Lactate Reduction in Clostridium propionicum PURIFICATION AND PROPERTIES OF LACTYL-COA DEHYDRATASE*

Clostridium propionicum converts lactate to pro- pionate (Cardon, B. P.;and Barker, €3. A. (1947) Arch. Biochem. Biophys. 12,166-171). We have obtained a soluble system that carries out this conversion as well as the hydration of acrylate to lactate and the reduction of acrylate to propionate. 3-Pentynyl-CoA inhibits re- duction of acrylate and lactate to propionate, but not hydration of acrylate to lactate by cell extracts. The conversion probably involves CoA esters. When [/3-2Hs] lactate is used as a substrate, the rate of propionate formation is reduced 1.8-foldy and the methyl group of the resulting propionate has lost 1.4 deuterium atoms. These results are consistent with the intermediate formation of acrylate (acrylyl-CoA) in the conversion of wlactate to propionate. Two proteins, which we designate E I and E 11, were purified to ~ 9 0 % homogeneity. Together, they catalyze the hydration of acrylyl-CoA to lactyl-CoA. E I has an apparent molecular mass of 27,000 daltons and is rapidly and irreversibly inactivated by OS. E I1 consists of two subunits of molecular mass 41,000 and 48,000 daltons and contains equal amounts of riboflavin and flavin mononucleotide. Hydration of acrylyl-CoA to lactyl-CoA requires M@+ and catalytic quantities of ATP. GTP can replace ATP, but ADP and adenylyl

the P-carbon of lactate ends up solely in the @-carbon o propionate (3). These organisms do not ferment any of thc intermediates of the randomizing pathway to propionate no. do they require CO, for lactate reduction (4).
In 1947, it was demonstrated that whole cells of C. propion icum rapidly ferment acrylate, as well as either lactate 0. alanine, to acetate and propionate (5). It was proposed tha lactate reduction involves acrylate as an intermediate, a shown in Scheme 2.
It was later shown that C. propionicum contains a pro pionyl-CoA dehydrogenase which can catalyze the second hal of Scheme 2; therefore, the reactions in Scheme 2 were pro posed to occur at the level of CoA thioesters (2). Furthe evidence for the occurrence of acrylyl-CoA was provided b the purification of acrylyl-CoA aminase (acrylyl-CoA + NH , " P-alanyl-CoA) (6). However, no evidence for the conversiol of acrylyl-CoA to either lactate or alanine was obtained Recently, it was found that in whole cells of C. propionicun incubated with lactate and 3-butynoic acid, which when acti, vated to 3-butynyl-CoA is an inactivator of acyl-CoA dehy drogenase, small amounts of acrylate accumulate (7). How ever, it was not clearly shown that acrylate was derived fron lactate (7). A partial purification of a lactyl-CoA dehydratasd from M. ekdenii was reported, although the activity detectec was extremely low and dehydration could not be demonstrater (8). Thus, there is no conclusive evidence for the dehydratior of lactate (lactyl-CoA) to acrylate (acrylyl-CoA).
In other clostridia, however, there is more convincing evi. dence for the dehydration of an a-hydroxy acid to the a,@. unsaturated acid. In a cell-free extract of Clostridium micro. sporum, the accumuIation of (E)-[14C]glutaconate from (R).
The dehydrations of lactate, 2-hydroxyglutarate, and 3. phenyl lactate are unusual elimination reactions. The hydro. gen which is eliminated is not activated, and the hydroxy; group is a very poor leaving group. This is in contrast to the majority of biological elimination reactions where the proton removed is a to a carbonyl group or other activating grou~ and the leaving nucleophile-8 to the carbonyl group. We now report the properties of the enzyme system isolated from C. propionicum which catalyzes the dehydration of lactyl-CoA.

EXPERIMENTAL PROCEDURES
Muteriak-All materials were reagent grade or better. Acetyl phosphate, CoA, ATP, ADP, and AMP were from Sigma. ~-['~C]Lactate was purchased from New England Nuclear, and DL-[*4cC]lactate was from Arnersham Corp. Pantethine was purchased from Calbiochem-Behring and was reduced to pantetheine by a standard procedure (10).
