Chemicals were obtained from Fluka (Neu-Ulm, Germany), Merck (Darmstadt, Germany), Sigma-Aldrich (Deisenhofen, Germany), or Roth (Karlsruhe, Germany). Biochemicals were purchased from Roche Diagnostics (Mannheim, Germany), Applichem (Darmstadt, Germany), or Gerbu (Craiberg, Germany). Materials and equipment for protein purification were obtained from Amersham Biosciences (Freiburg, Germany) or Millipore (Eschborn, Germany). Materials for cloning and expression were purchased from MBI Fermentas (St. Leon-Rot, Germany), MWG Biotech AG (Ebersberg, Germany), or Peqlab Biotechnologie (Erlangen, Germany). [2-¹⁴C]propionate was obtained from Hartmann Analytic (Braunschweig, Germany).

l-Malyl-CoA was chemically synthesized according to the methods of Eggerer and Grünewälder (10), with slight modification. The synthesis intermediate l-malylcaprylcysteamine [S-(β-hydroxysuccinyl)-N-caprylcysteamine] was synthesized by Richard Krieger (Institut für Organische Chemie, Freiburg, Germany) as described elsewhere (10, 28). l-Malyl-CoA was stored as a freeze-dried powder at −20°C. It contained 70% CoA-thioester and 30% free CoA as determined by high-performance liquid chromatography (HPLC) separation and detection at 260 nm (see below).

Succinyl-CoA, acetyl-CoA, and propionyl-CoA were synthesized as described elsewhere (17), and the dry powders were stored at −20°C.

[2-¹⁴C]propionyl-CoA was synthesized according to the protocol described previously for synthesis of [1,2-¹⁴C]acetyl-CoA (17), using [2-¹⁴C]propionate instead of [1,2-¹⁴C]acetate.

β-Methylmalyl-CoA was synthesized enzymatically from propionyl-CoA and glyoxylate using a preparation of recombinant l-malyl-CoA/β-methylmalyl-CoA lyase protein. A reaction mixture (50 ml) containing 200 mM morpholinepropanesulfonic acid (MOPS)-K⁺ buffer (pH 7.7), 2.5 mM glyoxylate, 2 mM MgCl₂, 2.5 mM propionyl-CoA, 36.7 kBq of [2-¹⁴C]propionyl-CoA as tracer, and 6.3 ml of recombinant l-malyl-CoA/β-methylmalyl-CoA lyase protein fraction (14.4 mg of protein; 2 U) was incubated at 55°C. After 60 min of incubation, the mixture was adjusted to pH 1.8 by addition of 1 M HCl. Protein was removed by centrifugation. The supernatant was extracted twice with diethyl ether to remove remaining glyoxylate. The volume of the supernatant was reduced to 5 ml by flash evaporation at 30°C (3 kPa) and subjected to a reversed-phase column (250 by 20 mm, 10 μm; Grom-Sil 120 ODS-4 HE; Crom, Herrenberg-Kayh, Germany), which was developed by a step gradient (64 ml each) of 2.9, 4.8, 5.7, 6.7, 8.6, and 10.5% acetonitrile (vol/vol) in 50 mM potassium phosphate buffer, pH 6.7, with a flow rate of 8 ml min⁻¹. The effluent was monitored by a radiomonitor and photometrically (260 nm). A radioactive fraction that eluted at a retention volume of 200 ml was collected and its pH was adjusted to pH 1.8 by addition of 1 M HCl. The acetonitrile was evaporated by flash evaporation at 30°C (2 kPa), the sample was lyophilized, and the powder was stored at −20°C. It contained 92% CoA-thioester and 8% free CoA as determined with 5,5′-dithiobis(2-nitrobenzoate) (Ellman's reagent) and detection at 412 nm (₄₁₂ = 13,600 M⁻¹ cm⁻¹ [9]). The reaction was also used to determine the equilibrium constant k_eq.

l-Malyl-CoA/β-methylmalyl-CoA lyase was tested at 55°C in the lyase or condensation direction.

