Eur. J. Biochem. 267, 3874±3884 (2000) q FEBS 2000
The involvement of coenzyme A esters in the dehydration of (R )-phenyllactate to (E )-cinnamate by Clostridium sporogenes Sandra Dickert1, Antonio J. Pierik1, Dietmar Linder2 and Wolfgang Buckel1 1
Laboratorium fuÈr Mikrobiologie, Fachbereich Biologie, Philipps-UniversitaÈt, Marburg, Germany; 2Fachbereich Humanmedizin, Justus von Liebig-UniversitaÈt, Gieûen, Germany
Phenyllactate dehydratase from Clostridium sporogenes grown anaerobically on l-phenylalanine catalyses the reversible syn-dehydration of (R)-phenyllactate to (E )-cinnamate. Purification yielded a heterotrimeric enzyme complex (130 ^ 15 kDa) composed of FldA (46 kDa), FldB (43 kDa) and FldC (40 kDa). By re-chromatography on Q-Sepharose, the major part of FldA could be separated and identified as oxygen insensitive cinnamoyl-CoA:phenyllactate CoA-transferase, whereas the transferase depleted trimeric complex retained oxygen sensitive phenyllactate dehydratase activity and contained about one [4Fe-4S] cluster. The dehydratase activity required 10 mm FAD, 0.4 mm ATP, 2.5 mm MgCl2, 0.1 mm NADH, 5 mm cinnamoyl-CoA and small amounts of cell-free extract (10 mg protein per mL) similar to that known for 2-hydroxyglutaryl-CoA dehydratase from Acidaminococcus fermentans. The N-terminus of the homogenous FldA (39 amino acids) is homologous to that of CaiB (39% sequence identity) involved in carnitine metabolism in Escherichia coli. Both enzymes are members of an emerging group of CoA-transferases which exhibit high substrate specificity but apparently do not form enzyme CoA-ester intermediates. It is concluded that dehydration of (R)-phenyllactate to (E )-cinnamate proceeds in two steps, a CoA-transfer from cinnamoyl-CoA to phenyllactate, catalysed by FldA, followed by the dehydration of phenyllactyl-CoA, catalysed by FldB and FldC, whereby the noncovalently bound prosthetic group cinnamoyl-CoA is regenerated. This demonstrates the necessity of a 2-hydroxyacyl-CoA intermediate in the dehydration of 2-hydroxyacids. The transient CoA-ester formation during the dehydration of phenyllactate resembles that during citrate cleavage catalysed by bacterial citrate lyase, which contain a derivative of acetyl-CoA covalently bound to an acyl-carrier-protein (ACP). Keywords: phenyllactate dehydratase; CoA-transferase; phenyllactyl-CoA; cinnamoyl-CoA; phenylalanine metabolism. Several clostridia ferment l-phenylalanine either as single amino acid or as a partner of a Stickland type reaction to ammonia, CO2, phenylacetate and 3-phenylpropionate [1±4]. The pathway has been elucidated by Helmut Simon and his coworkers in Clostridium sporogenes (clostridial cluster I [5]). In this strict anaerobic bacterium the amino group of Correspondence to W. Buckel, Laboratorium fuÈr Mikrobiologie, Fachbereich Biologie, Philipps-UniversitaÈt, D-35032 Marburg, Germany. Fax: 1 49 64212828979, Tel.: 1 49 64212821527, E-mail:
[email protected] Abbreviations: FMN, riboflavin-5 0 -phosphate; PVDF, poly(vinylidene difluoride); FldA, cinnamoyl-CoA:(R)-phenyllactate CoA-transferase; DTNB, 5,5 0 -dithiobis-(2-nitrobenzoate); FldH, phenyllactate dehydrogenase; ACP, acyl carrier protein. Enzymes: Acyl-CoA dehydrogenase (EC 1.3.99.2), aromatic amino acid aminotransferase (EC 2.6.1.57), carnitine dehydratase (EC 4.2.1.89) 2-enoate reductase/cinnamate reductase (EC 1.3.1.31), glutaconate CoA-transferase (EC 2.8.3.12), glutamate dehydrogenase (EC 1.4.1.2), histidine ammonia-lyase (EC 4.3.1.3), 2-hydroxyglutaryl-CoA dehydratase (EC 4.2.1.-), d-2-hydroxyisocaproate dehydrogenase (EC 1.1.1.-), phenylalanine ammonia-lyase (EC 4.3.1.5), phenylpyruvate:ferredoxin 2-oxidoreductase (CoA-phenylacetylating; EC 1.2.7.-), phenylacetate kinase (EC 2.7.2.-), phenyllactate dehydratase (EC 4.2.1.-), phenyllactate dehydrogenase (EC 1.1.1.-), phenyllactate CoA-transferase/cinnamoylCoA:phenyllactate CoA-transferase (EC 2.8.3.-), phosphate phenylacetyltransferase (EC 2.3.1.-), serine-hydroxymethyltransferase (EC 2.1.2.1). (Received 23 February 2000, accepted 25 April 2000)
