Properties of R-Citramalyl-Coenzyme A Lyase and Its Role in the Autotrophic 3-Hydroxypropionate Cycle of Chloroflexus aurantiacus

Abstract

The autotrophic CO₂ 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 CO₂ acceptor molecule acetyl-coenzyme A (CoA) requires condensation with propionyl-CoA (derived from one molecule of acetyl-CoA and one molecule of CO₂) to β-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/β-methylmalyl-CoA lyase. This promiscuous enzyme is involved in the CO₂ fixation pathway, forms acetyl-CoA and glyoxylate from l-malyl-CoA, and condenses glyoxylate with propionyl-CoA to β-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 Mg² or Mn²⁺ 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 CO₂ 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 γ-proteobacterium Congregibacter litoralis. This may indicate that part of the reactions may be involved in a different metabolic process.

Chloroflexus aurantiacus, a phototrophic green nonsulfur bacterium, uses an autotrophic CO₂ fixation cycle termed the 3-hydroxypropionate cycle (1, 9, 11, 14-19, 22, 37, 38). This organism grows optimally under heterotrophic conditions at 55°C and a pH of around 8, but it can also grow in mineral salt medium under autotrophic conditions using CO₂ as the sole carbon source (6, 18, 30, 31, 35). In thermal springs, filamentous Chloroflexus spp. and cyanobacteria form microbial mats that probably thrive photoautotrophically (39).

Each turn of the 3-hydroxypropionate cycle starts with acetyl-coenzyme A (CoA) and results in the net fixation of two molecules of bicarbonate to form one molecule of l-malate. l-Malate is converted to l-malyl-CoA by a CoA transferase (SmtAB) (11) using succinyl-CoA as the CoA donor. In the last step, l-malyl-CoA is cleaved by l-malyl-CoA lyase (Mcl) (14) into acetyl-CoA and glyoxylate (Fig. 1). Acetyl-CoA can serve as the substrate for another CO₂ fixation cycle and glyoxylate is assimilated in a second pathway (15).

FIG. 1.

Part of the bicyclic autotrophic CO₂ assimilation pathway in C. aurantiacus. The scheme shows the last two reactions of the first cycle and the known reactions of the second cycle. In the first cycle (3-hydroxypropionate cycle), two HCO₃⁻ are fixed to form glyoxylate as the first net fixation product. In the second cycle (glyoxylate assimilation cycle), glyoxylate and propionyl-CoA are condensed to erythro-β-methylmalyl-CoA (probably [2R,3S]-2-methylmalyl-CoA), which is converted to acetyl-CoA and pyruvate. SmtAB, succinyl-CoA:l-malate CoA transferase; Mcl, l-malyl-CoA lyase/erythro-β-methylmalyl-CoA lyase; Mcd, β-methylmalyl-CoA dehydratase; Sct, succinyl-CoA:R-citramalate CoA transferase; Ccl, R-citramalyl-CoA lyase.

In this second cycle, glyoxylate and propionyl-CoA are converted to acetyl-CoA and pyruvate, which can be used as precursors for biosynthesis. In a first reaction, propionyl-CoA and glyoxylate are condensed to β-methylmalyl-CoA by the same lyase (Mcl) (14) which catalyzes the last reaction of the first cycle (Fig. 1). β-Methylmalyl-CoA is dehydrated to mesaconyl-CoA by a β-methylmalyl-CoA dehydratase (Mcd) (unpublished data). The reactions converting mesaconyl-CoA to citramalate are unknown. Citramalate is activated by CoA transfer to the corresponding thioester in a succinyl-CoA-dependent reaction, followed by the cleavage of citramalyl-CoA into acetyl-CoA and pyruvate (15). Two CoA transferases, Sct and SmtAB, which were specific for the R and S stereoisomers of citramalate, respectively (11, 12), were found.

In the present work, we analyzed the cleavage of citramalyl-CoA into pyruvate and acetyl-CoA, the last step in the glyoxylate assimilation route. Extracts contained both S- and R-citramalyl-CoA lyase activities and both lyases were up-regulated under autotrophic conditions. We show that the l-malyl-CoA lyase Mcl also catalyzes S-citramalyl-CoA cleavage. The R-citramalyl-CoA lyase reaction is catalyzed by a new lyase, R-citramalyl-CoA lyase (Ccl), whose gene was identified and expressed in Escherichia coli. The roles and occurrences of these and other enzymes of the 3-hydroxypropionate cycle will be discussed.

MATERIALS AND METHODS

Bacteria and growth conditions.

Chloroflexus aurantiacus strain OK-70-fl (DSM 636) was grown in 2-, 5-, or 12-liter glass fermentors to an optical density at 578 nm (1-cm light path) of 3.5 to 4.0 at 55°C and a pH of around 8. The light exposure was 10,000 to 12,000 lx. Autotrophic growth occurred under anaerobic conditions on a minimal medium supplemented with trace elements and vitamins. The cultures were gassed with a mixture of H₂ and CO₂ (80%:20% [vol/vol]) as described elsewhere (37). Cells were also grown anaerobically under photoheterotrophic conditions on a modified minimal medium D (6) supplemented with 0.25% (wt/vol) Casamino Acids, 0.1% (wt/vol) yeast extract, and trace elements. The medium was buffered with 0.05% glycylglycine-Na⁺ buffer. Cells were stored under liquid nitrogen until use. Escherichia coli strain SURE from Stratagene (Heidelberg, Germany) was grown at 37°C in Luria-Bertani medium (33). Ampicillin was added to E. coli cultures to a final concentration of 100 μg/ml. Growth was measured photometrically at 578 nm as optical density using cuvettes with a 1-cm light path.

Materials.

