein. Glyoxylate started the reaction. The pH optima of the reactions were determined with 100 mM potassium phosphate buffer at different pHs (6.0 to 7.8) and 100 mM Tris-HCl buffer (pH 8.5). The apparent K_ms were determined at saturating concentrations of the second substrate using 1 to 10 mM glyoxylate and 0.03 to 0.3 mM NADH or NADPH.

(vii) Acetyl-CoA and propionyl-CoA carboxylases. Acetyl-CoA and propionyl-CoA carboxylases were assayed as described previously (40), with slight modifications. The acetyl-CoA- or propionyl-CoA- and MgATP-dependent fixation of ¹⁴C from [¹⁴C]bicarbonate into acid-stable labeled products was monitored. The assay mixture (1 ml) contained 100 mM Tris-HCl buffer (pH 7.8), 5 mM MgCl₂, 4 mM ATP, 2 mM NADPH, and 0.4 mM acetyl-CoA or propionyl-CoA. In addition, 10 mM [¹⁴C]KHCO₃ (36.7 kBq; specific radioactivity, 2.5 Bq nmol¹), 5 mM DTE, and 3.0 to 4.0 mg of protein were added. Cell extract started the reaction.

(viii) PEP carboxylase. The PEP-dependent fixation of ¹⁴C from [¹⁴C]bicarbonate into acid-stable labeled products was monitored. The assay mixture (1 ml) contained 100 mM Tris-HCl (pH 7.8), 5 mM MgCl₂, 2 mM PEP, 2 mM NADH, 10 mM [¹⁴C]KHCO₃ (36.7 kBq; specific radioactivity, 2.5 Bq nmol¹), 5 mM DTE, and 2 mg of protein. Cell extract started the reaction.

(ix) Pyruvate carboxylase. The pyruvate- and MgATP-dependent fixation of ¹⁴C from [¹⁴C]bicarbonate into acid-stable labeled products was monitored. The assay mixture contained 100 mM Tris-HCl-buffer (pH 7.8), 5 mM MgCl₂, 2 mM pyruvate, 5 mM ATP, 2 mM NADH, 10 mM [¹⁴C]KHCO₃ (36.7 kBq; specific radioactivity, 2.5 Bq nmol¹), 5 mM DTE, and 2 mg of protein. Cell extract started the reaction. The indirect fixation of label via pyruvate water dikinase and PEP carboxylase could be distinguished from biotin-dependent pyruvate carboxylation by the addition of 1 nM avidin to the assay mixture, completely inhibiting pyruvate carboxylation.

(x) Pyruvate phosphate dikinase. The pyruvate-, MgATP-, and phosphate-dependent fixation of ¹⁴C from [¹⁴C]bicarbonate into acid-stable labeled products was monitored. PEP formed was carboxylated by endogenous PEP carboxylase to oxaloacetate. The assay mixture (1 ml) contained 100 mM Tris-HCl buffer (pH 7.8), 5 mM MgCl₂, 2 mM pyruvate, 5 mM ATP, 5 mM K₂HPO₄, 2 mM NADH, 10 mM [¹⁴C]KHCO₃ (36.7 kBq; specific radioactivity, 2.5 Bq nmol¹), 5 mM DTE, and 2 mg of protein. Cell extract started the reaction.

(xi) Malonyl-CoA reduction to 3-hydroxypropionate. The malonyl-CoA-dependent oxidation of NADPH was monitored spectrophotometrically at 365 nm. The assay mixture (0.5 ml) contained 100 mM Tris-HCl buffer (pH 7.8), 0.3 mM NADPH, 1 mM malonyl-CoA, and 0.5 to 1.0 mg of protein. The addition of malonyl-CoA started the reaction.

(xii) Reduction of 3-hydroxypropionate to propionyl-CoA. The 3-hydroxypropionate-, CoA-, and MgATP-dependent oxidation of NADPH was monitored spectrophotometrically at 365 nm. The assay mixture contained 100 mM Tris-HCl buffer (pH 7.8), 10 mM KCl, 5 mM MgCl₂, 3 mM ATP, 0.5 mM CoA, 0.3 mM NADPH, 1 mM 3-hydroxypropionate, and 0.2 to 1.0 mg of protein. The addition of 3-hydroxypropionate started the reaction.