phenylalanine is transferred to 2-oxoglutarate and the resulting phenylpyruvate is reduced to (R)-phenyllactate, followed by dehydration to (E )-cinnamate and a second reduction to phenylpropionate. Both reductive steps are catalysed by NAD1-dependent dehydrogenases (R)-phenyllactate dehydrogenase and 2-enoate (cinnamate) reductase [4]. The latter enzyme has been purified and extensively characterized from Clostridium tyrobutyricum. In contrast to acyl-CoA dehydrogenases, the substrate is the carboxylate rather than the CoA-ester [6]. In the oxidative branch of the pathway, phenylpyruvate is converted to phenylacetyl-CoA, from which phenylacetate and ATP are formed via substrate level phosphorylation (Fig. 1). The reversible syn-dehydration of (R)-phenyllactate to (E )-cinnamate [3] is the mechanistically most demanding step of the pathway of phenylalanine fermentation. This elimination of water requires the cleavage of a not activated C-H-bond in the b-position and the removal of the a-hydroxy group adjacent to the electron withdrawing carboxylate. Nature has two solutions to this chemical problem, either activation of the b-hydrogen by electrophilic catalysis as shown for the anti elimination of ammonia mediated by phenylalanine ammonialyase, or activation of the a-hydroxyl group by nucleophilic catalysis as proposed for the syn elimination of water from (R)-2-hydroxyglutaryl-CoA to (E )-glutaconyl-CoA in the strict anaerobic bacterium Acidaminococcus fermentans [7]. In phenylalanine ammonia-lyase [8] and histidine ammonia-lyase [9], a novel prosthetic group derived from an alanine-serine-glycine
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Clostridial phenyllactate dehydratase (Eur. J. Biochem. 267) 3875
Fig. 1. Fermentation of l-phenylalanine. (1) Aromatic amino acid aminotransferase (EC 2.6.1.57). (2) Phenyllactate dehydrogenase (EC 1.1.1.-). (3) Phenyllactate dehydratase. (4) Cinnamate reductase (EC 1.3.1.31). (5) Glutamate dehydrogenase (EC 1.4.1.2); Glu, glutamate; 2-OG, 2-oxoglutarate. (6) Phenylpyruvate:ferredoxin 2-oxidoreductase (CoA-acetylating) (EC 1.2.7.-). (7) Phosphate phenylacetyltransferase (EC 2.3.1.-) and phenylacetate kinase (EC 2.7.2.-). DG 8 0 has been calculated after Thauer et al. [40] using the data for alanine instead of phenylalanine.
sequence motif attacks the aromatic ring as an electrophile in a Friedel-Crafts-type reaction. Thereby the b-hydrogen becomes acidic, whereas the carboxylate seems not to be important for catalysis. The substrate for 2-hydroxyglutaryl-CoA dehydratase, however, is the CoA-ester of the carboxylate adjacent to the a-hydroxyl group. It has been proposed that the electrophilic thiolester carbonyl undergoes an Umpolung by transient reduction to a ketyl radical anion, which acts as nucleophile and expels the adjacent hydroxyl group [11,12]. Although the stereochemical course of the syn-dehydration of (R)-phenyllactate [3] and experiments in cell-free extracts from C. sporogenes using (R)-phenyllactyl-CoA and (E )-cinnamoyl-CoA as substrates [12] favour the nucleophilic mechanism, a more definite answer requires the purification of the involved enzymes, which will be described in this work.
M AT E R I A L S A N D M E T H O D S Chemicals (R)-Phenyllactic acid [(R)-2-hydroxy-3-phenylpropionic acid] (S )-phenyllactic acid and 5-phenylvaleric acid were purchased from Fluka (Buchs, Switzerland); 2-phenylacetic acid, 4-phenylbutyric acid, 4-hydroxyphenylacetic acid and 3-(4hydroxyphenyl)-propionic acid were from Merck (Darmstadt, Germany); coenzyme A dilithium salt dihydrate was from ICN (Eschwege, Germany); (E )-cinnamic acid, 3-phenylpropionic acid (E )-cinnamoylimidazole and all other biochemicals were from Sigma (Deisenhofen, Germany). Experiments under anaerobic conditions For work with the oxygen sensitive enzymes, phenyllactate dehydratase and cinnamate reductase, an anaerobic chamber from Coy Laboratories (Ann Arbor, MI, USA) with a nitrogen atmosphere containing 5% H2 at 20 8C was used. Oxygen-free buffers for enzyme purification were prepared by boiling and cooling under vacuum. Afterwards the buffers were flushed with nitrogen, transferred to the anaerobic chamber, where 2 mm dithiothreitol was added under stirring which was continued overnight. Activities of oxygen sensitive enzymes were determined with an Ultrospec 4000 spectrophotometer
from Pharmacia Biotech (Freiburg, Germany) located inside the anaerobic chamber. Synthesis of CoA-esters (E )-Cinnamoyl-CoA was synthesized directly from cinnamoylimidazole following the method of Kawaguchi [14]. (R)-Phenyllactyl-CoA and phenylpropionyl-CoA were prepared from cinnamoyl-CoA and the corresponding free acids using the here described CoA-transferase FldA. All CoA-derivatives were purified by HPLC. HPLC The HPLC system was from Sykam (Gilching, Germany). Metabolites derived from l-phenylalanine were analysed on a LiChrospher 100 RP-8 (5 mm), 125±4 column from Merck (Darmstadt, Germany). A linear gradient from 5% to 20% (v/v) acetonitrile in 0.1% (v/v) trifluoroacetic acid within 10 min and up to 60% (v/v) acetonitrile in 