Chemicals were obtained from Fluka (Neu-Ulm, Germany), Sigma-Aldrich (Deisenhofen, Germany), Merck (Darmstadt, Germany), or Roth (Karlsruhe, Germany). Biochemicals were from Roche Diagnostics (Mannheim, Germany), Applichem (Darmstadt, Germany), or Gerbu (Gaiberg, Germany). Materials for cloning and expression were purchased from MBI Fermentas (St. Leon-Rot, Germany), New England Biolabs (Frankfurt, Germany), Genaxxon Bioscience GmbH (Biberach, Germany), MWG Biotech AG (Ebersberg, Germany), or QIAGEN (Hilden, Germany). Materials and equipment for protein purification were obtained from Amersham Biosciences (Freiburg, Germany) or Millipore (Eschborn, Germany).

Syntheses. (i) Succinyl-CoA, acetyl-CoA, and propionyl-CoA.

The CoA thioesters of succinate, acetate, and propionate were synthesized from their anhydrides (34, 36) by a slightly modified method described previously (14), and the dry powders were stored at −20°C.

(ii) Malyl-CoA.

l-Malyl-CoA was chemically synthesized from l-malylcapryloyl-cysteamine (S-[β-hydroxysuccinyl]-N-capryloylcysteamine) as described previously (8, 29), with a slight modification (14). l-Malyl-CoA was stored as freeze-dried powder at −20°C. It contained 80% CoA thioester and 20% CoA, as determined by high-pressure liquid chromatography (HPLC) separation and detection at 260 nm.

(iii) R-/S-citramalyl-CoA.

R- and S-citramalyl-CoA were synthesized enzymatically from R- and S-citramalate, respectively, and succinyl-CoA using a preparation of recombinant succinyl-CoA:R-citramalate CoA transferase (12) or succinyl-CoA:l-malate CoA transferase (11), respectively. A reaction mixture (1 ml) containing 200 mM morpholinopropanesulfonic acid (MOPS)-KOH (pH 6.5), 100 mM R-citramalate or S-citramalate, and 10 mM succinyl-CoA, succinyl-CoA:R-citramalate CoA transferase protein fraction (1 μmol/min), or succinyl-CoA:l-malate CoA transferase protein fraction (0.75 μmol/min) was incubated at 55°C. After 10 min of incubation, the mixture was adjusted to a pH of 2 by the addition of HCl. Precipitated protein was removed by centrifugation. The supernatant was subjected in 100-μl portions to a reversed-phase column (LiChrospher 100; endcapped; 5 μm, 125 by 4 mm; Merck, Darmstadt, Germany) which was developed with a 40-ml gradient from 2 to 10% acetonitrile in 50 mM ammonium acetate buffer, pH 4.0, with a flow rate of 1 ml min⁻¹. CoA and CoA esters were photometrically detected at 260 nm. R-Citramalyl-CoA and S-citramalyl-CoA were collected, the samples were lyophilized, and the dry powder was stored at −20°C.

Preparation of cell extract.

Frozen C. aurantiacus and E. coli cells were suspended in a twofold volume of 50 mM MOPS-KOH (pH 7.0) containing 0.2 mg DNase I per ml of cell suspension and passed twice through a chilled French pressure cell at 137 kPa. The lysate was ultracentrifuged at 100,000 × g at 4°C for 1 h.

Heterologous expression and purification of recombinant enzymes.

The succinyl-CoA:R-citramalate CoA transferase gene (sct) from C. aurantiacus was heterologously expressed in E. coli, and the recombinant enzyme was purified in two purification steps, by heat precipitation and size exclusion chromatography, as described elsewhere (12). The succinyl-CoA:l-malate CoA transferase gene (smtAB) from C. aurantiacus was heterologously expressed in E. coli, and the recombinant enzyme was purified in three purification steps by heat precipitation, MonoQ chromatography, and reactive green 19 agarose affinity chromatography, as described elsewhere (11).

Heterologous expression and purification of recombinant l-malyl-CoA lyase.

mcl from C. aurantiacus was heterologously expressed in E. coli, and the recombinant l-malyl-CoA lyase (Mcl) was purified in three steps, by heat precipitation, DEAE-Sepharose fast-flow chromatography, and size exclusion chromatography, as described elsewhere (14).

Enzyme assays.

R-Citramalyl-CoA lyase was tested at 55°C, routinely in the lyase direction.

(i) Coupled spectrophotometric assay.

The succinyl-CoA- and R-citramalate-dependent formation of pyruvate in the presence of excess recombinant succinyl-CoA:R-citramalate CoA transferase (12) was monitored photometrically at 324 nm with phenylhydrazine in a continous assay (ɛ₃₂₄ for pyruvate-phenylhydrazone, 10,400 M⁻¹ cm⁻¹). The assay mixture (0.5 ml) contained 200 mM MOPS-KOH buffer (pH 7.0), 4 mM MnCl₂, 4 mM dithioerythritol (DTE), 3.5 mM phenylhydrazinium chloride, 1 mM succinyl-CoA, 10 mM R-citramalate, and 0.5 U succinyl-CoA:R-citramalate CoA transferase. Either substrate (succinyl-CoA or R-citramalate) could be used to start the reaction. The buffers used to determine the optimum pH were 200 mM 2-(N-morpholino)ethanesulfonic acid (MES)-KOH buffer (pH 5.5 to 6.0) and 200 mM MOPS-KOH buffer (pH 6.0 to 8.0).

(ii) Uncoupled spectrophotometric assay.

The cleavage of R-citramalyl-CoA into pyruvate and acetyl-CoA was monitored photometrically at 324 nm with phenylhydrazine in a continuous assay (ɛ₃₂₄ for pyruvate-phenylhydrazone, 10,400 M⁻¹cm⁻¹). The assay mixture (0.5 ml) contained 200 mM MOPS-KOH buffer (pH 7.0), 4 mM MnCl₂, 4 mM DTE, 3.5 mM phenylhydrazinium chloride, and different concentrations of R-citramalyl-CoA and protein. The apparent K_m value was determined using 0.01 to 0.2 mM R-citramalyl-CoA. This assay was used for determining the stoichiometries of the reaction with 0.096 mM and 0.144 mM R-citramalyl-CoA.

(iii) Coupled HPLC assay.

