Bacteria and growth conditions. C. aurantiacus
strain OK-70-fl (DSM 636) was grown anaerobically at 55°C and pH 8 in 12-liter glass fermentors under autotrophic or heterotrophic conditions as described earlier (17
). Cells were stored in liquid nitrogen until use. E. coli
strain XL1-Blue recA1 endA1 gyrA96 thi-1 hsdR17 supE44 relA1 lac
(F′ proAB lacIqZΔM15
]) was grown at 37°C in Luria-Bertani (LB) medium (34
). Ampicillin was added to E. coli
cultures to a final concentration of 50 μg/ml. Cell extracts were prepared aerobically as described previously (17
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).
Syntheses. (i) l-Malyl-CoA. l
-Malyl-CoA was chemically synthesized according to the methods of Eggerer and Grünewälder (10
), with slight modification. The synthesis intermediate l
-caprylcysteamine] was synthesized by Richard Krieger (Institut für Organische Chemie, Freiburg, Germany) as described elsewhere (10
-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).
(ii) Succinyl-CoA, acetyl-CoA, and propionyl-CoA.
Succinyl-CoA, acetyl-CoA, and propionyl-CoA were synthesized as described elsewhere (17
), and the dry powders were stored at −20°C.
C]propionyl-CoA was synthesized according to the protocol described previously for synthesis of [1,2-14
), using [2-14
C]propionate instead of [1,2-14
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 [2-14
C]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
]). The reaction was also used to determine the equilibrium constant keq
l-Malyl-CoA/β-methylmalyl-CoA lyase was tested at 55°C in the lyase or condensation direction.
(i) Lyase reaction.
-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
for glyoxylate phenylhydrazone = 17,000 M−1
]). 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.
(ii) Condensation reaction.
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 Km 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.
Formation of acetyl-CoA and β-methylmalyl-CoA from l-malyl-CoA and propionyl-CoA.
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).
Cloning and expression of a putative l-malyl-CoA lyase (mlcA) gene in E. coli XL1-Blue.
Standard protocols were used for preparation, cloning, transformation, amplification, and purification of DNA (3
). 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
-malyl-CoA lyase protein EC 18.104.22.168; 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 Pag
I restriction site) partially corresponding to nucleotides 7026 to 7045 of contig 965 of the genome database of C. aurantiacus
); and (ii) 5′-CTTGCTGCAGCGTCACAGACC-3′ (21-mer, primer contained Pst
I 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 Nco
I and Pst
I 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
Purification of recombinant l-malyl-CoA lyase from E. coli.
The purification was performed at 4°C, followed by measuring the l-malyl-CoA lyase reaction.
(i) Heat precipitation.
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.
(ii) DEAE-Sepharose fast flow chromatography.
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.
(iii) Size exclusion chromatography.
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.
Purification of β-methylmalyl-CoA lyase from C. aurantiacus.
The purification was performed at 4°C or room temperature and followed by measuring the condensation reaction of glyoxylate with propionyl-CoA.
(i) Heat precipitation.
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.
(ii) DEAE-Sepharose fast flow chromatography.
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.
(iii) Phenyl-Sepharose chromatography.
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.
(iv) Size exclusion chromatography.
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.
(v) Resource Q chromatography.
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.
Determination of molecular mass.
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.
Cell extracts were prepared as described previously (17
). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE; 12.5% polyacrylamide) was performed as described by Laemmli (26
). Proteins were stained with Coomassie blue according to the method of Zehr et al. (39
). Protein levels were determined by the method of Bradford (5
), using bovine serum albumin as the standard. Determination of the N-terminal amino acid sequence of the purified β-methylmalyl-CoA lyase protein from C. aurantiacus
after blotting on polyvinylidene difluoride membrane was performed by TopLab (Martinsried, Germany) using an Applied Biosystems Procise 492 sequencer (Weiterstadt, Germany). The phenylthiohydantoin derivatives were identified with an online Applied Biosystems Analyzer 140 C. Optical absorption spectra of the purified β-methylmalyl-CoA lyase from C. aurantiacus
extract (0.44 mg of protein ml−1
) and of the purified recombinant l
-malyl-CoA lyase from E. coli
(2.4 mg of protein ml−1
) were collected at room temperature using a Perkin Elmer Life Science Lambda 2S spectrometer and the same buffer as a blank.