0.1% (v/v) trifluoroacetic acid within 17 min was applied at a flow rate of 1 mL´min21. Phenylalanine typically eluted at 3.8 min, phenylpyruvate at 5.8 min, phenyllactate at 7.0 min, phenylacetate at 9.2 min, phenylpropionate at 13.3 min and cinnamate at 14.8 min. The compounds were detected by their absorbance at 210 nm. CoA-esters were purified on a LiChrosorb RP18, 7 mm, Hibar 250±10 column from Merck using a linear gradient from 15 to 40% acetonitrile in 0.1% trifluoroacetic acid at 5 mL´min21; the absorbance was measured at 260 nm (R)-phenyllactyl-CoA typically eluted at 5.5 min, phenylpropionyl-CoA at 10.2 min and cinnamoyl-CoA at 11.8 min. The CoA-esters were lyophilized and stored in small portions at 2 20 8C, which were thawed just before use. Purity was determined by UV-visible spectroscopy with a Uvikon 943 double-beam spectrophotometer (Kontron, MuÈnchen, Germany). All CoA-esters showed the characteristic absorbance maximum at l 254 nm. For cinnamoyl-CoA, which had an additional maximum at 307 nm, the 260 nm/307 nm absorbance ratio was determined as 0.94. Although D'Ordine [14] found the second maximum at 309 nm (: 18.7 mm21´cm21), the 260/309 absorbance ratio was identical. Therefore the extinction
3876 S. Dickert et al. (Eur. J. Biochem. 267)
coefficient of D'Ordine was used for calculation of enzyme activities at 309 nm. CoA-esters and the free carboxylates were analysed on a LiChrospher 100 RP-18 (5 mm), 250±4 column from Merck. A linear gradient from 15% to 40% (v/v) acetonitrile in 0.1% (v/v) trifluoroacetic acid within 15 min and up to 100% (v/v) acetonitrile in 0.1% (v/v) trifluoroacetic acid within 1 min was applied at a flow rate of 1 mL´min21. (R)-PhenyllactylCoA typically eluted at 5.0 min (R)-phenyllactate at 7.9 min (E )-cinnamoyl-CoA at 13.2 min, phenylpropionate at 14.5 min and (E )-cinnamate at 15.4 min. The compounds were detected by their absorbance at 210 nm. Cell growth Clostridium sporogenes ATCC 3584 was obtained from the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ, Braunschweig, Germany). The organism was grown anaerobically at 37 8C for 16 h in a broth containing 60 mm l-phenylalanine, 0.5% yeast extract, 0.1% sodium thioglycolate, 10 mm potassium phosphate pH 7.2, 2 mm biotin, 1000-fold diluted trace element solution SL10 [15], 60 nm Na2SeO3 and 300 nm Na2WO4 . Determination of enzyme activity Cinnamoyl-CoA: phenyllactate CoA-transferase was measured aerobically in 50 mm potassium phosphate pH 7.0 and 15 mm (E )-cinnamoyl-CoA in a total volume of 1.0 mL at 25 8C. After addition of the enzyme the reaction was initiated with 1.5 mm 3-phenylpropionate. The decrease of the absorbance of cinnamoyl-CoA was measured at 309 nm, : 18.7 mm21´cm21 [14]. The unit of enzymatic activity (U) is defined as conversion of 1 mmol substrate per min. The back reaction was measured under the same conditions with 15 mm phenyllactyl-CoA and 1.5 mm cinnamate. Phenyllactate dehydratase was measured anaerobically in 50 mm Tris/HCl pH 8, 2.5 mm MgCl2, 0.4 mm ATP, 0.1 mm NADH, 10 mm FAD, 5 mm cinnamoyl-CoA, cell-free extract (5±20 mg protein) and approximately 0.1 U partially purified cinnamate reductase (eluate from phenyl-Sepharose) as auxiliary enzyme in a total volume of 1.0 mL at 20 8C. After addition of the dehydratase the assay was started with 1 mm (R)-phenyllactate and after a lag phase of up to 20 min the decrease of the absorbance of NADH was followed at 340 nm, : 6.2 mm21´cm21. The phenyllactate dehydratase activity of the cell-free extract was determined in parallel and was subtracted from the activity of the purified dehydratase. Phenyllactyl-CoA dehydratase was measured anaerobically in 50 mm Tris/HCl pH 8, 2.5 mm MgCl2, 0.4 mm ATP, 10 mm FAD, 20 mm (R)-phenyllactyl-CoA and cell-free extract (1 mg protein) in a total volume of 1.0 mL. After addition of the enzyme the assay was incubated at 20 8C for 30 min. For analysis of the products, the incubations were acidified to pH 2±3 by addition of trifluoroacetic acid (0.5%) and the denatured protein was removed by centrifugation. The supernatants were subjected to HPLC (see above). Cinnamate reductase was measured anaerobically in 50 mm Tris/HCl pH 8.0, 0.1 mm NADH and 0.5 mm cinnamate in a total volume of 1.0 mL at 20 8C. After addition of enzyme the decrease of absorbance was followed at 340 nm. Phenyllactate dehydrogenase was measured aerobically in 50 mm Tris/HCl pH 8.0 and 0.1 mm NADH in a total volume of 1.0 mL at 25 8C. After addition of the enzyme, the reaction was started with 0.1 mm phenylpyruvate; the decrease of absorbance was followed at 340 nm.