The coupled HPLC assay mixture (0.5 ml) contained 200 mM MOPS-KOH buffer (pH 7.0), 4 mM DTE, 1 mM succinyl-CoA, 10 mM R-citramalate, 0.5 U of purified recombinant succinyl-CoA:R-citramalate CoA transferase, and 0.1 U of purified recombinant R-citramalyl-CoA lyase, respectively. The reaction was started with the addition of R-citramalate. Samples of 110 μl were taken after 1 and 5 min of incubation at 55°C, and the reaction was stopped by the addition of 3 μl of 25% HCl. Precipitated protein was removed by centrifugation, and samples were analyzed for CoA thioesters by HPLC. A reversed-phase column (LiChrospher 100; endcapped; 5 μm, 125 by 4 mm; Merck) was used for the separation of CoA thioesters. A 30-min gradient from 1 to 8% acetonitrile in 50 mM potassium phosphate buffer, pH 6.7, with a flow rate of 1 ml min⁻¹ was used. CoA and CoA thioesters were detected at 260 nm. Retention times were 2 min (free organic acids), 10.8 min (R-citramalyl-CoA), 11.6 min (succinyl-CoA and free CoA), and 17.8 min (acetyl-CoA).

Cloning and expression of a putative R-citramalyl-CoA lyase (ccl) gene in E. coli.

Standard protocols were used for the preparation, cloning, transformation, amplification, and purification of DNA (4, 33). Plasmid DNA was isolated with the QIAprep spin miniprep kit (QIAGEN).

Heterologous expression of ccl from C. aurantiacus.

Two oligonucleotides were designed upstream (5′-GCAGATGACCATGGAAGCAGTAACG-3′; 25-mer; the NcoI restriction site is italicized) and downstream (5′-TCTGGATCCAGTAGCTTATGACAGG-3′; 25-mer; the BamHI restriction site is italicized). PCR was performed with Pfunds polymerase (Genaxxon) for 30 cycles with an annealing temperature of 55°C and extension at 72°C for 2 min. The 979-bp PCR product was purified and cloned into the pTrc 99A vector (accession number U13872; Amersham Biosciences) by using the NcoI and BamHI restriction sites of the multiple cloning site. The nucleotide sequence of the PCR product was confirmed to ensure that no errors had been introduced. The recombinant plasmid pSF1 was transformed into E. coli SURE, and the expression of the ccl gene was induced at an optical density at 578 nm of 0.7 (12-liter fermentor; 37°C) by the addition of 0.5 mM isopropyl-β-d-thiogalactopyranoside to the Luria-Bertani medium containing 100 μg of ampicillin ml⁻¹. After additional growth for 4 h, the cells were harvested and stored in liquid nitrogen until use.

DNA sequencing and computer analysis.

The DNA sequence determination for the purified plasmids was performed by G. L. Igloi (Institut Biologie III, Universität Freiburg, Germany). DNA and amino acid sequences were analyzed with the BLAST network service at the National Center for Biotechnology Information (Bethesda, MD) and the local C. aurantiacus server (http://genome.jgi-psf.org/draft_microbes/chlau/chlau.home.html) at the Department of Energy Joint Genome Institute (Walnut Creek, CA). The protein sequence alignment and the similarity tree of protein sequences were constructed using the MultAlin multialignment program (http://prodes.toulouse.inra.fr/multalin/multalin.html) (7).

Purification of recombinant R-citramalyl-CoA lyase from E. coli.

The purification was performed at 4°C and the activity was measured with the coupled-spectrophotometric assay with recombinant succinyl-CoA:R-citramalate CoA transferase.

(i) Heat precipitation.

Cell extract (100,000 × g centrifugation supernatant) from 6 g of cells (wet mass) of E. coli with recombinant R-citramalyl-CoA lyase was incubated at 65°C for 10 min to precipitate unwanted protein from E. coli cells, followed by centrifugation (21,000 × g) at 4°C for 10 min. The supernatant was incubated again at 75°C for 10 min, followed by centrifugation (21,000 × g) at 4°C for 10 min.

(ii) Ammonium sulfate precipitation.

After heat precipitation, the supernatant (10 ml) was brought to 40% (NH₄)₂SO₄ saturation by the addition of saturated ammonium sulfate solution, pH 7.0, with slow stirring on ice. The mixture was stirred slowly at 4°C for an additional one-half hour. The precipitated protein was centrifuged. The pellet was dissolved in 5 ml of 50 mM Tris-HCl buffer, pH 7.0.

(iii) Gel filtration chromatography.

After ammonium sulfate precipitation, the protein solution was applied to a Superdex 200 gel filtration column (bed volume, 320 ml; Amersham Biosciences) equilibrated with 20 mM Tris-HCl buffer, pH 7.0, containing 100 mM KCl with a flow rate of 2.5 ml min⁻¹. Active protein was eluted with a retention volume of 190 to 220 ml. Active fractions were immediately pooled, desalted, and concentrated to a final volume of 14.5 ml by ultrafiltration (Amicon YM 30 membrane; Millipore).

(iv) MonoQ chromatography.

After size exclusion chromatography, the concentrated sample was applied to a MonoQ 5/5 column (Amersham Biosciences) which had been equilibrated with 20 mM Tris-HCl buffer (buffer A), pH 8.0, containing 100 mM KCl with a flow rate of 1 ml min⁻¹. The column was washed with 5 bed volumes of buffer A containing 100 mM KCl and developed with a 30-ml linear gradient of 100 mM to 300 mM KCl in buffer A. Active fractions were pooled (11 ml), concentrated, and stored at −20°C with 10% glycerol.

Other methods.