The l-malyl-CoA- or β-methylmalyl-CoA-dependent formation of glyoxylate was monitored photometrically at 324 nm with phenylhydrazine in a continuous assay as described previously (15, 17) (₃₂₄ for glyoxylate phenylhydrazone = 17,000 M⁻¹ cm⁻¹ [33]). The assay mixture (0.5 ml) contained 200 mM MOPS-K⁺ buffer (pH 7.7), 4 mM MgCl₂, 3.5 mM phenylhydrazinium chloride, 1 mM l-malyl-CoA or β-methylmalyl-CoA, and protein. The reaction was started by addition of CoA-thioester. Phenylhydrazine had no effect on the stability of CoA derivatives.

The propionyl-CoA- or acetyl-CoA-dependent consumption of glyoxylate was monitored photometrically with phenylhydrazine in a discontinuous assay. The assay mixture (0.2 ml) contained 200 mM MOPS-K⁺ buffer [pH 7.7], 4 mM MgCl₂, 2 mM glyoxylate, 4 mM concentration of propionyl-CoA or acetyl-CoA, and protein. The reaction was started by the addition of protein. After 20 min of incubation, samples (25 μl) were retrieved, diluted, and cooled down to room temperature by addition of 0.975 ml of 200 mM MOPS-K⁺ buffer (pH 7.4) containing 3.5 mM phenylhydrazinium chloride. After 15 min of incubation at room temperature, the formed glyoxylate phenylhydrazone was detected at 324 nm. In additional experiments, glyoxylate was replaced by pyruvate or oxaloacetate (2 mM concentration each), and acetyl-CoA or propionyl-CoA were replaced by succinyl-CoA. When the apparent K_m values were to be determined, the concentration of the CoA-thioester or glyoxylate was varied between 0.05 and 20 mM; glyoxylate was either omitted (lyase reactions) or its concentration remained fixed at 2 mM, and the samples (25 μl) were developed after 5 min of incubation at 55°C (condensation reaction). Buffers used to determine the pH optimum were 200 mM MOPS-K⁺, pH 6.0 to 8.9, at room temperature, which corresponded to pH 5.7 to 8.6 at 55°C; the ΔpH of 0.3 was experimentally determined. The dependence of the reaction on divalent metal ions was investigated by addition of 1 mM EDTA to the reaction mixture in the absence of a divalent cation. The temperature optimum of the l-malyl-CoA lyase reaction was determined by varying the reaction temperature between 45 and 75°C.

The test mixture (0.2 ml) contained 200 mM MOPS-K⁺ buffer (pH 7.7), 4 mM MgCl₂, 2.5 mM l-malyl-CoA, 0.75 mM CoA (present as an impurity of l-malyl-CoA), 2.5 mM propionyl-CoA, and 24 μg of purified recombinant l-malyl-CoA lyase. l-Malyl-CoA was omitted in a control experiment. The addition of recombinant enzyme started the reactions. Samples of 50 and 150 μl were taken after 0 and 20 min of incubation at 55°C, and the reaction was stopped by addition of 10 and 30 μl of 1 M HCl, respectively. Protein was removed by centrifugation, and samples were analyzed for glyoxylate and for CoA thioesters by HPLC. A reversed-phase column (LiChrospher 100; end-capped; 5 μm, 125 by 4 mm; Merck, Darmstadt, Germany) was used for separation of CoA-thioesters. A gradient from 2 to 10% acetonitrile in 200 mM sodium phosphate buffer, pH 4.8, with a flow rate of 1 ml min⁻¹ over 40 min was used. CoA-thioesters were detected at 260 nm. Retention times were 2 min (free organic acids), 8 min (l-malyl-CoA), 10 min (β-methylmalyl-CoA; free CoA), 16 min (acetyl-CoA), and 24 min (propionyl-CoA).