(xiii) Succinate dehydrogenase. Succinate dehydrogenase activity was assayed anaerobically by spectrophotometrically measuring the succinate-dependent reduction of oxidized 2,6-dichlorophenolindophenol at 546 nm in membrane fractions (E₅₄₆, 13,800 M¹ cm¹; this value was extrapolated from the UV-visible spectrum of 2,6-dichlorophenolindophenol [E₆₀₀, 22,000 M¹ cm¹]) (8). The assay mixture (0.5 ml) contained 100 mM Tris-HCl buffer (pH 7.8), 2.5 mM 2,6-dichlorophenolindophenol, 1 mM phenazine methosulfate, 0.5 mM succinate, and 11 to 15 µg of protein (solubilized membrane fraction). Succinate started the reaction.

(xiv) Fumarate hydratase. Fumarate hydratase activity was monitored spectrophotometrically at 240 nm (E₂₄₀, 2,440 M¹ cm¹) (2). The assay mixture (0.5 ml) contained 100 mM Tris-HCl buffer (pH 7.8), 0.2 mM fumarate, and 0.2 to 0.5 mg of protein. The addition of fumarate started the reaction.

(xv) Malate dehydrogenase. Malate dehydrogenase activity was assayed spectrophotometrically by measuring the oxaloacetate-dependent oxidation of NADPH or NADH at 365 nm. The assay mixture (0.5 ml) contained 100 mM Tris-HCl buffer (pH 7.8), 0.3 mM NADH or NADPH, 1 mM oxaloacetate, and 0.1 to 0.2 mg of protein. Oxaloacetate started the reaction.

(xvi) Citrate synthase. Citrate synthase activity was monitored spectrophotometrically at 412 nm with DTNB as the CoA-detecting agent. The assay mixture (0.5 ml) contained 100 mM Tris-HCl buffer (pH 7.8), 0.25 mM DTNB, 0.2 mM acetyl-CoA, 2 mM oxaloacetate, and 0.3 mg of protein. Either acetyl-CoA or oxaloacetate started the reaction.

(xvii) Isocitrate dehydrogenase. Isocitrate dehydrogenase activity was monitored by measuring NADP⁺ reduction spectrophotometrically at 365 nm. The assay mixture (0.5 ml) contained 100 mM Tris-HCl buffer (pH 7.8), 5 mM MgCl₂, 0.125 mM FeCl₂, 1 mM NADP⁺, 5 mM mercaptoethanol, 10 mM mixture of D- and L-isocitrate, and 0.6 to 0.8 mg of protein. The addition of isocitrate started the reaction.

(xviii) Aconitase. Aconitase activity was determined by coupling the reaction to endogenous isocitrate dehydrogenase activity, which reduces NADP⁺. The assay mixture (0.5 ml) was identical to the isocitrate dehydrogenase assay mixture, except for 10 mM citrate instead of isocitrate and 1.0 mg of protein. Citrate started the reaction.

(xix) 2-Oxoglutarate and pyruvate dehydrogenases. 2-Oxoglutarate and pyruvate dehydrogenase activities were monitored spectrophotometrically by measuring NAD⁺ or NADP⁺ reduction at 365 nm. The assay mixture (0.5 ml) contained 100 mM Tris-HCl buffer (pH 7.8), 5 mM MgCl₂, 1 mM NAD⁺ or NADP⁺, 0.5 mM CoA, 10 mM mercaptoethanol, 0.5 mM thiamine diphosphate, 5 mM 2-oxoglutarate or pyruvate, and up to 2.9 mg of protein. The addition of 2-oxoglutarate or pyruvate started the reaction.

Formation of acetyl-CoA from L-malyl-CoA. The assay mixture (0.5 ml) contained 200 mM MOPS-K⁺ buffer (pH 7.7), 10 mM MgCl₂, 0.9 mM L-malyl-CoA, 0.3 mM CoA (as an impurity of L-malyl-CoA), and 0.3 mg of protein. The addition of cell extract started the reaction. Samples of 100 µl were taken after different periods of incubation, and adding 20 µl of 1 M HCl stopped the reaction. Protein was removed by centrifugation, and samples were analyzed by HPLC using system 2 (see below).