Cell Growth and Media-C. propwnicum (American Type Culture SCHEME 1. Stickland reaction.

SCHEME 2. Lactate reduction.
Collection 25522) were stored in solid agar containing 3 g of alanine, 3 g of Bacto-peptone, 29 g of Difco thioglycolate media, 15 g of agar, 2.5 ml of saturated CaS04, 50 mg of MgSO4,20 mg of FeS04, and 1 mg of methylene blue in 1 liter of 10 mM potassium phosphate, pH, 7.5. Cells were transferred from agar to 15 ml of the above medium minus the agar and grown at 37 "C. Cells were grown sequentially to 125 ml, 2 liters, and 100 liters in a previously described medium (5), with 1 mg liter-' methylene blue and 0.03% sodium sulfide added. Four liters of cells were used to inoculate 100 liters of medium. After sterilizing the medium, air was purged from the fermenter with Nz. The cells were harvested in a Delaval continuous flow centrifuge. After harvesting, the cells were washed once with 50 mM potassium phosphate, pH 7.5, containing 1 mg liter-' methylene blue and 0.06% NazSz04 (Buffer A). The cells were collected by centrifugation and stored frozen covered with Buffer A in tightly capped vials.
Enzyme Preparation-All operations except cell disruption, centrifugations,,concentration of E I1 and FPLC' were performed in a Coy anaerobic chamber with. an atmosphere of 10% H1 in Nz. All operations were performed at 4 "C. Frozen cells (10-15 g) were resuspended in a 25-ml flask filled with Buffer A. They were broken by passing twice through a French press at 1000 p.s.i. and collected in a 50-ml flask containing 5 ml of Buffer A. Approximately 20 mg of NazSz04 were added and the volume increased to 60 ml with Buffer A. This was centrifuged for 20 min at 25,000 X g and the pellet discarded. The volume of the supernatant fluid was increased to 200 ml with Buffer A. Ammonium sulfate was added to a concentration of 0.23 g ml-' while the pH was maintained at 7.5 with KOH. After 30 min, the suspension was centrifuged for 10 min at 10,000 X g and the pellet discarded. To the supernatant fluid, ammonium sulfate was added to give a final concentration of 0.34 g ml-' . After 30 min the suspension was centrifuged for 15 min at 10,000 X g. The pellet was saved and resuspended in approximately 50 ml of Buffer A. This was twice dialyzed for 12 h against 1 liter of 10 mM potassium phosphate, pH 7.5, 0.06% NazS204, and 1 mg liter-' methylene blue (Buffer B). This dialyzed material will be referred to as Fraction A.S. The solutions were kept anaerobic with NazSz04 at all times. The methylene blue served as an indicator of anaerobiosis. Fraction A.S. was loaded onto a Whatman DE32 column (30 X 2.5 cm) equilibrated with Buffer B. Fraction A.S. was pumped from the anaerobic chamber to the column with a peristaltic pump. The protein was loaded onto the DE32 by layering it under the Buffer B that covered the column. The column was loaded in this manner to prevent oxygen inactivation. The proteins were eluted with -adient of 30-200 mM potassium phosphate, pH 7.5, 0.06% NazSzO,, and 1 mg liter" methylene blue over 1.5 liters. Fractions were collected in the anaerobic chamber, and the solvent was pumped from the chamber to the column with a peristaltic pump. Fractions I and I1 were found with acrylate hydration assays described in Fig. 2. Each fraction was pooled and stored in the anaerobic chamber.