q FEBS 2000
Purification of the enzyme complex phenyllactate dehydratase Wet packed cells of C. sporogenes were suspended in a buffer containing 20 mm Mops pH 7.5, 5 mm ATP, 5 mm MgCl2, 2 mm dithiothreitol, 1±2 mm dithionite and 10 mm FAD. Cells were broken by sonification (Branson Sonifier, Emerson Technologies, Dietzenbach, Germany). Cell walls and membranes were removed by ultracentrifugation at 90 000 g for 45 min and filtering through a 0.45-mm filter. An equal volume of 2.0 m (NH4)2SO4 solution was added to the cell-free extract, which was then loaded on a phenyl-Sepharose column (3 10 cm) equilibrated with 1.0 m (NH4)2SO4 solution in 20 mm Mops pH 7.5. After washing the column with 80 mL equilibration buffer, the enzyme complex was eluted at a rate of 4 mL min21 with a step gradient to 0.85 m (NH4)2SO4 directly followed by a linear gradient of 0.85±0.6 m (NH4)2SO4 over 220 mL, both in 20 mm Mops pH 7.5. The enzyme complex eluted around 0.8 m (NH4)2SO4. After concentrating with an Amicon PM 30 cell (Millipore, Eschborn, Germany) and desalting with 20 mm Mops pH 7.5, the enzyme was pumped on a Q-Sepharose column (1.8 10 cm) equilibrated with 20 mm Mops, pH 7.5. After a washing step with 30 mL 20 mm Mops pH 7.5, the enzyme complex eluted around 0.3 m NaCl with a linear gradient from 0.2 to 0.4 m NaCl in 20 mm Mops pH 7.5 in 250 mL at a rate of 2 mL´min21. After concentration by ultrafiltration (Amicon PM 30) the enzyme was chromatographed on a Superose 12 column (2 48 cm) in 0.15 m NaCl 1 20 mm Mops pH 7.5 at 0.5 mL´min21. The purified enzyme complex was desalted by ultrafiltration and transferred to the same Q-Sepharose column as used above. A less steep linear gradient from 0.2 to 0.35 m NaCl in 20 mm Mops pH 7.5 in 250 mL was applied at a rate of 2 mL´min21. Purification of cinnamate reductase Wet packed cells of C. sporogenes were suspended in 100 mm potassium phosphate pH 7 which contained 2 mm dithiothreitol. The suspension was treated as described above. The filtered 90 000 g supernatant was pumped on a DEAE/Sepharose (3 10 cm) equilibrated with 50 mm potassium phosphate pH 7.0. After washing the column with 80 mL equilibration buffer, a linear gradient up to 1.0 m (NH4)2SO4 in 50 mm sodium phosphate pH 7 over 600 mL was applied at a rate of 4 mL´min21. The reductase eluted around 0.25 m (NH4)2SO4 immediately followed by phenyllactate dehydrogenase (see below). An equal amount of 2.0 m (NH4)2SO4 solution was added to the protein solution, which was then loaded on a phenyl-Sepharose column (3 10 cm) equilibrated with 1.0 m (NH4)2SO4 solution in 50 mm potassium phosphate pH 7. After washing the column with 80 mL equilibration buffer, a linear gradient over 220 mL down to 20 mm Mops pH 7.5 without (NH4)2SO4 was applied at a rate of 4 mL´min21 . The reductase eluted at buffer without (NH4)2SO4 and was loaded directly on a Mono-Q column (1.4 10 cm) equilibrated with 50 mm potassium phosphate pH 7. The reductase eluted isocratically upon 10 mL equilibration buffer. Purification of phenyllactate dehydrogenase The fraction from the DEAE / Sepharose chromatography described above, which contained phenyllactate dehydrogenase activity, was further purified by phenyl-Sepharose analogous to the purification of the reductase; it eluted at 0.45 m (NH4)2SO4. The enzyme was desalted, concentrated by ultrafiltration
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Clostridial phenyllactate dehydratase (Eur. J. Biochem. 267) 3877
(Amicon PM 30) and transferred to a Mono-Q column equilibrated with 50 mm potassium phosphate pH 7.0. After a 20-mL washing step with the equilibration buffer the dehydrogenase (38 kDa) eluted at 0.3 m NaCl together with a 42-kDa protein by applying a linear gradient over 50 mL up to 0.5 m NaCl in equilibration buffer. The two proteins comigrated on native PAGE (data not shown). Determination of the molecular mass
Fig. 2. Analysis of the purified phenyllactate dehydratase (FldABC). SDS/PAGE (10%): phenyllactate dehydratase FldABC (3 mg protein), lane 1; molecular mass marker; lane 2.