Formation of [¹⁴C]malyl-CoA from [1,4(2,3)-¹⁴C]malate and ATP or GTP. The assay mixture (0.5 ml) contained 1 mM L-[¹⁴C]malate (36.7 kBq), 10 mM MgCl₂, 0.3 mM CoA, 3 mM ATP or GTP, and 0.3 mg of protein. The addition of cell extract started the reaction. After incubation for 1, 10, 20, and 50 min, samples of 100 µl were taken; the reaction was stopped by adding 20 µl of 1 M HCl. Protein was removed by centrifugation, and samples were analyzed by HPLC using system 2 (see below).

Formation of [¹⁴C]malyl-CoA and [¹⁴C]glyoxylate from [1,4(2,3)-¹⁴C]malate and succinyl-CoA. The assay mixture (0.5 ml) contained 100 mM potassium phosphate buffer (pH 6.7), 10 mM KCl, 10 mM MgCl₂, 0.5 mM L-[¹⁴C]malate (36.7 kBq), 1 mM succinyl-CoA, and 0.5 mg of protein. The addition of cell extract started the reaction. After incubation at 45°C for various time periods, samples of 100 µl were taken; the reaction was stopped by adding 12 µl of concentrated HCl. Protein was removed by centrifugation, and samples were analyzed by HPLC using systems 1 and 2 (see below). A control reaction was carried out in which succinyl-CoA was omitted. For detection of [¹⁴C]glyoxylate formed from L-[¹⁴C]malate, a 50 µl-sample was retrieved after 20 min of incubation at 55°C; the reaction was stopped by adding 5 µl of concentrated HCl. Protein was removed by centrifugation, and 30 µl of 2,4-dinitrophenylhydrazine (0.1% [wt/vol] in 2 M HCl) was added to the supernatant. After incubation at 30°C for 30 min, the developed 2,4-dinitrophenylhydrazones were extracted twice in ethyl acetate and separated by thin-layer chromatography (TLC) on cellulose F₂₅₄ plates with solvent system 3 (see below). The supernatant was analyzed by HPLC using system 2 (see below) to identify [¹⁴C]malyl-CoA. The radioactive peak eluting at 11 min was collected and incubated at pH 12 and 70°C for 30 min to hydrolyze CoA-esters. The sample was acidified by adding solid NaHSO₄, and the free acids were extracted overnight by Kutscher-Steudel extraction with diethyl ether. TLC on cellulose plates with solvent system 1 (see below) was used to analyze part of the sample. The ¹⁴C-labeled product and the malate standard cochromatographed with an R_f of 0.32.

Formation of [¹⁴C]malyl-CoA from [1,2-¹⁴C₂]acetyl-CoA and glyoxylate. The assay mixture (0.5 ml) contained 100 mM potassium phosphate buffer (pH 7.0), 10 mM MgCl₂, 5 mM glyoxylate, 0.5 mM [¹⁴C]acetyl-CoA (16.7 kBq), and 0.8 mg of protein. The addition of cell extract started the reaction. Samples of 125 µl were taken at intervals, and the reaction was stopped by adding 12 µl of 1 M HCl. Protein was removed by centrifugation, and samples were analyzed by HPLC using system 2 (see below). In a control experiment, glyoxylate was omitted.

Conversion of [1,2-¹⁴C₂]glyoxylate to products. [¹⁴C]Glyoxylate (1.1 mM, 36.7 kBq) was incubated with and without 0.5 mM NADH or NADPH and cell extract (0.5 to 1.0 mg of protein). In addition, the assay mixture (0.2 ml) contained 100 mM potassium phosphate buffer (pH 7.5), 10 mM KCl, and 0.3 mM thiamine diphosphate. Cell extract started the reaction, and the mixture was then incubated at 45°C for 40 min. The addition of 12 µl of concentrated HCl stopped the reaction. Protein was removed by centrifugation, and the supernatant was analyzed by HPLC. When 0.19 mM [¹⁴C]glyoxylate (1.7 kBq) was incubated with 2 mM glycine or 2 mM acetyl-CoA, the assay mixture (0.1 ml) additionally contained 2 mM glyoxylate, 100 mM NH₄HCO₃ (pH 7.8), 5 mM MgCl₂, and 0.15 mg of protein. After 50 min of incubation, the addition of 0.5 ml of ethanol stopped the reaction. Protein was removed by centrifugation, and samples were analyzed by TLC on cellulose plates with solvent system 2 (see below).