The pooled Fraction I1 was concentrated using a Diaflow PM-10 membrane. This was loaded onto a Sepharose 6B column (95 X 2.5 ' The abbreviations used are: FPLC, fast protein liquid chromatography; HPCL, high performance liquid chromatography; TLC, thin layer chromatography; ADP-PNP, adeylyl-imidodiphosphate. cm) equilibrated with Buffer B, and the column was eluted with Buffer B. Fraction 11 was loaded onto the column with a syringe under the approximately 5 cm of Buffer B which covered the column. Fractions were collected in the anaerobic chamber. The enzyme active in acrylate hydration in Fraction I1 will be referred to as E 11. Fractions containing E I1 were located with assays identical to those used to locate Fraction I1 from the DE32 column and then pooled. E I1 was further purified using FPLC (Pharmacia The enzyme required for acrylate hydration in Fraction I will be referred to as E 1. Fractions containing E I were located as described for the DE32 column. These were pooled, and loaded onto a hydroxylapatite column (Bio-Rad DNA grade, 2 X 5 cm) equilibrated with Buffer B. Proteins were eluted with a 300-ml gradient of 10-200 mM potassium phosphate, pH 7.5,0.05% NazSz04, and 1 mg liter-' methylene blue. To each 10-ml fraction, 1 mg of NazS2O4 was added.
Fractions containing E I were located as above and pooled.
Protein Concentration-Protein concentrations were measured by the method of Bradford (11) with bovine albumin as a standard.
Radioactiuity-Radioactivity was measuredusing either a Beckman LS 100C or LS 1800 scintillation counter. Samples were dissolved in Amersham ACS scintillation fluid. UV-visible Spectroscopy-Spectra were obtained with a Perkin-Elmer 559 UV-Vis spectrophotometer.
HPLC-Three HPLC systems were used. HPX-87H Organic Acids Column (Bio-Rad) was used with a precolumn (5 X 0.3 cm) containing Aminex Q-15 S resin (Bio-Rad) and with a mobile phase of 5 mM HzS04. The effluent was monitored with a Waters Refractive Index detector. The CIS (Waters) and PRP (Hamilton) reverse phase columns were used with mobile phases as noted. CoA thioesters and flavins were detected with a Beckman UV detector at 254 nM.
Enzyme Assays (Fraction AS.)-Lactate reduction was measured by incubating Fraction A.S. with 10 pmol of ~~-[ '~C ] l a c t a t e (approximately 10,000 cpm pmol"), 10.8 pmol of acetyl phosphate, 10 pmol of MgS04, 4 mg of NazSz04, and 0.5 pmol of CoA in a volume of 1.5 ml of 50 m M potassium phosphate, pH 7.5, and 1 mg liter" methylene blue. After 10 min at 37 "C, the reaction was terminated by the addition of 20 pl of concentrated HClO, and the precipitate removed by centrifugation. Lactate and propionate were separated by HPLC on an HPX-87H column, and the amount of propionate formed was determined either by refractive index or by measuring radioactivity in lactate and propionate. Refractive index was used only when at least 0.5 pmol of propionate was formed.
Acrylate reduction was measured by incubating Fraction AS. with 10 pmol of sodium acrylate, 0.5 pmol of CoA, 10.8 pmol of acetyl phosphate, and 4 mg of NazS20, in a volume of 1.5 ml of 50 mM potassium phosphate, pH 7.5, and 1 mg liter-' methylene blue. After 10 min at 37 "C, 20 pl of concentrated HClO, were added, and the precipitate was removed by centrifugation. Acrylate and propionate were separated by HPLC on the HPX-87H column, and the amount of propionate formed was determined by refractive index.
Acrylate hydration by Fraction A.S. was measured in assays identical to those for lactate reduction except the lactate was replaced with 20 pmol of sodium acrylate and the NaZSzO4 halved. After removal of the precipitate, the amount of lactate formed was determined colorimetrically.
Identification of Propionate-Propionate formed from lactate by Fraction A.S. under standard assay conditions was isolated directly by HPLC on an HPX-87H column. The fractions containing propionate were adjusted to pH > 7 with KOH, and the solvent was removed under reduced pressure. The residue was taken up in 1 ml of HzO and concentrated HC10, added to pH < 1. The precipitate was removed by centrifugation and the propionate reisolated by HPLC. Fractions containing propionate were adjusted to pH 7 with KOH and lyophilized to dryness. The residue was taken up in 0.75 ml of 2H20 and its NMR spectrum determined. 'H NMR = 2.21 ppm (9, J = 7.6 Hz, 2H) and 1.07 ppm (t, J = 7.6 Hz, 3H).