Apparent molecular masses of enzymes were determined by gel filtration on a Superose 12 column (2 48 cm) in 0.15 m NaCl with 20 mm Mops, pH 7.5 at a flow rate of 0.5 mL´min21. Cytochrome c, chymotrypsinogen, hen egg albumin, bovine serum albumin, aldolase, catalase and ferritin were used for calibration. The molecular mass standards were obtained from Roche Molecular Biochemicals (Mannheim, Germany). The molecular mass of the transferase depleted trimer was also determined via density gradient centrifugation in a glycerol gradient from 10 to 35% (v/v). The enzyme (15 mg) was centrifuged at 220 000 g for 20 h at 4 8C in a swing-out rotor (TST 60.4, Sorvall, Bad Homburg, Germany). In parallel, hen egg albumin (43 kDa), bovine serum albumin (68 kDa) and catalase (240 kDa) were centrifuged as standards. After centrifugation the gradients were fractionated and analysed by SDS/PAGE. Other biochemical methods Protein concentration was determined with the Bio-Rad Protein Assay. Bovine serum albumin was used as standard [16]. After separation of the subunits of phenyllactate dehydratase by SDS/PAGE, gels were stained with Coomassie Brilliant Blue and scanned with a ScanJet 6100 C/ T (Hewlett Packard). The
Fig. 3. Separation of CoA-transferase (FldA) and dehydratase (FldBC). The elution profile from Q-Sepharose chromatography (solid line) is shown together with the SDS/PAGE (10%) and the volume activities of the corresponding fractions (X, phenyllactate CoA-transferase activity; W, phenyllactyl-CoA dehydratase activity 15).
3878 S. Dickert et al. (Eur. J. Biochem. 267)
q FEBS 2000
be reduced by NADH to 3-phenylpropionate. Therefore a freshly prepared cell-free extract from C. sporogenes was incubated anaerobically with 1.0 mm (R)-3-phenyllactate and 0.1 mm NADH. The decrease in absorbance at 340 nm was followed spectrophotometrically. Significant rates (20 mU´mg21) were only obtained, if 2.5 mm MgCl2 and 0.4 mm ATP were added. NADH could not be replaced by NADPH. HPLC analysis revealed the formation of 3-phenylpropionate and phenylacetate under similar conditions. In the absence of NADH cinnamate could be detected. On the other hand cinnamate was completely reduced to phenylpropionate and oxidized to traces of up to 7% phenylacetate. The cell-free extract also catalysed the ATP- and MgCl2-independent reduction of cinnamate by NADH to phenylpropionate at a rate of 0.3 U´mg21 protein (see below). Fig. 4. UV-visible spectra of phenyllactate CoA-transferase and the FldA-depleted phenyllactate dehydratase. The spectra of the transferase (solid line) and the phenyllactate dehydratase (thin line) are shown together with a threefold enlargement of the dehydratase absorbance shoulders.
data were processed with the imagequant program from Pharmacia Biotech. Iron was determined after treatment with 0.3 m HCl, using ferene, 3-(2-pyridyl)-5,6-bis(5-sulfo-2-furyl)1,2,4-triazine, disodium salt, as iron chelator [17]. Mohr's salt (NH4)2Fe(SO4)2, was used as standard. The N-termini of polypeptides were sequenced by Edman degradation [18] after separation by SDS/PAGE followed by blotting on poly(vinylidene difluoride) (PVDF) membranes [19]. For inactivation studies 0.5 mm FldA were incubated with 10 mm sodium borohydride in the presence of 5 mm and 45 mm cinnamoyl-CoA as described in [20]. After an incubation time of 15 min the activity was determined with the standard assay. Inactivation studies with hydroxylamine were performed after preincubation of 0.8 mm FldA with 190 mm cinnamoylCoA for 10 min. Then neutralized hydroxylamine was added to a final concentration of 200 mm. After an incubation time of 20 min the activity was determined with the standard assay.
R E S U LT S NADH dependent reduction of (R )-3-phenyllactate to 3-phenylpropionate According to the pathway proposed by Simon and coworkers (Fig. 1) (E )-cinnamate formed from (R)-3-phenyllactate should
Purification and properties of the phenyllactate dehydratase complex, FldABC After the first purification step, chromatography on phenylSepharose, phenyllactate dehydratase activity could only be measured by re-addition of a small amount of cell-free extract (100±200 mg protein per mL). Furthermore, 10 mm FAD had to be added, which could not be replaced by FMN or riboflavin. Only after lag phases of up to 20 min, the decrease in absorbance became linear with time. Chromatography on Q-Sepharose followed by Superose 12 led to an active preparation (10 mU´mg21 ) which comprised three major bands with apparent molecular masses of 46 kDa (FldA), 43 kDa (FldB) and 40 kDa (FldC) as revealed by SDS/PAGE (Fig. 2). The three proteins probably form a heterotrimeric enzyme complex, whose size has been determined by gel filtration on Superose 12 as 130 ^ 15 kDa. MALDI-TOF mass spectrometry confirmed the molecular mass of FldA (46.44 kDa) but gave values for the two smaller subunits slightly different from those obtained by SDS/PAGE, FldB (45.49 kDa) and FldC (37.34 kDa). Later it was shown that inclusion of 5 mm cinnamoyl-CoA or phenylpropionyl-CoA into the assay increased the specific activity 10±15-fold whereby the amount of cell-free extract could be reduced to 10 mg protein/mL. Hence, this rate enhancement showed that the actual substrate for the dehydration was most likely (R)-phenyllactyl-CoA rather than the carboxylate. This was confirmed by the use of enzymatically prepared phenyllactylCoA, which was converted by the enzyme complex and cinnamate reductase to phenylpropionate in the presence of
Fig. 5. N-terminal sequences and alignment of the CoA-transferase. The N-terminal sequences of the enzymes described in this paper that have been purified from C. sporogenes are shown together with the alignment of the CoA-transferase FldA with an ORF of the C. difficile contig 910 (Sanger Institute), the CaiB gene product from E. coli and the CaiB-1 gene product from Archaeoglobus fulgidus. Uncertain amino acids are in brackets.