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE; 12.5%) was performed by the Laemmli method (26). The following proteins were used as molecular mass standards: rabbit phosphorylase b, 97 kDa; bovine serum albumin, 67 kDa; egg ovalbumin, 45 kDa; lactate dehydrogenase, 34 kDa; carbonic anhydrase, 29 kDa; and lysozyme, 14 kDa. Proteins were visualized by Coomassie blue staining (40). Protein was determined by the Bradford method (5) using bovine serum albumin as the standard. Purified recombinant R-citramalyl-CoA lyase (3.8 mg ml⁻¹) was dialyzed against 2 liters metal-free buffer (20 mM Tris-HCl, pH 7.0) and analyzed for metals by inductively coupled plasma emission spectroscopy by R. Auxier, Chemical Analysis Laboratory, University of Georgia, Athens (GA), using buffer as a blank.

RESULTS

R- and S-citramalyl-CoA lyase activity in cell extracts of C. aurantiacus.

Extracts of photoautotrophically grown cells of C. aurantiacus catalyzed the S- and R-citramalate- and succinyl-CoA-dependent formation of pyruvate and acetyl-CoA (15). This reaction is due to a combination of two enzyme steps, (1) succinyl-CoA + citramalate → succinate + citramalyl-CoA and (2) citramalyl-CoA → acetyl-CoA + pyruvate (15). In previous studies, the citramalyl-CoA lyase activity and its regulation could not be studied in detail, because R- and S-citramalyl-CoA were not available. Here, citramalyl-CoA lyase activity was measured in a coupled spectrophotometric assay using an excess of purified recombinant succinyl-CoA:R-citramalate CoA transferase (12) and succinyl-CoA:S-citramalate CoA transferase (11). Succinyl-CoA:R-citramalate CoA transferase catalyzes R-citramalate + succinyl-CoA → R-citramalyl-CoA + succinate. Succinyl-CoA:l-malate CoA transferase also acts on S-citramalate, catalyzing S-citramalate + succinyl-CoA → S-citramalyl-CoA + succinate (succinyl-CoA:S-citramalate CoA transferase activity). In this way, the lyase activity in cell extracts could be determined independent of the corresponding CoA transferase activity. R-Citramalyl-CoA lyase activity was Mn²⁺ and DTE dependent; S-citramalyl-CoA lyase activity was Mg²⁺ dependent. The formation of the phenylhydrazone of pyruvate was monitored at 55°C, the optimal growth temperature of this bacterium.

Extracts of autotrophically grown cells catalyzed the R-citramalyl-CoA and S-citramalyl-CoA cleavage at rates of 13 nmol min⁻¹ mg protein⁻¹ and 160 nmol min⁻¹ mg protein⁻¹, respectively. In photoheterotrophically grown cells, the specific activities were 2 nmol min⁻¹ mg protein⁻¹ for R-citramalyl-CoA cleavage and 20 nmol min⁻¹ mg protein⁻¹ for S-citramalyl-CoA cleavage. Hence, both activities are up-regulated severalfold under autotrophic conditions.

Identification of R-citramalyl-CoA lyase gene of C. aurantiacus and expression in E. coli.

Contig NZ_AAAH02000037 of the C. aurantiacus genome contains the gene coding for succinyl-CoA:R-citramalate CoA transferase (Sct) (12). The gene for a putative R-citramalyl-CoA lyase (Ccl) with a molecular mass of 34 kDa is located downstream of sct and was annotated as 3-hydroxy-3-methylglutaryl (HMG)-CoA lyase (has similarity to the Rhodospirillum rubrum HMG-CoA lyase gene [GenBank accession number AAB50182] with an E value of 1e-48). 3-Hydroxy-3-methylglutaryl-CoA lyase catalyzes a similar reaction, the cleavage of 3-hydroxy-3-methylglutaryl-CoA into acetoacetate and acetyl-CoA. Furthermore, (R)-citramalyl-CoA and (S)-HMG-CoA, the substrate of HMG lyase, have the same stereochemistry, which fits well with the high similarities of the amino acid sequences of the two lyases.

The DNA fragment containing the putative R-citramalyl-CoA lyase gene was amplified by PCR and cloned in E. coli SURE and the protein was expressed. The E. coli cell extract was heat precipitated for 10 min at 65°C, followed by 10 min at 75°C. After this treatment, the supernatant was tested for R-citramalyl-CoA lyase activity at 55°C. The specific activity was 0.32 μmol min⁻¹ mg protein⁻¹; this activity was missing in the heat-treated cell extract from recombinant E. coli cells lacking the DNA insert.

Purification and characterization of recombinant R-citramalyl-CoA lyase.

The R-citramalyl-CoA lyase was further purified from the heat-treated extract of 6.5 g E. coli cells in three steps, with a yield of 52% (Table 1). The specific activity of the purified enzyme was 1.52 μmol min⁻¹ mg protein⁻¹. A specific activity of 13 nmol min⁻¹ mg protein⁻¹ observed in cell extracts of autotrophically grown C. aurantiacus indicates that the R-citramalyl-CoA lyase represents approximately 1% of the soluble protein of autotrophically grown cells. SDS-PAGE showed a 34-kDa protein and two additional very faint bands (<5%) (Fig. 2). The properties of the protein are summarized in Table 2. Size-exclusion chromatography indicated a native molecular mass of 65 ± 10 kDa, suggesting that the native protein was a homodimer. The protein was colorless and exhibited an absorption maximum at 280 nm (ɛ₂₈₀ = 37.2 mM⁻¹ cm⁻¹ [α₂]), indicating that it contained no UV-visible light-absorbing cofactor. The molar absorption coefficient at 280 nm, calculated based on the amino acid composition derived from the ccl gene and assuming a dimeric structure, is ɛ₂₈₀ = 37.3 mM⁻¹ cm⁻¹, which agrees well with the absorption coefficient determined. The enzyme could be stored for two weeks at 4°C and a pH of 7.0 without significant loss of activity or kept frozen for months in the presence of 10% glycerol (vol/vol).

TABLE 1.