Synthesis of CoA Thwesters-Acetyl-coA was synthesized from acetic anhydride by a modification of the procedure of Ochoa (15). CoA (20 mg) was dissolved in 2 ml of 0.2 M KHCO, at 0 "C, and acetic anhydride (30 pl) was added with stirring. After 5 min, the solution was acidified to pH 3 with HCl, and 0.5 ml of 1 M KC1 was added. The acetyl-coA was purified using a Waters Cl8 Sep-Pak. The Sep-Pak was equilibrated with 1 mM HCl, and one portion of the acetyl-coA solution was added to the Sep-Pak. The Sep-Pak was then washed with 5 ml of 1 mM HCl to remove KC1 and acetate, and the acetyl-coA was eluted with 5 ml of 20% MeOH in 1 mM HCl. The remaining acetyl-coA was purified in this manner. The MeOH was removed under reduced pressure and acetyl-coA lyophilized to dryness. This was redissolved in 1 mM HC1 and stored frozen. The concentration of acetyl-coA was determined with Ellman's reagent (16). P-Lactyl-CoA was synthesized using @-propiolactone (17) and purified identically to the acetyl-coA. 3-Pentynyl-CoA was synthesized by a modifcation of a previously described procedure for the synthesis of 3-pentynyl pantetheine (10). 3-Pentynoic acid (30 mg, 30.6 pmol) (a kind gift of G. Fendrich) was dissolved in 1 ml of acetone and 66 mg (32 pmol) of dicyclohexylcarbodiimide were added. After stirring for 30 min at 0 "C, 20 mg of CoA dissolved in 2 ml of 0.2 M KHco3 were added, and this was stirred for an additional 10 min. This was adjusted to pH 3 with HCl and the acetone removed under reduced pressure. To this solution 0.5 ml of 1 M KC1 was added and the 3pentynyl-CoA purified identically to the acetyl-coA. The concentration of 3-pentynyl-CoA was determined with Ellman's reagent.
Acrylyl-CoA was synthesized by a modification of a previously described procedure (16). CoA (20 mg) was dissolved in 2 ml of 0.2 M KHC03 at 0 "C, and 65 pl of acrylyl chloride were added with rapid stirring. After 5 min, the solution was titrated to approximately pH 3 with 1.0 M KZCOa. To the acrylyl-CoA solution, 1 ml of 1 M KC1 was added. This was twice passed through a CIS Sep-Pak equilibrated with 1 mM HC1 and the eluent saved. The Sep-Pak was washed with 5 ml of 1 mM HCl, the first 3 ml of which were saved and combined with the previously saved material. This material will be referred to as Fraction A. Fraction A contains approximately 20% of the acrylyl-CoA and was saved in order to conserve material. The acrylyl-CoA was eluted with 3 ml of 25% MeOH in 1 mM HCI. Purified acrylyl-CoA will be referred to as Fraction B. After washing the Sep-Pak with MeOH and re-equilibrating it with 1 mM HC1, the acrylyl-CoA in Fraction A was purified as above, except only the 25% MeOH wash was saved and combined with Fraction B. MeOH was removed under reduced pressure, and the acrylyl-CoA used within 6 h. A large excess of acrylyl chloride to CoA was used in order to maximize the rate of formation of acrylyl-CoA, thereby minimizing the amount of CoA present with the acrylyl-CoA, as thiols rapidly react with acrylyl thioesters in a Michaels addition (2).
The concentration of acrylyl-CoA was estimated by determining its absorbance at 260 nm and assuming the €260 of acrylyl-CoA and of crotonyl-CoA (€260 = 22,600 M" cm") (18) are identical. The amount of acrylyl-CoA measured by this method represents an upper limit on the amount of acrylyl-CoA actually present, due to the instability of acrylyl-CoA.