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Clostridial phenyllactate dehydratase (Eur. J. Biochem. 267) 3879
Table 1. Purification of FldA. Specific activity Step
Protein (mg)
Activity (U)
(U mg21)
(s21)
Enrichment (-fold)
Yield (%)
Cell-free extract Phenyl-Sepharose Q-Sepharose 1 Superose 12 Q-Sepharose 2
587 80 15 12 0.5
63 52 33 21 3
0.11 0.65 2.2 1.8 6
0.2 1.4 4.7 3.9 4.6
1 6 20 16 55
100 83 52 33 5
only 1 mg cell-free extract. Furthermore, the results supported the idea of a relationship to the [4Fe-4S] cluster and FMN-containing component D of 2-hydroxyglutaryl-CoA dehydratase from Acidaminococcus fermentans, which requires initivation by ATP, MgCl2, a reducing agent and catalysis by the extremely oxygen-sensitive component A [11,12]. During re-chromatography of the purified phenyllactate dehydratase on the Q-Sepharose column, most of the FldA protein eluted at 115±125 mL just before the trimeric complex at 135±155 mL, which thereby became depleted of the largest subunit. Interestingly, FldA exhibited cinnamoyl-CoA: phenylpropionate CoA-transferase activity (Fig. 3), whereas the residual complex retained phenyllactate dehydratase activity, which could be stimulated by cinnamoyl-CoA. By scanning of the Coomassie stained SDS/PAGE, the ratio of FldA: FldB: FldC in the five fractions between 135 and 155 mL was determined as (11 ^ 1):(42 ^ 0.7):(46 ^ 2), which suggested that only about 25% of the protein existed as heterotrimer and the remaining 75% of a FldBC heterodimer. In another experiment a complete separation of FldA from FldBC was achieved. In this case the dehydratase activity could not be reconstituted by addition of FldA. With enzymatically synthesized (R)-phenyllactyl-CoA, however, FldBC showed dehydratase activity, whereby only 1 mg cell-free extract was required as shown for the trimeric complex (see above). The FldA-depleted dehydratase was subjected to density gradient centrifugation in a 10±35% glycerol gradient at 220 000 g for 20 h, whereby the three subunits sedimented together and were detected in the same fractions as the marker protein bovine serum albumin dimer (136 kDa) indicating still a trimeric
complex. The UV-visible spectrum of the isolated CoAtransferase (FldA) did not reveal any significant absorption besides that of the protein at 279 nm. In contrast, the trimeric complex showed additional absorbance shoulders at 330 nm and around 400 nm, which are indicative for one [4Fe-4S]21 cluster (:390 14.5 mm21´cm21 [21]) and consistent with its iron content of 4.8 mol per trimer (Fig. 4). The acid-labile sulfur was only qualitatively detected by its odour (H2S) after addition of 100 mm HCl. Phenyllactate dehydratase is an oxygen sensitive enzyme with a half-life of approximately 12 h under air. Upon precipitation of the protein with trifluoroacetic acid, no flavin was detectable by UV-VIS spectroscopy of the supernatant. Nevertheless, it has been found, that the presence of FAD in the assay is beneficial for dehydratase activity. It can therefore be concluded that active dehydratase contains FAD, which is lost during purification.
Cinnamoyl-CoA:(R )-phenyllactate CoA-transferase (FldA) The CoA-transferase can be easily measured with 1.5 mm phenylpropionate and 15 mm cinnamoyl-CoA following the decrease in the absorbance of cinnamoyl-CoA at 309 nm which is absent in phenylpropionyl-CoA, cinnamate and phenylpropionate. In the reverse direction with 1.5 mm cinnamate and 15 mm phenyllactyl-CoA as substrates an
Table 2. Substrate specificity of phenyllactate CoA-transferase. Each assay contained 50 mm potassium phosphate pH 7.0, 1.5 U CoA-transferase, 15 mm (E )-cinnamoyl-CoA and phenylpropionate (standard assay) or another CoA-acceptor as indicated (100% activity corresponds to 1.7 U´mg21, determined with 3-phenylpropionate as CoA-acceptor). CoA-Acceptor
Concentration (mm)
Activity (%)
3-Phenylpropionate 3-(4-Hydroxyphenyl)-propionate (R)-Phenyllactate 4-Phenylbutyrate 5-Phenylvalerate (S )-Phenyllactate Benzoate (4-Hydroxyphenyl)-acetate 2-Phenylacetate Propionate Acetate
2.0 2.1 2.5 1.7 1.8 1.9 1.9 2.2 2.0 1.6 2.4
100 91 88 81 16 13 6 1 , 0.5 , 0.5 , 0.5
Fig. 6. Double reciprocal plot of phenyllactate CoA-transferase activity vs. phenylpropionate concentration. Each assay contained 50 mm potassium phosphate pH 7.0, 1.2 U CoA-transferase, phenylpropionate as indicated and 14 mm (A), 28 mm (K) and 51 mm () cinnamoyl-CoA.