Standard protocols were used for preparation, cloning, transformation, amplification, and purification of DNA (3, 34). Plasmid DNA preparation was performed according to the method of Birnboim and Doly (4). On the basis of a DNA and protein sequence alignment of the mlcA gene (l-malyl-CoA lyase protein EC 4.1.3.24; accession number AAB58884) from Methylobacterium extorquens, a region on the C. aurantiacus genome (contig 965) showed one highly conserved putative l-malyl-CoA lyase gene. Two oligonucleotides were designed: (i) 5′-GAGCATCATGAAGGGTATTC-3′ (20-mer; primer contained PagI restriction site) partially corresponding to nucleotides 7026 to 7045 of contig 965 of the genome database of C. aurantiacus (http://www.jgi.doe.gov/JGI_microbial/html/chloroflexus/chloro_homepage.html); and (ii) 5′-CTTGCTGCAGCGTCACAGACC-3′ (21-mer, primer contained PstI restriction site) partially corresponding to nucleotides 9351 to 9372. These primers were used in a PCR containing 0.5 μg of chromosomal DNA of C. aurantiacus and 2.5 U of Pwo polymerase (Peqlab Biotechnologie), and the Peqlab DNA amplification kit was used. An annealing temperature of 60°C was used to amplify a 2,347-bp genomic region which contained the putative mlcA gene. The PCR product was purified and cloned into pTrc 99A (accession number U13872; Amersham Biosciences) by using the NcoI and PstI restriction sites of the multiple cloning site. Plasmid PCR and colony PCR confirmed the correct integration of the PCR fragment into the vector. The recombinant plasmid was transformed into E. coli XL1-Blue, and the expression of the putative mlcA gene was induced at an optical density at 578 nm of 0.7 (12-liter fermentor, 37°C) by addition of 0.3 mM isopropyl-β-d-thiogalactopyranoside to the LB-ampicillin medium. After induction, the culture was incubated for 4 h at room temperature. Cells were harvested at 4°C and stored in liquid nitrogen prior to purification of recombinant protein. Transformed E. coli XL1-Blue cells containing the pTrc-vector but no PCR fragment served as a control for the expression of the putative mlcA gene.

The BLAST program was used to search protein databases at the National Center for Biotechnology Information (Bethesda, Md.) and the local C. aurantiacus server (http://spider.jgi-psf.org/JGI_microbial/bin/psf_blast?PROJEKT_ID = 2351478) at the Department of Energy (DOE) Joint Genome Institute (Walnut Creek, Calif.).

The purification was performed at 4°C, followed by measuring the l-malyl-CoA lyase reaction.

Cell extract (100,000 × g supernatant) from 25 g of cells (wet weight) was incubated at 65°C for 20 min to precipitate unwanted protein from E. coli cells, followed by ultracentrifugation (100,000 × g) at 4°C for 60 min.

The supernatant after heat precipitation (30 ml) was applied to a 50-ml DEAE-Sepharose Fast Flow column (Amersham Biosciences; flow rate, 3 ml min⁻¹) which had been equilibrated with 20 mM MOPS-K⁺ buffer (pH 7.2) containing 10% (vol/vol) glycerol (buffer A). The column was washed with four bed volumes of buffer A and three bed volumes of buffer A containing 100 mM KCl and developed with a linear gradient from buffer A to buffer A plus 200 mM KCl over 250 ml. Active fractions (120 to 180 mM KCl) were pooled (150 ml) and stored at −80°C.

The volume of the active pool obtained from DEAE-Sepharose chromatography was reduced to 4 ml by ultrafiltration (Amicon YM 10 membrane; Millipore) and applied to a 120-ml HiLoad Superdex 200 16/60 column (Amersham Biosciences; flow rate, 1 ml min⁻¹). The column was developed with 20 mM MOPS-K⁺ buffer (pH 7.6) containing 10% (vol/vol) glycerol and 150 mM KCl. The active protein eluted with a retention volume of 59 to 76 ml, and active fractions were pooled (18 ml) and stored at −80°C.

The purification was performed at 4°C or room temperature and followed by measuring the condensation reaction of glyoxylate with propionyl-CoA.

Cell extract (100,000 × g supernatant) from 25 g of cell mass (wet weight) of autotrophically grown C. aurantiacus was incubated at 65°C for 10 min to precipitate unwanted protein, carotenoids, and other colored compounds, followed by ultracentrifugation (100,000 × g) at 4°C for 30 min.