NMR Experiments"[3-'H3]Lactate (60 pmol) was incubated with
Fraction A.S. (42 mg of protein), 4 mg of CoA, 30 pmol of MgSO,, 1 mg of ATP, 12 mg of NaZS204, and 10 mg of acetyl phosphate in a final volume of 7.7 ml of 50 mM potassium phosphate, pH 7.5, and 1 mg liter" methylene blue. After incubation for 30 min at 37 "C, 50 pl of concentrated HClO, were added. The precipitate was removed by centrifugation, and propionate was isolated by HPLC on the HPX-87H column. The propionate containing solution was titrated to pH 7 and then lyophilized to dryness. To the residue, 5 ml of 'Hz0 were added, and the sample was lyophilized to dryness again. After 0.8 ml of 'H2O was added, the NMR spectrum was determined at 90 MHz.
[2-*H]Lactate (150 pmol) was incubated with Fraction AS. (17 mg of protein), 4 mg of CoA, 20 pmol of MgSO,, 8 mg of NazSz04, and 4 mg of acetyl phosphate in 3.5 ml of 50 mM potassium phosphate, pH 7.5, containing 1 mg liter" methylene blue. This was incubated for 20 min at 37 "C and then the reaction was stopped with 50 pl of concentrated HC104. Propionate was isolated and the NMR spectrum determined as above, except a 500 MHz NMR spectrometer was used.
Gels-Discontinuous sodium dodecyl sulfate-polyacrylamide gel electrophoresis was performed according to the procedure of Laemmli (21), with a 4% spacing and 10% running gel. Coomassie Brilliant blue was used to visualize proteins. Nondenaturing gel electrophoresis was performed similarly, except the sodium dodecyl sulfate and 2mercaptoethanol were omitted. A 5% spacing gel and 7.5% running gel were used.
Isolation and Identifieation of Lactyl-CoA-The products of 10 acrylyl-CoA hydration assays as described in Table IV were chromatographed on the PRP reverse phase HPLC column with a mobile phase of 3.5% CH&N in 10 mM ammonium acetate, pH 5.8. Fractions were collected and lactate detected colorimetrically (12). A portion of each fraction was dried at 100 "C, and the residue was dissolved in 1.0 ml of the precipitating solution. Concentrated H2S04 (6 ml) was added, and the rest of the assay was performed as described (12). Fractions containing lactyl-CoA were acidified to pH 3 with HCI and then lyophilized to dryness. The residue was resuspended in 1 mM HC1 and chromatographed on the PRP HPLC column as described above. The fractions containing lactyl-CoA were acidified to pH 3 with HCl and saved at -20 "C.
Lactyl-CoA was hydrolyzed by incubating it for 30 min at 37 "C with 0.1 N KOH and then acidifying to pH 3 with HCI. Ellman's reagent was used to determine the concentration of sulfhydryl groups (16).
Removal of Cofactors from E 11-E I1 (0.5-1.0 mg ml-') was airoxidized by shaking and then chilled to 0 "C. HC104 was added to give a final concentration of 0.29 M. After 30-60 s, the solution was titrated back to pH 7.5 with KOH. The precipitate was removed by centrifugation, and the supernatant fluid was chromatographed on a CIS HPLC column. The flow rate was 1.6 ml min-l. The column was equilibrated with 20% MeOH, 10 mM ammonium acetate, pH 5.8. After injecting the sample, the column was washed for 5 min with the starting solvent and then a linear gradient to 100% MeOH over TABLE I 72 min was applied. All HPLC of flavins and Factors I and I1 were done using this solvent system. Compounds of interest were collected, and the MeOH was removed by slightly warming the solution and blowing Nz over the surface. For this and all subsequent procedures, exposure to light was minimized. The compounds were lyophilized to dryness and stored at -20 "C. Flavins were also removed by incubating E I1 for at least 24 h at room temperature in the presence of 0 2 . Three volumes of MeOH were added, and precipitated protein was removed by centrifugation. To prepare the supernatant fluid for HPLC, MeOH was removed by blowing a stream of Nz over the surface of the solution.