Determination of glyoxylate carboligase and tartronate semialdehyde reductase activities. A slight modification of the method of Chang et al. (6) was used for the detection of ¹⁴CO₂ released from [1,2-¹⁴C₂]glyoxylate. The reaction mixture (0.5 ml) contained 100 mM potassium phosphate buffer (pH 7.5), 0.3 mM thiamine diphosphate, 10 mM KCl, 1 mM DTE, 2.8 mM [¹⁴C]glyoxylate (35.5 kBq), and 1 mg of protein. The reaction was started with cell extract, and the mixture was shaken for 10 to 50 min at 180 rpm and 45°C in closed vials coated with alkaline Whatman paper. The reaction was stopped by the addition of 20 µl of concentrated HCl, and the mixture was then shaken again for 15 min at 45°C. The radioactivity trapped by the Whatman paper was measured. An assay mixture without cell extract was used as a control.

HPLC. The following systems were used.

(i) System 1. For separation of free organic acids, a Polyspher OA HY column (300 by 6.5 mm; Merck, Darmstadt, Germany) was used with 5 mM H₂SO₄ as an eluent and a flow rate of 0.8 ml min¹. Retention times of organic acids detected at 210 nm were 3.7 min (oxalate), 5.8 min (glyoxylate), 6.5 min (glycerate), and 7.2 min (glycolate).

(ii) System 2. An RP-C₁₈ column (LiChrospher 100, end capped, 5 µm, 125 by 4 mm; Merck) was used for the separation of CoA-thioesters. A gradient of 1 to 8% acetonitrile in 50 mM potassium phosphate buffer (pH 6.7) and a flow rate of 1 ml min¹ over 30 min were used. CoA-esters were detected at 260 nm. Retention times were 2 min (malate), 10 min (L-malyl-CoA), 13 min (CoA and succinyl-CoA), and 19 min (acetyl-CoA). Simultaneous detection of standard compounds and ¹⁴C-labeled reaction products was possible by using two detectors (UV and radioactivity) in series.

TLC. Cellulose or cellulose F₂₅₄ plates (0.1 mm; Merck) were used for product separation. The solvent systems used were as follows: 1pentan-1-ol-formate-water (48.8 ml:48.8 ml:2.4 ml); 2butan-1-ol-formate-water (10:2:15 [vol/vol]); and 3butan-1-ol-ethanol-NH₃ (35%)-water (140:20:1:39 [vol/vol]). 2,4-Dinitrophenylhydrazones were separated on cellulose F₂₅₄ plates with solvent system 3 as described previously (26, 34). The corresponding R_fs for the glyoxylate-2,4-dinitrophenylhydrazones were 0.41 and 0.62 (cis and trans isomers, respectively). The radioactive products cochromatographed with nonradioactive glyoxylate-2,4-dinitrophenylhydrazones. The standard was detected by UV light and sprayed with 10% (wt/vol) NaOH solution (8). The plates were analyzed with a Fujix BAS1000 imaging analyzer (Fuji Film, Tokyo, Japan). Other samples cochromatographed with radioactive or nonradioactive standard compounds. Organic acids were detectable by spraying the plates with a 0.05% bromocresol green solution (Fluka Chemie, Buchs, Switzerland) at an acidic pH (8).

Determination of acid-stable ¹⁴C. The fixation of ¹⁴C from [¹⁴C]bicarbonate into nonvolatile acid-stable products was measured with samples (200 µl) incubated for 1, 2, and 5 min at 55°C (see above). The addition of 50 µl of 5 M H₂SO₄ (to pH 1 to 2) stopped the reaction. Volatile ¹⁴CO₂ (nonfixed) was removed from the samples by vigorously shaking the samples in scintillation vials for 2 h. The remaining radioactivity in the samples was determined by liquid scintillation counting. The radioactivity in samples from two control experiments, in which the substrate and the extract were omitted, served as blanks and controls.

Determination of amount of ¹⁴C. The amount of ¹⁴C in liquid samples (up to 200 µl) and in Whatman paper was determined by liquid scintillation counting using 3 ml of Rotiszint 2200 scintillation cocktail (Roth). The counting efficiency (75 to 85%) was determined via the channel ratio method.