The supernatant after heat precipitation (30 ml) was applied to a 50-ml DEAE-Sepharose Fast Flow column (Amersham Biosciences; flow rate, 3 ml min⁻¹) which had been equilibrated with 20 mM M-K⁺ buffer, pH 7.4, containing 10% (vol/vol) glycerol (buffer B). The column was washed with two bed volumes of buffer B and three bed volumes of buffer B containing 100 mM KCl and developed with a linear gradient from buffer B to buffer B plus 200 mM KCl over 250 ml. Active fractions (100 to 160 mM KCl) were pooled (94 ml) and stored at −80°C.

The DEAE fraction was adjusted to a final concentration of 1 M ammonium sulfate by using a 2 M ammonium sulfate solution. The protein fraction was then centrifuged and the supernatant was directly applied to a 20-ml phenyl-Sepharose column (Amersham Biosciences; flow rate, 1 ml min⁻¹). The column had been equilibrated with 20 mM MOPS-K⁺ buffer (pH 7.9) containing 1 M (NH₄)₂SO₄ and 10% (vol/vol) glycerol. After washing the column with one bed volume of this buffer, the column was developed with 120 ml of a decreasing linear gradient of 1 to 0 M ammonium sulfate, at a flow rate 1 ml min⁻¹. The activity eluted with 500 to 250 mM salt and the pooled fractions (35 ml) were concentrated and desalted immediately by ultrafiltration to a final volume of 2.2 ml.

The sample obtained from phenyl-Sepharose chromatography and ultrafiltration was applied to a 120-ml HiLoad Superdex 200 16/60 column, with a flow rate of 1 ml min⁻¹. The column was developed with 20 mM MOPS-K⁺ buffer (pH 7.6) containing 10% (vol/vol) glycerol and 150 mM KCl. The active protein eluted with a retention volume of 62 to 72 ml, and fractions were pooled (11 ml) and stored at −80°C.

The sample obtained from size exclusion chromatography was reduced to 1.9 ml and desalted by ultrafiltration using 20 mM MOPS-K⁺ buffer (pH 7.6) containing 10% glycerol (vol/vol) (buffer C). The protein solution was applied in two runs each onto a 1-ml Resource Q column (Amersham Biosciences; flow rate, 5 ml min⁻¹) which had been equilibrated with buffer C. The column was washed with two bed volumes of buffer C and developed with a gradient from buffer C to buffer C plus 1 M KCl over 20 ml. Active fractions (130 to 250 mM KCl) were pooled (4 ml) and stored at −20°C.

The native molecular masses of the purified recombinant protein and the purified protein from C. aurantiacus were estimated using a 120-ml HiLoad Superdex 200 16/60 column (Amersham Biosciences; flow rate, 1 ml min⁻¹). The column was developed with 20 mM MOPS-K⁺ buffer (pH 7.6) containing 10% (vol/vol) glycerol and 150 mM KCl and calibrated with the following molecular mass standards: ferritin (450 kDa), bovine serum albumin (67 kDa), and ovalbumin (45 kDa). A native gel (8% polyacrylamide) was performed to obtain further information about the molecular masses of the proteins. Bovine serum albumin served as a molecular standard as follows: monomer, 67 kDa; dimer, 134 kDa; trimer, 201 kDa; tetramer, 268 kDa.