Compounds were dissolved in 50 mM Tris.HC1, pH 8.1, prior to treatment with phosphodiesterase (Sigma, Crotalus Atrox) or alkaline phosphatase (Sigma, Type I11 R). Phosphodiesterase (0.2 mg) was added to <20 nM of the compound and incubated for 30 min at room temperature. If the compound was not going to be further treated with alkaline phosphatase, 2 volumes of MeOH were added and the sample chilled to 0 "C. To digest with alkaline phosphatase, 0.014 mg of alkaline phosphatase was added and incubated for 60 min at room temperature. Two volumes of MeOH were added, and the solution was chilled to 0 "C. To prepare samples for HPLC, the precipitated protein was removed by centrifugation and then MeOH removed with a stream of Nz.
For UV-visible sDectroscopv and KIOo cleavage, the compounds were dissolved in 56 mM ammonium acetate, pH-5:8. To cleave the sample with KIO,, a 7.5-fold molar excess of KIO, was added. The amount of Factors I and I1 was estimated by assuming c2m = 32,500 M" cm". The reaction was kept at room temperature for 20 min, and the products were purified by HPLC.

RESULTS
Conversion of Lactate to Propionate and Reduction of Acrylate to Propionate in a Cell-free System-Initial experiments were carried out in a cell-free system obtained from C. propionicum which has been subjected to (NH&S04 fractionation (Fraction A.S.). This system carries out the conversion of [l-14C]lactate to [14C]propionate (Table I). There is an absolute requirement for acetyl phosphate, CoA, and Mg?. ADP and ATP stimulate the reaction significantly, but AMP has no effect. ATP at concentrations up to 1 mM does not eliminate the requirement for acetyl phosphate. Pantetheine cannot replace CoA. @-Lactate was not reduced to propionate, nor was it converted to lactate? In this system, Na2S204 serves both as reducing agent and also to keep the system anaerobic.
In the early stages of these experiments, it became apparent that the ability of Fraction A.S. to catalyze the conversion of lactate to propionate was rapidly lost upon exposure to air. The O2 sensitivity of the enzyme system was further investigated. Aliquots of Fraction AS. containing methylene blue were made aerobic by shaking until the dye became oxidized and then reduced with a small amount of Na2S204 at various times thereafter. The ability of these aliquots to reduce lactate to propionate was then assayed. After a 1-min exposure to oxygen, lactate reduction was inhibited >go%. This loss of catalytic activity is irreversible, since dialysis of Fraction A.S. inactivated by O2 against Buffer B for 24 h at 4 "C did not restore activity.
The stereoisomer of lactate which is reduced to propionate is D-lactate, in accordance with previously reported results (7). When ~-['~C]lactate was added to standard assays containing 10 pmol DL-lactate, no radiolabeled propionate was found, while when DL-[ 14C]lactate was used [14C]propionate was found.
Fraction A.S. also catalyzes the hydration of acrylate to In some preparations of Fraction A.S., there was reduction of 0lactate of propionate, and acrylate was hydrated to 8-lactate. The cells that had this activity had grown to stationary phase. In cells that were harvested in late exponential growth, this activity was never observed.
Cofactor requirement for the conversion of Iactate to propionate The complete reaction contained 0.3 mg ml-l Fraction A.S., 6  The ability of the extract to reduce acrylate to propionate was significantly less sensitive to O2 exposure than the conversion of lactate to propionate or the hydration of acrylate. Exposure to O2 for 10 min had no effect on the acrylate reductase activity. However, exposure of Fraction A.S. to O2 for 24 h resulted in complete loss of the acrylate reductase activity. Effect of 3-Pentynyl-CoA-3-Pentynyl-CoA, a mechanism based inactivator of butyryl-CoA dehydrogenase, was examined as an inhibitor of acrylate reduction in fraction A.S. (10). The data in Fig. 1 show that the ability to convert acrylate to propionate is rapidly lost after addition of 3-pentynyl-CoA to Fraction A.S. Loss of activity is irreversible, since activity is not regained after 24-h dialysis against Buffer B.
To ascertain that the CoA thioester of 3-pentynoic acid is the inactivating species, 2 mM 3-pentynoic acid, 4.4 mM acetyl phosphate, and 0.4 mM CoA was incubated with Fraction A.S. at 37 "C for 30 min. Acrylate reduction was inhibited 18%, whereas incubation with 0.3 mM 3-pentynyl-CoA resulted in complete inactivation. Therefore, 3-pentynyl-CoA is probably the inactivating species, and Fraction A.S. does not efficiently catalyze the activation of 3-pentynoic acid to the CoA thioester.