Purification of recombinant R-citramalyl-CoA lyase from 6.5 g of E. coli (fresh cell mass)^a

Purification step	Total enzyme activity (μmol min−1)	Total protein (mg)	Sp act (μmol min−1 mg−1)	Amt recovered (%)	Purification achieved (fold)
Heat precipitation (65°C, 10 min; 75°C, 10 min)	12.8	40	0.32	100	1
Ammonium sulfate precipitation	9.2	16	0.58	72	1.8
Gel filtration	8.3	5.8	1.43	65	4.5
MonoQ	6.7	4.4	1.52	52	4.8

^aThe purification process started with 400 mg of total protein.

FIG. 2.

Results of denaturing PAGE (12.5%) of enzyme fractions obtained during the purification of recombinant R-citramalyl-CoA lyase of C. aurantiacus from E. coli (10 μg each). Lanes: 1, cells before induction; 2, cells after induction; 3, cell extract after heat precipitation (10 min at 65°C, followed by 10 min at 75°C); 4, sample after ammonium sulfate precipitation; 5, size exclusion fraction; 6, MonoQ fraction; 7, molecular mass markers. The arrows indicate the molecular mass markers (rabbit phosphorylase b, 97 kDa; bovine serum albumin, 67 kDa; egg ovalbumin, 45 kDa; lactate dehydrogenase, 34 kDa; carbonic anhydrase, 29 kDa; lysozyme, 14 kDa). The gel was stained with Coomassie brilliant blue R-250.

TABLE 2.

Molecular and catalytic properties of recombinant R-citramalyl-CoA lyase(Ccl)^a

Property	Value(s)
Sp act	1.5 ± 0.1 μmol min−1 mg protein−1
Apparent Km value	70 μM ± 20 μM (R-citramalyl-CoA)
Catalytic no. for α2	1.7 s−1
Optimum pH	7.0 (55°C)
Native molecular mass	65 kDa ± 10 kDa
Subunit molecular mass	34 kDa
Suggested composition	α2
Influence of divalent
cations (mM)b	Mn2+ (4), 100%; Co2+ (0.2), 100%; Ni2+ (0.2), 75%; Mg2+ (4), 60%; Zn2+ (0.2), 20%; no addition, 40%
Inhibitor (mM)b	Iodoacetamide (4), <1%; EDTA (3-h preincubation with 10 mM) (4), 24%

^aR-Citramalyl-CoA is the substrate and pyruvate and acetyl-CoA are the products of recombinant R-citramalyl-CoA lyase.

^b100% activity refers to the enzyme activity found in the assay containing DTE.

Catalytic properties.

The catalytic properties of the purified recombinant enzyme were studied at 55°C. The optimal pH for the enzyme activity was 7.0, with half-maximal activities at pHs of 6.3 and 7.6. The catalytic number for R-citramalyl-CoA cleavage was 1.7 s⁻¹, assuming α₂ composition. The enzyme showed high affinity for its substrate, with an apparent K_m for R-citramalyl-CoA of 70 μM. The stoichiometry of the reaction was 0.97 mol pyruvate phenylhydrazone formed per mol of purified R-citramalyl-CoA added.

R-citramalyl-CoA lyase was highly specific for its substrate (Table 2). It was inactive with S-citramalyl-CoA, d-malyl-CoA, or l-malyl-CoA. No cofactor was required; however, enzyme activity was stimulated by divalent cations in the order Mn²⁺ ≈ Co²⁺ > Ni²⁺ > Mg²⁺. No stimulation was obtained after the addition of Ca²⁺. Zn²⁺ inhibited the enzyme. When Mn²⁺ (routinely 4 mM) was omitted, approximately 40% residual activity was obtained when DTE was included in the assay. R-Citramalyl-CoA lyase activity in the presence of 4 mM EDTA and after a 3-h incubation of the enzyme with 10 mM EDTA was reduced to 25% of the remaining activity (compare reference 28). The activity was completely restored by the addition of excess Mn²⁺.

The recombinant enzyme was stimulated by exogenous thiols. The addition of DTE (routinely 4 mM) enhanced the enzyme activity about threefold. In the absence of DTE, the addition of iodoacetamide (4 mM) resulted in complete loss of activity. The addition of both 4 mM DTE and 4 mM Mn²⁺ enhanced the enzyme activity approximately 10-fold compared to the results of an assay lacking DTE and Mn²⁺.

Metal analysis of recombinant R-citramalyl-CoA lyase.

After MonoQ-Sepharose chromatography, the purified recombinant R-citramalyl-CoA lyase was dialyzed and analyzed for the presence of metals. A comprehensive metal analysis (20 elements) was done by plasma emission spectroscopy and revealed that the recombinant enzyme contains 0.5 mol of zinc per mol of native enzyme (α₂). Additionally, 0.2 mol copper per mol native protein and traces of iron were detected.

Cleavage of S-citramalyl-CoA.

Extracts of autotrophically grown cells cleaved S-citramalyl-CoA in addition to the R stereoisomer. Since R-citramalyl-CoA lyase was specific for the R stereoisomer, we searched for the induced enzyme acting on the S stereoisomer. A bifunctional enzyme (l-malyl-CoA lyase/β-methylmalyl-CoA lyase) from C. aurantiacus, which catalyzes the cleavage of l-malyl-CoA into glyoxylate and acetyl-CoA and the synthesis of erythro-β-methylmalyl-CoA from glyoxylate and propionyl-CoA, has been previously characterized and shown to be involved in CO₂ fixation (14). This enzyme was heterologously expressed in E. coli and purified 3.4-fold from 2.5 g E. coli cells in three steps, with a yield of 52%. The enzyme also was found to act on S-citramalyl-CoA, which is cleaved into pyruvate and acetyl-CoA. These reactions were Mg²⁺ dependent. The specific lyase activity at 55°C was 4.1 μmol min⁻¹ mg protein⁻¹ for l-malyl-CoA cleavage and 31 μmol min⁻¹ mg protein⁻¹ for S-citramalyl-CoA cleavage.

Similar genes in other bacteria.