The recombinant l-malyl-CoA lyase was purified in two steps from heat-treated extract of 25 g of E. coli cells with a yield of 48% (Table ​(Table1).1). The specific lyase activity at 55°C was 2.3 μmol of l-malyl-CoA cleaved min⁻¹ mg of protein⁻¹. The ratio of lyase activity/condensation activity remained virtually unchanged during purification. SDS-PAGE showed a 38-kDa protein and three additional very faint protein bands (<5%) (Fig. ​(Fig.3A)3A) which were also present in E. coli cells lacking the DNA insert (data not shown). The properties of the recombinant enzyme are summarized in Table ​Table2.2. The protein was colorless and exhibited an absorption maximum near 280 nm, indicating that it contained no UV-visible light-absorbing cofactor. Determination of the native molecular mass by size exclusion chromatography indicated a native molecular mass of approximately 210 ± 20 kDa. In native PAGE gels, the protein migrated with an estimated molecular mass of ≥210 kDa (Fig. ​(Fig.3B).3B). This indicates that the native enzyme has an α₅ or α₆ composition. The catalytic number (55°C, pH 7.1) for l-malyl-CoA consumption was 8.8 s⁻¹, assuming an α₆ native enzyme of the calculated 228 kDa, and the apparent K_m for l-malyl-CoA was 64 μM. The optimal pH for enzyme activity at 55°C was 7.1 (corresponding to 7.4 at room temperature). The enzyme was stable at room temperature for days and could be stored at −80°C. Enzyme activity required the presence of Mg²⁺, which could partially be replaced by Mn²⁺ or Co²⁺. Consequently, complexing agents such as EDTA completely inhibited the enzyme, and activity could be restored by adding excess Mg²⁺. Pyruvate was not condensed with acetyl-CoA, whereas the rate of oxaloacetate condensation was 40% of the rate observed with glyoxylate; the product of that reaction may be citryl-CoA. The optimal temperature for activity was 70°C, as determined in the continuous spectrophotometric lyase reaction assay using l-malyl-CoA as substrate.

Extract of autotrophically grown C. aurantiacus cells contained a propionyl-CoA-dependent glyoxylate-consuming activity of 0.017 μmol min⁻¹ mg of protein⁻¹, which was down-regulated in heterotrophically grown cells by a factor of 4. This β-methylmalyl-CoA lyase was purified 30-fold from 25 g of autotrophically grown cells of C. aurantiacus in five steps, with a yield of 7% (Table ​(Table3).3). SDS-PAGE revealed one strong band corresponding to 38 kDa and two additional protein bands (Fig. ​(Fig.4).4). The UV-visible spectrum, native molecular mass (Fig. ​(Fig.3B),3B), and enzyme stability properties were indistinguishable from those of recombinant l-malyl-CoA lyase. β-Methylmalyl-CoA synthesis required glyoxylate and propionyl-CoA, the reaction depended on Mg²⁺, and EDTA reversibly inhibited the enzyme (Table ​(Table2).2). The specific condensation activity was 0.51 μmol min⁻¹ mg of protein⁻¹ (55°C, pH 7.4), corresponding to a catalytic number of 1.9 s⁻¹ (assuming a 228-kDa α₆ holoenzyme). The determined N-terminal amino acid sequence (MRKLAHNFYK) was identical to the deduced N-terminal sequence of recombinant l-malyl-CoA lyase, and the apparent K_m value for l-malyl-CoA (57 μM) was similar to that of recombinant l-malyl-CoA lyase. The apparent K_m values for propionyl-CoA and acetyl-CoA were 1.8 and 0.5 mM, respectively, and 2.0 mM for glyoxylate. In the condensation reaction with propionyl-CoA, pyruvate was inactive, whereas oxaloacetate was transformed at 14% of the rate observed with glyoxylate; the product of that reaction may be methylcitryl-CoA.

Contig 965 of the C. aurantiacus genome (Fig. ​(Fig.2)2) contains some other open reading frames (ORFs), besides the one representing the l-malyl-CoA lyase gene studied here (mclA), that may play a role in autotrophic carbon metabolism. Three ORFs (ytf1, ytf2, and ytf3) likely represent succinyl-CoA-dependent CoA transferases, and ORF ytf3 is possibly succinyl-CoA:l-malate CoA transferase (for a review, see reference 14). Unfortunately, expression of ORF ytf3 in E. coli did not result in formation of a functional protein and the expected CoA transferase activity could not be shown (unpublished results). Searching for enzymes catalyzing Claisen-type condensation or retrocondensation reactions revealed an ORF on contig 967, which is similar to 3-hydroxy-3-methylglutaryl-CoA lyase (EC 4.1.3.4) and is next to another putative CoA transferase. We are currently testing the possibility that these two ORFs actually represent citramalyl-CoA lyase and succinyl-CoA:citramalate CoA transferase, respectively. These two enzymes have been postulated to play a role in the glyoxylate assimilation cycle (Fig. ​(Fig.1,1, reactions 13 and 14).