We also examined the effect of 3-pentynyl-CoA on lactate reduction. While the inactivation of acrylate reduction was rapid, there was no inhibition of lactate reduction until >90% of the acrylate reduction was inactivated (Fig. 1). The rate of acrylate reduction was always greater than the rate of lactate reduction, consistent with acrylate being an intermediate in lactate reduction.
To ascertain that the inhibition of lactate reduction by 3- pentynyl-CoA was caused by inactivation of acrylate reduction, and not by inhibition of any activities involved in lactate/acrylate interconversion, the effect of 3-pentynyl-CoA on acrylate hydration was examined. Complete inactivation of acrylate reduction by 3-pentynyl-CoA increased the rate of acrylate hydration 2-fold. Thus, 3-pentynyl-CoA does not inhibit any enzyme involved in acrylate hydration. The increase in the rate of acrylate hydration suggests that there is a common intermediate that can partition between hydration to lactate and reduction to propionate.
Attempts pmol") and 6.7 mM acrylate (unlabeled) were added to assays as in Table I containing Fraction A.S. which had been exposed to sufficient 3-pentynyl-CoA to eliminate the ability to reduce acrylate. After incubating the assays for either 10 or 30 min, no [14C]acrylate was detected. Therefore, during lactate reduction, free acrylate is either not formed or released into solution.

Isotope Effect in the Conversion of Lactate to Propiomte-
The effect of substitution of 'H in the p position of lactate on its conversion to propionate was examined. Assay conditions were as in Table I trum was a triplet at 1.14 ppm (J = 7.6 Hz, 1.36H) and a multiplet at 2.24 ppm @.OH). The splitting of the &hydrogens requires two a-hydrogens. Therefore, no 'H from the / 3 position is transferred to the a position. Splitting of the ahydrogens into a multiplet is due to the presence of /3-lH1-, 6-'HZ-, and possibly p-lH3-propionate. From the relative peak areas and assuming two a-hydrogens, 1.36 solvent hydrogens were incorporated into the ,8 position. This shows that at least one 6-hydrogen is lost during reduction. The loss of slightly more than one @-hydrogen is probably due to equilibration occurring between lactate and acrylate and/or acrylate and propionate.
The fate of the a-hydrogen of lactate was determined. The Purification of Enzymes Involved in Acrylate Hydratwn-To purify the enzyme which catalyzes the hydration of acrylate, advantage was taken of the fact that this activity can be destroyed by exposure to 02. Thus, initially, we attempted to find a fraction which could restore activity to Fraction A.S. inactivated with 02. The elution profile obtained when Fraction A.S. was chromatographed on DE32 is shown in Fig. 2. Addition of Fraction I to 02-inactivated Fraction A.S. restores the ability of the Fraction A.S. to convert acrylate to lactate. In assays as described in Fig. 2

FIG. 2. Separation of Fractions I and I1 on DE32. Fraction
A.S. was chromatographed on a DE32 column, and proteins were eluted as described under "Experimental Procedures." Acrylate hydration assays as described under "Experimental Procedures" for Fraction AS. were used to assay Fraction I (A), except each assay contained 0.32 mg of 02-inactivated Fraction AS. Standard acrylyl-CoA hydration assays as described in Table I1 were used to assay Fraction I1 (A), except the final volume was 2.0 ml. Protein concentration (0) was determined as described under "Experimental Procedures." for at least 2 months with little loss of activity when stored at 4 "C in the anaerobic chamber.
A second fraction, Fraction 11, was found that when combined with Fraction I could hydrate acrylate to lactate (Fig.   2). However, in some preparations, the combination of Fraction I and Fraction I1 did not convert acrylate to lactate. We thought it likely that acrylate was first activated to acrylyl-CoA, and that in some preparations the enzyme(s) required for acrylate activation were separated from the enzyme(s) that were responsible for hydration. This line of reasoning led us to try acrylyl-CoA as substrate. In the presence of ATP and M$+, the combination of Fractions I and I1 always converted acrylyl-CoA to lactate. As will be shown later, the actual product in lactyl-CoA.