Long-term labeling of growing cells with [1,2-¹³C₂]glycine. For the long-term labeling of an autotrophically grown culture (5 liters) with glycine, [1,2-¹³C₂]glycine was proffered from a sterile solution (50 ml) containing 0.2 g of [1,2-¹³C₂]glycine and 0.18 MBq of [1,2-¹⁴C₂]glycine by use of a peristaltic pump. The feeding rate during the 140-h feeding period (five generations) was adjusted to the cell density. Less than 10% of cell carbon should be proffered by glycine. Aliquots were retrieved at intervals, and protein was determined by a modified Lowry method (25). The radioactivity in the cells and in the cell-free supernatant was determined by liquid scintillation counting.

NMR analyses. The isolation of amino acids and ribonucleosides from cell mass and the experimental setup for ¹H and ¹³C nuclear magnetic resonance (NMR) experiments were previously described (12, 39).

	RESULTS

Top Abstract Introduction Materials and Methods Results Discussion References

Conversion of L-malyl-CoA to acetyl-CoA and glyoxylate. From previous work, it has been postulated that C. aurantiacus possesses malyl-CoA lyase to release the CO₂ acceptor acetyl-CoA and glyoxylate as the CO₂ fixation product. The indirect evidence was based on the spectrophotometric detection of glyoxylate-phenylhydrazone when both succinyl-CoA and L-malate were present. We synthesized L-malyl-CoA to prove the existence of L-malyl-CoA lyase and to study the enzyme reaction in more detail at the growth temperature of 55°C.

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View larger version (18K): [in this window] [in a new window]   FIG. 2.   Spectrophotometric assay of glyoxylate-phenylhydrazone formation from L-malyl-CoA by cell extracts (0.16 mg of protein) of autotrophically grown C. aurantiacus at 55°C. L-Malyl-CoA (0.2 mM) started the reaction. In control experiments, the addition of 10 mM L-malate and 0.3 mM CoA could not start the reaction. The assay mixture (0.5 ml) additionally contained MOPS-K+ buffer (pH 7.7), 10 mM MgCl2, and 3.5 mM phenylhydrazinium chloride (initial rate: 139 nmol min1 mg of protein1).

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View larger version (19K): [in this window] [in a new window]   FIG. 3.   L-Malyl-CoA cleavage to acetyl-CoA and glyoxylate. (A) HPLC detection of acetyl-CoA formation from L-malyl-CoA by extracts of autotrophically grown cells after 10 min of incubation at 55°C. The reaction mixture (0.5 ml) contained 200 mM MOPS-K+ buffer (pH 7.7), 10 mM MgCl2, 0.9 mM L-malyl-CoA, and 0.3 mg of protein. (B) Time course of the reaction: circles, L-malyl-CoA; triangles, acetyl-CoA; and squares, CoA. Note that the L-malyl-CoA preparation contained 28% CoA as an impurity. Therefore, besides 0.9 mM L-malyl-CoA, 0.3 mM CoA was also initially present in the assay mixture. The 77% peak area corresponds to 0.9 mM CoA-thioester or free CoA.

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View larger version (21K): [in this window] [in a new window]   FIG. 4.   Formation of malyl-CoA from acetyl-CoA and glyoxylate. (A) HPLC separation of 14C-labeled products formed from [14C]acetyl-CoA and glyoxylate after 1 min of incubation at 55°C with extracts of autotrophically grown cells. (B) Time course of the reaction: circles, [14C]acetyl-CoA; squares, [14C]malyl-CoA; triangles, [14C]succinyl-CoA; diamonds, 14C-polar products; and hexagons, [14C]acetyl-CoA in a control reaction in which glyoxylate was omitted. The reaction mixture (0.5 ml) contained 100 mM potassium phosphate buffer (pH 7.0), 10 mM MgCl2, 5 mM glyoxylate, 0.5 mM [14C]acetyl-CoA (16.7 kBq), and 0.8 mg of protein. The 100% peak area corresponds to a 0.5 mM concentration.