A similar gene cluster containing genes possibly encoding succinyl-CoA:R-citramalate CoA transferase (sct) and R-citramalyl-CoA lyase (ccl) is present in the genome of another member of the Chloroflexaceae, Roseiflexus sp. strain RS-1 (Fig. 3) (GenBank accession number NZ_AAQU00000000). In C. aurantiacus, the sct-ccl gene cluster contains genes for two subunits of a protein homologous to acetone carboxylase, whose function is unknown (Fig. 3) (12). The Roseiflexus gene cluster containing the sct and ccl genes does not harbor the genes for the acetone carboxylase-like enzyme, but this set of genes is located elsewhere on the genome. In addition, a gene cluster is present in the genome of Roseiflexus sp. strain RS-1 which contains genes likely encoding three key enzymes of the 3-hydroxypropionate cycle: three genes which probably encode subunits of acetyl-CoA/propionyl-CoA carboxylase, the main carboxylating enzyme of the pathway; the gene for malonyl-CoA reductase; and the gene for propionyl-CoA synthase (Fig. 3). A third gene cluster with genes homologous to those for succinyl-CoA:S-(citra)malate CoA transferase, l-malyl-CoA lyase/β-methylmalyl-CoA lyase, β-methylmalyl-CoA dehydratase (unpublished results), and a CoA transferase with unknown substrate specificity is almost identical to the one found in C. aurantiacus. These findings suggest that Roseiflexus sp. strain RS-1 may also use the 3-hydroxypropionate cycle for CO₂ fixation.

FIG. 3.

Important gene clusters for autotrophic growth in Chloroflexus aurantiacus and similar genes or gene clusters in other organisms. Corresponding ORFs are shown in the same color. Putative products or ORFs: red, CoA transferase; medium blue, R-citramalyl-CoA lyase; gray, malonyl-CoA reductase; magenta, propionyl-CoA synthase; dark blue, l-malyl-CoA lyase/β-methylmalyl-CoA lyase; dark green, β-methylmalyl-CoA dehydratase; medium green, acetyl-CoA carboxylase; violet, ORFs located on the gene cluster on contig NZ_AAAH02000037 together with the genes for Sct and Ccl; black, ORFs which show no similarities to known proteins required for CO₂ fixation in C. aurantiacus. sct encodes succinyl-CoA:R-citramalate CoA transferase; ccl encodes R-citramalyl-CoA lyase; mcr encodes malonyl-CoA reductase; pcs encodes propionyl-CoA synthase; smtA encodes succinyl-CoA:l-malate CoA transferase, subunit A; smtB encodes succinyl-CoA:l-malate CoA transferase, subunit B (note that the smtAB gene product also has succinyl-CoA:S-citramalate CoA transferase activity); ctr encodes putative CoA transferase; mcl encodes l-malyl-CoA lyase/erythro-β-methylmalyl-CoA lyase (note that the mcl gene product also has S-citramalyl-CoA lyase activity); mcd encodes β-methylmalyl-CoA dehydratase; and accD accA accC encodes subunits of acetyl-CoA carboxylase.

Furthermore, we found open reading frames (ORFs) for some putative enzymes of the 3-hydroxypropionate cycle in members of the proteobacteria, the α-proteobacterium Erythrobacter sp. strain NAP-1 and the γ-proteobacterium Congregibacter litoralis. Both bacteria have ORFs for a putative CoA-transferase, for R-citramalyl-CoA lyase, and for propionyl-CoA synthase. Erythrobacter sp. strain NAP-1 contains ORFs for a putative propionyl-CoA synthase and a putative malonyl-CoA reductase together in one cluster. However, in both bacteria, genes coding for l-malyl-CoA lyase/β-methylmalyl-CoA lyase and β-methylmalyl-CoA dehydratase are missing.

DISCUSSION

Postulated role of R-citramalyl-CoA lyase, required enzyme activity, and regulation.

The 3-hydroxypropionate cycle of Chloroflexus aurantiacus forms glyoxylate as its first CO₂ fixation product. This intermediate needs to be converted into a precursor molecule for biosynthetic reactions. In a second cycle, glyoxylate and propionyl-CoA are condensed to β-methylmalyl-CoA (14), which is further converted to citramalate and activated to citramalyl-CoA (15). The cleavage of citramalyl-CoA into pyruvate and acetyl-CoA is the last reaction in this pathway. Here we have characterized a new lyase whose function is to cleave R-citramalyl-CoA into acetyl-CoA and pyruvate. Pyruvate is the cellular carbon precursor, and acetyl-CoA is the CO₂ acceptor molecule that is regenerated.

The specific activity of R-citramalyl-CoA lyase in cell extracts of autotrophically grown C. aurantiacus (13 nmol min⁻¹ mg protein⁻¹) is high enough to meet the requirements of growing cells. The generation time of autotrophically growing cultures was 26 h, which corresponds to a calculated minimal enzyme rate of 12 nmol min⁻¹ mg protein⁻¹ (16). R-Citramalyl-CoA lyase activity is up-regulated severalfold under autotrophic conditions, and it has a low apparent K_m value for R-citramalyl-CoA and a high specificity for its substrate, strongly favoring the role of the enzyme in CO₂ fixation.

One intriguing result needs explanation. Extracts of autotrophically grown cells also catalyzed the cleavage of S-citramalyl-CoA into pyruvate and acetyl-CoA. The specific activity of this reaction was even higher than the R-citramalyl-CoA lyase activity and is also up-regulated severalfold under autotrophic conditions. This apparent discrepancy may be explained as follows. We showed that S-citramalyl-CoA cleavage is catalyzed by l-malyl-CoA lyase/β-methylmalyl-CoA lyase (Mcl). The up-regulation of S-citramalyl-CoA cleavage activity under autotrophic conditions is explained by the promiscuity of Mcl. The presence and regulation of an enzyme acting specifically on R-citramalyl-CoA are taken as an indication that the R isomer of this dicarboxylic acid may represent the natural intermediate in the glyoxylate assimilation cycle. R-Citramalyl-CoA lyase forms a transcriptional unit together with succinyl-CoA:R-citramalate CoA transferase, which activates R-citramalate to R-citramalyl-CoA (12). However, the possibility that both stereoisomers are formed in the course of glyoxylate conversion to acetyl-CoA cannot be excluded. A final determination requires the knockout of genes, which is a difficult task in this bacterium.