3880 S. Dickert et al. (Eur. J. Biochem. 267)
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Fig. 7. Arrangement of the ORFs in C. difficile and the genes of the 2-hydroxyglutarate operon in A. fermentans. Data have been obtained from the Sanger Centre C. difficile BLAST Server on 25 January 2000 (contig 910). For further explanation see Table 3.
increase of the absorption at 309 nm was observed. Since the enzyme turned out not to be oxygen-sensitive, all measurements could be performed under aerobic conditions. The CoA-transferase has been purified to homogeneity over 4 columns under anaerobic conditions (Fig. 3; 115±125 mL elution volume). Upon gel filtration on the calibrated Superose 12 column, the CoA-transferase behaved as a homodimer (97 ^ 12 kDa). The N-terminal sequence of 39 amino acids was obtained and revealed 39% sequence identity with that of the putative l-carnitine CoA-transferase CaiB from Escherichia coli [22], with an ORF from Clostridium difficile (51%, see below) and Archaeoglobus fulgidus (46%) [23] (Fig. 5). During purification the specific activity of phenyllactate CoA-transferase increased from 0.11 U´mg21 in the cell-free extract to 1.75 U´mg21 for the trimeric complex and to 6 U mg21 after separation from the residual phenyllactate dehydratase complex. The turnover number of 4.7 s21 was not influenced by the separation as would be expected if there was no direct or allosteric interaction between the active sites of transferase and dehydratase in the complex (Table 1). Although (R)-phenyllactate should be the natural CoA-acceptor for the CoA-transferase, analysis of the in vitro substrate specificity revealed that 3-phenylpropionate was even a better CoA-acceptor. Therefore phenyllactate CoA-transferase activity was routinely determined with 3-phenylpropionate and cinnamoyl-CoA. Further measurements with cinnamoyl-CoA and other cosubstrates showed a very high substrate specificity of the CoA-transferase (Table 2). In order to check the enzyme for CoA-ester hydrolase activity, all assays with the different substrates were repeated in the presence of 1 mm 5,5 0 -dithiobis-(2-nitrobenzoate) (DTNB). No
formation of free coenzyme A could be detected at 412 nm with any of these substrates. The CoA-transferase showed maximum activity between pH 7±8 in 50 mm potassium phosphate. In organic buffers like Tris/HCl, Mops, phosphate-borate-acetate or citrate the activity was much lower (# 50%). Within the temperature range from 20 8C to 50 8C the CoA-transferase activity increased with an activation energy of Ea < 130 kJ´mol21 as determined from an Arrhenius plot. Above 50 8C the activity decreased and became almost zero at 60 8C. Kinetic measurements with the CoA-transferase resulted in Vmax 7.2 U´mg21 and apparent Km values of 15, 12 and 9 mm for phenylpropionate with 14, 28 and 51 mm cinnamoyl-CoA as second substrate, respectively. In the reverse direction, Vmax was determined as 5.3 U´mg21 and the apparent Km values for cinnamoyl-CoA were 19, 9 and 3 mm with 3, 9 and 57 mm phenylpropionate as second substrate, respectively. Double reciprocal plots of activity vs. phenylpropionate concentration at three different cinnamoylCoA concentrations (Fig. 6) or vice versa intersected at 1/[ phenylpropionate] 0, which is indicative for a ternary complex of the CoA-transferase together with the two substrates, rather than a ping-pong mechanism with transient formation of a thiolester between a conserved glutamate residue and CoA as shown for glutaconate CoA-transferase from A. fermentans [20,24]. Attempts to reduce such a possible CoA-ester to the corresponding inactive alcohol gave ambiguous results. Incubation of phenyllactate CoA-transferase with 10 mm sodium borohydride and 49 mm cinnamoyl-CoA at pH 7 consistently resulted in approximately 50% inactivation after 15 min, whereas complete inactivation could not be achieved. Incubation of the CoA-transferase with 190 mm
Table 3. Products of the genes shown in Fig. 7. Abbreviation
Enzyme
Organism
Identity with orfs from C. difficile
d-LDH FldH CaiB FldA HgdC HgdAB
d-Lactate dehydrogenase Phenyllactate dehydrogenase Carnitine CoA-transferase Phenyllactate CoA-transferase Activator of 2-hydroxyglutaryl-CoA dehydratase Component D of 2-hydroxyglutaryl-CoA dehydratase
L. plantarum C. sporogenes E. coli C. sporogenes A. fermentans A. fermentans
Bcd GctAB GcdA
Butyryl-CoA dehydrogenase Glutaconate CoA-transferase a-Subunit of Glutaconyl-CoA decarboxylase
T. thermosaccharolyticum A. fermentans A. fermentans
orf orf orf orf orf orf orf orf ± ±
1, 1, 2, 2, 3, 4, 5, 6,
41% 31% 26% 51% 52% 39% 39% 57%
(N-terminus) (N-terminus) (HgdA) (HgdB)
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Clostridial phenyllactate dehydratase (Eur. J. Biochem. 267) 3881
cinnamoyl-CoA for 10 min followed by 200 mm hydroxylamine for 20 min did not result in any significant inactivation: When the experiment had been performed at 20 8C the residual activity was 85%; on ice even 108% was observed.
contig 910 (Fig. 7). There are four consecutive ORFs, which might code for all four proteins required for the phenyllactate dehydratase system. The deduced amino acid sequence of the first ORF shows 51% identity to the N-terminus of phenyllactate CoA-transferase from C. sporogenes and 26% identity to CaiB from E. coli (Table 3). The deduced amino acid sequences of the following three ORFs are homologous with HgdA, B and C of the 2-hydroxyglutaryl-CoA dehydratase from A. fermentans (40±50% identity). A fifth ORF, however, does not encode a 2-enoate reductase but an acyl-CoA dehydrogenase. Upstream of the CoA-transferase homologue, an ORF is located whose deduced amino acid sequence is similar to d-lactate dehydrogenase from Lactobacillus plantarum (41% identity) and shows 31% identity to the N-terminus of d- or (R)-phenyllactate dehydrogenase from C. sporogenes described in this paper.