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Formation of malyl-CoA from malate. The proposed 3-hydroxypropionate cycle assumes that malyl-CoA is cleaved torather than formed fromacetyl-CoA and glyoxylate; both directions would be possible from a thermodynamic point of view. The cleavage direction requires malyl-CoA synthesis from malate. There are two options for the synthesis of malyl-CoA: either in a nucleoside triphosphate-dependent synthetase reaction or in a CoA transferase reaction.

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View larger version (19K): [in this window] [in a new window]   FIG. 5.   Spectrophotometric assay of glyoxylate-phenylhydrazone formation dependent on succinyl-CoA (1 mM) and L-malate (5 mM). The assay mixture (0.5 ml) additionally contained 200 mM MOPS-K+ buffer (pH 6.5), 10 mM MgCl2, 3.5 mM phenylhydrazinium chloride, and 0.3 mg of protein. In the first experiment, L-malyl-CoA was added after the L-malate-dependent reaction had been recorded. This procedure served as a control to show that L-malyl-CoA lyase was not the rate-limiting step in the coupled assay.

View larger version (20K): [in this window] [in a new window]   FIG. 6.   Formation of malyl-CoA from succinyl-CoA and malate. (A) HPLC separation of 14C-labeled products formed from [14C]malate and succinyl-CoA after 4 min of incubation at 45°C with extracts of autotrophically grown cells. (B) Time course of the reaction: circles, [14C]malyl-CoA; squares, [14C]acetyl-CoA; triangles, [14C]succinyl-CoA; and hexagons, [14C]acetyl-CoA in a control reaction in which succinyl-CoA was omitted. The assay mixture (0.5 ml) contained 100 mM potassium phosphate buffer (pH 6.7), 10 mM KCl, 10 mM MgCl2, 0.5 mM L-[14C]malate (36.7 kBq), 1 mM succinyl-CoA, and 0.5 mg of protein. The lag phase was due to the low temperature of the assay at the beginning of the experiment, when the assay temperature unintentionally had not yet reached 45°C. The 100% peak area corresponds to a 0.5 mM concentration.

Comparative study of enzyme activities related to carbon assimilation pathways. Various enzyme activities involved in autotrophic carbon metabolism were investigated with cells grown autotrophically with H₂-CO₂ or heterotrophically with Casamino Acids-yeast extract, anaerobically, and exposed to light. The specific enzyme activities measured at the growth temperature of 55°C are given in Table 1. Many enzyme activities did not show a significant difference under the two growth conditions. However, there were some remarkable exceptions. Activities that were higher in autotrophically grown cells were NADPH-dependent reduction of malonyl-CoA; NADPH-, ATP-, and CoA-dependent reductive conversion of 3-hydroxypropionate; succinyl-CoA:L-malate CoA transferase; and L-malyl-CoA lyase. Down-regulation of the CoA transferase in heterotrophically grown cells is deduced from the fact that L-malyl-CoA lyase could easily be detected, whereas succinyl-CoA-dependent malate cleavage was virtually undetectable, suggesting very low CoA transferase activity. All of these enzyme activities are postulated to play a role in the autotrophic CO₂ fixation cycle. Succinate dehydrogenase activity was higher in heterotrophically grown cells, possibly in keeping with a higher demand under such conditions. Either no or very small amounts of NAD(P)⁺-dependent pyruvate and 2-oxoglutarate dehydrogenase activities were detectable. This finding indicates that the citric acid cycle is incomplete under autotrophic and heterotrophic growth conditions and that pyruvate may not be the precursor of acetyl-CoA. All enzymes forming 2-oxoglutarate from oxaloacetate and acetyl-CoA and those oxidizing succinate to oxaloacetate were present.