Comparison of similar gene clusters in different organisms.

C. aurantiacus and Roseiflexus sp. strain RS-1, two closely related bacteria, have similar gene clusters which contain the genes for the succinyl-CoA:R-citramalate CoA transferase (sct) and for the lyase (ccl) (Fig. 3). Roseiflexus sp. strain RS-1 also possesses a gene cluster coding for a putative acetyl-CoA carboxylase (accACD), a malonyl-CoA reductase (mcr), and a propionyl-CoA synthase (pcs). The localization of the genes coding for three essential enzymes of the 3-hydroxypropionate cycle in one cluster supports the idea of a common metabolic process, whereas these genes are located on different contigs in Chloroflexus aurantiacus. Yet, Roseiflexus sp. strain RS-1 has not been reported to be capable of autotrophic growth. Other CO₂ fixation pathways cannot operate because Roseiflexus sp. strain RS-1 does not possess the genes for key enzymes for these pathways, e.g., ribulose 1,5-bisphosphate carboxylase/oxygenase, carbon monoxide dehydrogenase, or ATP citrate lyase.

Furthermore, we found ORFs for some putative enzymes of the 3-hydroxypropionate cycle in members of the proteobacteria. Both the α-proteobacterium Erythrobacter sp. strain NAP-1 and the γ-proteobacterium Congregibacter litoralis have ORFs similar to sct and ccl, as well as the propionyl-CoA synthase (pcs), but lack the genes coding for l-malyl-CoA lyase/β-methylmalyl-CoA lyase and β-methylmalyl-CoA dehydratase. Erythrobacter sp. strain NAP-1 contains the ORFs for a putative propionyl-CoA synthase (pcs) and a putative malonyl-CoA reductase (mcr) located in one cluster. Erythrobacter sp. strain NAP-1 can coassimilate CO₂ in the presence of an additional organic carbon source (24), but it seems not to grow autotrophically. The genes encoding putative enzymes required to convert acetyl-CoA via propionyl-CoA to succinate may be used to coassimilate acetate and propionate via an incomplete 3-hydroxypropionate cycle. Erythrobacter litoralis, which is closely related to Erythrobacter sp. strain NAP-1, does not possess these genes. The other organism that contains some ORFs for the 3-hydroxypropionate cycle, the γ-proteobacterium Congregibacter litoralis, has not been characterized so far; notably, its capability for autotrophic growth and assimilation of organic acids has not been characterized. Furthermore, it may be possible that Erythrobacter strain NAP-1 and/or the γ-proteobacterium Congregibacter litoralis recruits other enzymes for specific reactions required for the 3-hydroxypropionate cycle, as has been shown for the malonyl-CoA reductase from Metallosphaera sedula (2).

Comparison with other enzymes.

R-Citramalyl-CoA lyase from C. aurantiacus represents a new member of the 3-hydroxy-3-methyl-glutaryl-CoA lyase (HMG-CoA lyase) protein family catalyzing a Claisen-aldol condensation (Table 3 and Fig. 4 and 5). This class of enzymes also contains R-citramalate synthase, which catalyzes the irreversible condensation of acetyl-CoA and pyruvate and is involved in threonine-independent isoleucine biosynthesis of some archaeal and eukaryotic microorganisms (21, 40). Based on sequence alignments between lyases and synthases of members of this class, a consensus sequence for the lyases, Gly-Cys-Pro-Tyr-Ala-Pro, was identified, which is absent in the synthases (Fig. 4) (10). One can speculate that the cysteine of this so-called G-loop is required to maintain the CoA thioester bound during catalysis. Variants of the human HMG-CoA lyase in which the conserved cysteine was replaced by alanine or serine showed large diminutions in catalytic efficiencies, with K_m values for the substrate unchanged (32). A function for this cysteine as a general base was assigned based on the crystal structure of HMG-CoA lyase (10). All members of the class of HMG-CoA lyases contain a divalent metal in the active site. The activator cation ligands of the protein are two histidine residues, an aspartate residue, and an asparagine residue, which are also conserved for R-citramalyl-CoA lyase from C. aurantiacus (Fig. 4). Metal analysis of recombinant R-citramalyl-CoA lyase produced in E. coli indicated 0.5 mol Zn²⁺ per mol of native dimeric enzyme. However, activity of the enzyme was inhibited by the addition of Zn²⁺ and stimulated by Mn²⁺ or Mn²⁺, neither of which was detected by metal analysis. The metal content of the recombinant enzyme may reflect the relative concentrations of the metals in the medium in which E. coli was grown. A possible explanation of the observed features is that the enzyme contains two metal binding sites. One site contains tightly bound zinc and the second Mn²⁺ or Mn²⁺. Occupation of the second site with Mn²⁺ or Mn²⁺ leads to the stimulation of activity. Whether zinc is also required for catalysis was not determined, and binding assays are needed to clarify this point. HMG-CoA lyases are assumed to contain Mg²⁺, 4-hydroxy-2-oxovalerate aldolase Mn²⁺, transcarboxylase Co²⁺, and isopropylmalate synthase Zn²⁺ as catalytically active cations (10, 13, 25, 27).

TABLE 3.