Purification of auxiliary enzymes Cinnamate reductase (2-enoate reductase, FldZ) from C. sporogenes could be anaerobically purified over three columns with a yield of 47%. SDS/PAGE showed one subunit of approximately 73 kDa, as it had been described for the 2-enoate reductase from Clostridium tyrobutyricum [6]. The specific activity of the 95% pure enzyme from C. sporogenes was about 5 U mg21 which is similar to that from C. tyrobutyricum (10.6 U´mg21). As the N-terminal sequences of both enzymes showed 60% identity, the enzyme from C. sporogenes was not characterized further. It was used as an auxiliary enzyme for the dehydratase assay (see above). Phenyllactate dehydrogenase (FldH) from C. sporogenes was aerobically purified 50-fold over three columns with a yield of 10%. Specific activity increased from 5 U´mg21 in the cell-free extract to 260 U´mg21. SDS/PAGE showed equal amounts of two proteins of 38 and 42 kDa, which migrated together in a nondenaturing PAGE (10% acrylamide). N-terminal sequencing displayed 51% identity of the 38 kDa protein to d-2hydroxyisocaproate dehydrogenase from Lactobacillus casei [25] and 31% identity to a deduced protein from Clostridium difficile (see below). The N-terminus of the comigrating 42 kDa protein showed 64% identity to a serine-hydroxymethyltransferase from Saccharomyces cerevisiae [26]. Hence the 38 kDa protein could represent the subunit of (R)-phenyllactate dehydrogenase. Maximum activity of phenyllactate dehydrogenase was found in 50 mm Tris/HCl, pH 8.0. The apparent Km values were determined in the presence of 100 mm of the second substrate as 20 mm for phenylpyruvate and 10 mm for NADH, respectively. A potential operon for the fermentation of phenyllactate in Clostridium difficile A BLAST [27] search with the N-terminal sequence of FldA in the at present unfinished genome project of C. difficile (Sanger Institute) mentioned above showed a significant hit in the
Fig. 8. Proposed mechanism for the dehydration of phenyllactate.
DISCUSSION The identification of the largest subunit of the phenyllactate dehydratase complex as cinnamoyl-CoA:phenyllactate CoAtransferase and the requirement of cinnamoyl-CoA for high activity demonstrates that the elimination of water from (R)-phenyllactate to yield (E )-cinnamate occurs at the thiolester level (Fig. 8). Furthermore, the requirement of ATP, MgCl2 and a not yet purified heat-labile, oxygen sensitive component of the cell-free extract reveals the necessity for initiation of the dehydration of (R)-phenyllactyl-CoA as found in the 2-hydroxyglutaryl-CoA dehydratase system from Acidaminococcus fermentans [11]. Notably, early experiments on the dehydration of (R)-2-hydroxyglutarate to (E )-glutaconate in cell-free extracts from A. fermentans revealed a lag-phase, which was also observed with phenyllactate dehydratase [7]. Like component D of 2-hydroxyglutaryl-CoA dehydratase, phenyllactate dehydratase contains about one [4Fe-4S] cluster/ FldBC and requires a flavin; in this case FAD rather than FMN. Taken together, both systems are very similar, despite differences in the formation of the CoA-esters. In contrast to the heterodimeric component D of 2-hydroxyglutaryl-CoA dehydratase, phenyllactate dehydratase has been characterized as a trimeric complex, which is composed of phenyllactate CoA-transferase (FldA) and a heterodimer (FldBC) most likely phenyllactyl-CoA dehydratase. The iron-sulfur
3882 S. Dickert et al. (Eur. J. Biochem. 267)
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Fig. 9. Reaction mechanism of the citrate lyase. The enzyme complex is composed of CitD, acyl carrier protein; CitE, citryl-ACP lyase and CitF, acetyl-ACP:citrate ACP-transferase. Nomenclature after Bott and Dimroth [41].
cluster-containing dehydratase part seems to be very unstable and after its inactivation/denaturation, the CoA-transferase can be isolated as a separate enzyme. The trimeric complex probably contains some noncovalently bound cinnamoyl-CoA, which could explain the activity obtained already in the absence of external cinnamoyl-CoA. In order to achieve maximum activity, only very low concentrations of this CoA-ester (5 mm) are required. Hence, cinnamoyl-CoA can be regarded as a noncovalently bound prosthetic group, like FAD in acyl-CoA dehydrogenases, which remains at the same enzyme during many catalytic cycles [28], rather than a coenzyme, which after one turnover dissociates and interacts with another enzyme (Fig. 8). This mechanism is related to that of citrate lyase from Klebsiella pneumoniae (formerly called K. aerogenes) [29] (Fig. 9), citramalate lyase from Clostridium tetanomorphum [30] and malonate decarboxylase from Malonomonas rubra [31] which contain a derivative of acetyl-CoA [32] covalently attached to a small acyl carrier protein (ACP; < 10 kDa). The additional subunits of citrate- and citramalate-lyases act as CoA-transferases (