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Long-term labeling of whole cells with [1,2-¹³C₂]glycine. Glyoxylate conversion to glycine and the assimilation of glycine via several hypothetical or existing reactions would be a possibility for the assimilation of glyoxylate into cell carbon. Some possible reactions were mentioned above. The possibility of glycine assimilation was tested by feeding an autotrophically grown culture [1,2-¹³C₂]glycine continuously for several generations. The feeding rate was exponentially increased and chosen so that less than 10% of cell carbon was derived from proffered glycine and more than 90% was derived from CO₂. The low rate of glycine supply ensured that glycine did not turn down autotrophic carbon fixation. This important prerequisite was found to be correct when the ¹³C content of cell constituents was compared with that of proffered glycine (less than 10%; Table 2). Biosynthetic building blocks, such as amino acids and nucleosides, were isolated from cellular polymers, and the amount and distribution of ¹³C in the individual carbon atoms were determined by NMR spectroscopic techniques. These techniques also allowed the determination of the degree of coupling of one carbon atom to another; 100% coupling between two carbon atoms meant that the two carbon atoms of glycine were incorporated as a C₂ unit without breakage of the C---C bond, rather than by rearrangement reactions involving C---C bond cleavage or by separate incorporation of C₁ units. From the observed labeling pattern, the patterns of labeling of central precursor metabolites was deduced by a retrobiosynthetic approach. This method has been described elsewhere (1, 12).

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion References

Required specific enzyme activities. A prerequisite for any proposed pathway is that the postulated enzyme activities can be measured in cell extracts. Furthermore, the in vitro rates must be at least as high as the minimal rates that can explain the growth rate, i.e., in this case, under autotrophic growth conditions. The generation times were 26 to 40 h for autotrophically grown cells and 3 to 10 h for heterotrophically grown cells. The calculated CO₂ fixation rate for autotrophically grown cells was 26 to 17 nmol of CO₂ fixed min¹ mg of protein¹; this value corresponds to 13 to 8.5 nmol of intermediates cycled in this pathway min¹ mg of protein¹, since two molecules of CO₂ are fixed per one turn of the cycle. The CO₂ fixation rate for growing cells was estimated from the equation dS/dt = (µ/y)x, correlating substrate S (moles of inorganic carbon) consumption per time (t) unit (minute) with the specific growth rate µ, the molar growth yield y, and the dry cell mass x of the culture. A generation time of 26 h corresponds to a µ of 0.00044 min¹, y is 24 g of dry cell mass formed per mol of CO₂ assimilated (assuming 50% of dry cell mass is carbon), and x is 1 g of dry cell mass, corresponding to 500 mg of total cell protein. All activities of the postulated cycle were detectable, and the rates met this requirement. Notably, the observed specific activity of the postulated enzyme L-malyl-CoA lyase, which was higher than the required minimal activity, the low apparent K_m for L-malyl-CoA, and the down-regulation of the activity in heterotrophically grown cells support the proposed role of this enzyme in CO₂ fixation. Similar arguments hold true for the CoA transferase, which activates L-malate by using succinyl-CoA.

Malate-forming reactions. The conversion of glyoxylate with acetyl-CoA to malate or malyl-CoA is not considered a meaningful reaction in autotrophically grown cells, since malyl-CoA cleavage to acetyl-CoA and glyoxylate is considered the physiological direction of L-malyl-CoA lyase (EC 4.1.3.24). Nonetheless, the in vitro formation of malate from acetyl-CoA and glyoxylate was observed, but only in extracts of autotrophically grown cells. Besides malate synthase (EC 4.1.3.2), several known enzyme reactions might have caused this reaction. Reaction (1) catalyzed by L-malyl-CoA lyase could be followed by a hydrolase activity (2):

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Citrate synthase (EC 4.1.3.7), in the absence of its substrates, shows high nonspecific L-malyl-CoA hydrolase activity (11, 41). Malate synthesis from acetyl-CoA and glyoxylate via the combined action of malyl-CoA lyase and citrate synthase mimicking an apparent malate synthase activity has been described previously by Cox and Quayle (7) for Pseudomonas sp. strain AM1 (now Methylobacterium extorquens). Also, an acyl-CoA thioesterase (EC 3.1.2.20) acting on L-malyl-CoA might be present (22). The situation in C. aurantiacus is very much the same as in M. extorquens, i.e., the simultaneous presence of active malyl-CoA lyase and citrate synthase under autotrophic growth conditions. We therefore believe that, in fact, citrate synthase is responsible for apparent malate synthase and enzymatic L-malyl-CoA hydrolysis activities under autotrophic growth conditions. Since L-malyl-CoA lyase activity was strongly down-regulated under heterotrophic growth conditions, only slight apparent malate synthase activity was measurable under these conditions.