Three enzyme classes and representative members catalyzing similar Claisen-aldol condensation reactions

Enzyme class	Enzyme	Substrate(s)	Product(s)
3-Hydroxy-3-methylglutaryl-CoA lyase family (COG 0119)	3-Hydroxy-3-methylglutaryl-CoA lyase (HMG-CoA lyase)	3-Hydroxy-3-methylglutaryl-CoA	Acetoacetate + acetyl-CoA
	Isopropylmalate synthase (LeuA)	α-Ketoisovalerate + acetyl-CoA	Isopropylmalate + CoA
	Homocitrate synthase (NifV)	α-Ketoglutarate + acetyl-CoA	Homocitrate + CoA
	4-Hydroxy-2-oxovalerate aldolase (DmpG)	4-Hydroxy-2-ketovalerate	Acetaldehyde + pyruvate
	Transcarboxylase 5S	Oxaloacetate	Pyruvate + enzyme-bound carboxylate (carbamylated lysine)
	R-Citramalate synthase (CimA)	Acetyl-CoA + pyruvate	R-Citramalate + CoA
	R-Citramalyl-CoA lyase (Ccl)	R-Citramalyl-CoA	Pyruvate + acetyl-CoA
Malyl-CoA lyase family (COG 2301)	Subunit of citrate lyase (CitE)	Citryl-S-R-ACP	Acetyl-S-R-ACP + oxaloacetate
	Malate synthase type A	Acetyl-CoA + glyoxylate	Malate + CoA
	Malate synthase type G	Acetyl-CoA + glyoxylate	Malate + CoA
	l-Malyl-CoA/β-methylmalyl-CoA/S-citramalyl-CoA lyase	Acetyl-CoA + glyoxylate, acetyl-CoA + pyruvate, and propionyl-CoA + glyoxylate	l-Malyl-CoA, S-citramalyl-CoA, and β-methylmalyl-CoA
Citrate synthase family (COG 0372)	ATP citrate lyase	Citrate + ATP + CoA	Acetyl-CoA + oxaloacetate + ADP + Pi
	Citryl-CoA lyase	Citryl-CoA	Acetyl-CoA + oxaloacetate
	Methylcitrate synthase	Propionyl-CoA + oxaloacetate	Methylcitrate + CoA
	Citrate synthase	Acetyl-CoA + oxaloacetate	Citrate + CoA

FIG. 4.

Sequence alignment of R-citramalyl-CoA lyase from Chloroflexus aurantiacus to different proteins of the same family. GenBank accession numbers are as follows: for Chloroflexus aurantiacus R-citramalyl-CoA lyase (Ccl), ZP_00768726; for Brucella melitensis 3-hydroxy-3-methylglutaryl-CoA lyase (HMG-CoA lyase), NP_540843; for Homo sapiens HMG-CoA lyase, AAA92733; for Rhodospirillum rubrum HMG-CoA lyase, AAB50182; for Bacillus subtilis HMG-CoA lyase, NP_389705; for Mycobacterium tuberculosis isopropylmalate synthase (LeuA), NP_218227; for Salmonella enterica serovar Typhimurium LeuA, AAL19077; for Leptospira interrogans LeuA, AAS70315; for Methanococcus jannaschii (R)-citramalate synthase (d-citramalate synthase) (CimA), Q58787; and for Azotobacter vinlandii homocitrate synthase (NifV), AAA64727. The numbers in parentheses at the start of some rows in the first sequence alignment block give the number of N-terminal residues in each protein that are omitted from this alignment. Strictly conserved residues are in bold. The circles indicate residues involved in the active site, and the triangle indicates an important residue in HMG-CoA lyases.

FIG. 5.

Similarity tree of homologues of R-citramalyl-CoA lyase (Ccl) from Chloroflexus aurantiacus based on amino acid sequences. The BLOSUM 62 matrix was used for the alignment. The PAM scale indicates the percentage of point-accepted mutations. GenBank accession numbers are as follows: for Methanococcus jannaschii (R)-citramalate synthase (CimA), Q58787; for Salmonella enterica serovar Typhimurium isopropylmalate synthase (LeuA), AAL19077; for Mycobacterium tuberculosis LeuA, NP_218227; for Leptospira interrogans LeuA, AAS70315; for Azotobacter vinelandii homocitrate synthase (NifV), AAA64727; for Bacillus subtilis 3-hydroxy-3-methylglutaryl-CoA lyase (HMG-CoA lyase), NP_389705; for Rhodospirillum rubrum HMG-CoA lyase, AAB50182; for Brucella melitensis HMG-CoA lyase, NP_540843; for Homo sapiens HMG-CoA lyase, AAA92733; and for Chloroflexus aurantiacus R-citramalyl-CoA lyase (Ccl), ZP_00768726.

In contrast to R-citramalyl-CoA lyase, l-malyl-CoA lyase/β-methylmalyl-CoA lyase from C. aurantiacus, which additionally catalyzes the reversible cleavage of S-citramalyl-CoA, belongs to the class of malyl-CoA lyases (Table 3) (14). Members of this class are also activated by divalent metal ions, usually Mg²⁺ or Mn²⁺. The crystal structure of malate synthase G, a member of this group that catalyzes the irreversible condensation of acetyl-CoA and glyoxylate, has been solved (20). Catalysis proceeds via formation of an enolate ion, as in the case of HMG-CoA lyase family enzymes. However, there is no sequence homology between these classes of enzymes. Even though they both use similar functional groups for catalysis, the active site arrangements are different, suggesting convergence from common chemistry (25). The genome of Clostridium tetani contains a gene annotated as citryl-CoA lyase. It is likely that this gene encodes S-citramalyl-CoA lyase, based on similar biochemical properties (5a).

A different catalytic strategy is used by the (si)-citrate synthase family of enzymes. This class of enzymes, in the absence of a metal cofactor, catalyzes the formation of a carbon-carbon bond via a neutral enol intermediate (23). ATP citrate lyases are chimeric proteins consisting of a succinyl-CoA synthetase domain and a citryl-CoA lyase domain (homologous to citrate synthase) in various primary structure arrangements (3). Members of this citrate synthase class are unrelated to R-citramalyl-CoA or S-citramalyl-CoA lyase from C. aurantiacus or to the other two Claisen-condensing enzyme classes mentioned above (Table 3); however, because all three catalyze similar types of reactions, it would not be surprising if, for example, a citramalyl-CoA lyase was identified within the family of (si)-citrate synthases. The genes coding for (re)-citrate synthases are not known.