Regulation of enzyme activities. The levels of various enzyme activities have been comparatively studied with cells grown autotrophically and heterotrophically. Interestingly, some reactions which are essential for the 3-hyproxypropionate cycle seem to be down-regulated in heterotrophically grown cells. These reactions concern the reduction of malonyl-CoA, the reductive conversion of 3-hydroxypropionate, and the conversion of L-malate to glyoxylate and acetyl-CoA. This regulation needs to be studied in more detail to elucidate the underlying mechanisms. Acetyl-CoA and propionyl-CoA carboxylase activities remain unchanged. High acetyl-CoA carboxylase activity under heterotrophic growth conditions may be required due to the four- to eightfold higher growth rate of heterotrophically grown cells, which in any case require acetyl-CoA carboxylase activity for fatty acid synthesis. In contrast, propionyl-CoA carboxylase activity normally is not required in carbon metabolism, and the unchanged activity in heterotrophically grown cells was unexpected. However, propionyl-CoA carboxylase activity and acetyl-CoA carboxylase activity may be due to one carboxylase enzyme. This assumption could explain the lack of down-regulation of propionyl-CoA carboxylase activity under heterotrophic growth conditions.

C₁ metabolism. Proffered [1,2-¹³C₂]glycine served as a precursor for cellular glycine and to a lesser extent for L-serine. This finding follows from the labeling pattern and ¹³C content of the amino acids isolated from cell material. Glycine C-2 obviously also served as a precursor for C₁ units. The strong labeling of C₁ units was deduced from the labeling of the methyl group of methionine and C-2 and C-8 of purine; even C-3 of serine was labeled. The specific ¹³C content of these C₁ units was approximately 50% lower than that of C-2 of glycine, which served as a C₁ unit precursor. This result indicates that approximately half of the C₁ units must have been derived from C-2 of glycine and supports glycine degradation by the reversible glycine cleavage system (formerly glycine synthase; EC 1.4.4.2 and EC 2.1.2.10), leading to 5,10-methylenetetrahydrofolate. This enzyme activity has been reported for C. aurantiacus (19, 20).

Possible glyoxylate assimilation reactions. The lack of labeling in the other amino acids excludes the possibility that glyoxylate is assimilated via glycine. Several possibilities have been discussed (19, 20, 39). (i) The lack of labeling of aspartate excludes the possibility that glyoxylate was assimilated via the conversion of one molecule of glyoxylate to glycine and, following the condensation of glycine with another molecule of glyoxylate, to -hydroxyaspartate. -Hydroxyaspartate could be converted to oxaloacetate by NH₃ elimination; the respective two enzymes are 3-hydroxyaspartate aldolase (EC 4.1.3.14) and 3-hydroxyaspartate dehydratase (EC 4.2.1.38). (ii) The minimal labeling of threonine excludes the possible assimilation of glycine by condensation with acetaldehyde to form threonine. Either threonine aldolase (EC 4.1.2.5) or serine hydroxymethyltransferase (EC 2.1.2.1) catalyzes this reaction. (iii) An interesting possibility is glycine reduction to acetylphosphate catalyzed by glycine reductase. This process would result in the labeling of all carbon compounds, notably, the positions derived from acetyl-CoA. This possibility can also be excluded. (iv) Glycine conversion to serine obviously takes place, but the lack of labeling of the other cell compounds shows that serine formation is a dead end.

Anaplerotic reactions. The organism seems to use pyruvate as a precursor for PEP and oxaloacetate synthesis, rather than the reverse option, the formation of pyruvate from these precursors. PEP carboxylase was quite active; pyruvate phosphate dikinase was preliminarily identified based on the pyruvate-, MgATP-, and phosphate-dependent fixation of ¹⁴C from [¹⁴C]bicarbonate. The presence of pyruvate carboxylase activity was excluded based on the lack of inhibition of pyruvate-dependent CO₂ fixation by avidin. If acetyl-CoA is the starting compound of the CO₂ fixation cycle, then there must be a way to convert acetyl-CoA to pyruvate. Pyruvate synthase activity catalyzing the ferredoxin-dependent reductive carboxylation of acetyl-CoA to pyruvate is rather low or absent in C. aurantiacus strain OK-70-fl (42). Alternatively, 3-hydroxypropionate or propionyl-CoA, intermediates of the postulated cycle, could be converted to pyruvate, possibly by condensation with glyoxylate. This possibility is currently being tested in our laboratory.