Fraction I1 was purified as decribed under "Experimental Procedures." Purified Fraction 11, E 11, was stored at 4 "C in the anaerobic chamber in the presence of NazSz04. The specific activity and yield of E I1 during the purification are shown in Table 11. Under these conditions, E I1 remains active for at least 1 month. Purified E I1 migrates as a single peak upon chromatography on a Mono-Q FPLC column. Upon electrophoresis on polyacrylamide under nondenaturing conditions, a single band was detected. On a denaturing gel, however, two bands were detected (Fig. 3). The intensity of each band was measured with a densitometer. The amount of Purification of E 11 E I1 was purified from 14 g of cells as described under "Experimental Procedures." E I1 was measured in standard acrylyl-CoA hydration assays containing 0.56 mM acrylyl-CoA, 0.17 mM CoA, 0.2 mg m1" Fraction I, and either 0.23 mg ml-' DE32, 0.058 mg ml-' Sepharose 6B or 0.034 mg ml-l FPLC-purified E 11.  The time course of lactate formation from acrylyl-CoA catalyzed by Fraction I and E I1 was determined. The rate of acrylate hydration is nonlinear as, after 4 min, the rate was only 18% of the initial rate. This is probably due to: 1) a significant portion of the acrylyl-CoA is converted to lactyl-CoA (After 4 min, 22% of the acrylyl-CoA was hydrated) during the assay; 2) product inhibition by the lactyl-COG; and 3) acrylyl-CoA is destroyed nonenzymatically via a Michaels addition. The half-life of acrylyl-CoA under the assay conditions is approximately 90 s (data not shown).
Neither Fraction I nor E I1 alone can convert acrylyl-CoA to lactyl-CoA. To determine if either Fraction I or E I1 converts acrylyl-CoA to an intermediate that is subsequently converted to lactate, complete assays as described in Table I1 munus either Fraction I or E 11, or both, were incubated at 37 "C for 5 min. The missing component(s) was added to each assay, and they were incubated for an additional 5 min. The amount of lactyl-CoA formed when either Fraction I or E I1 was initially omitted was less than or equal to the amount formed when both E I1 and Fraction I were omitted. The experiment was repeated, except the initial incubation was only 1 min, and the same results were obtained. Thus, neither

Lactyl-CoA
Dehydratase 13187 E I1 nor Fraction I converts acrylyl-CoA to a stable intermediate. Fraction I was further purified as described under "Experimental Procedures'' and stored in the anaerobic chamber at 4 "C. The purified protein will be referred to as E I. The specific activity and yield of E I during the purification are shown in Table 111. The specific activity and total activity of the E I are lower limits, since the assay is not linear in E I, i.e. increasing the amount of E I 2-fold results in a less than %fold increase in the amount of lactyl-CoA formed. The amount of E I present in assays at each stage of purification was adjusted such that the same amount of lactyl-CoA was formed in each assay. Therefore, the relative specific activities at each stage are comparable. The results of denaturing gel electrophoresis of E I are shown in Fig. 3. The protein with an apparent molecular weight of 27,000 represents 90% of the protein in purified E I. Thus, it is likely E I consists of a single polypeptide with a molecular weight of 27,000.
The cofactor requirements for acrylyl-CoA hydration by E I and E I1 are shown in Table IV. ATP and M e are absolutely required. The amount of lactyl-CoA formed is greater than the amount of ATP added; therefore, stoichiometric ATP hydrolysis does not occur. While GTP can replace ATP, ADP, and ADP-PNP, a nonhydrolyzable ATP analogue cannot. Thus, it is likely that ATP hydrolysis is required to "activate" the system. Acrylyl-CoA is the actual substrate, since acrylate and CoA (+-acetyl-coA and acetyl phosphate) did not substitute for acrylyl-CoA. The addition of 0.26 mM CoA resulted in 22% inhibition of acrylyl-CoA hydration. 8-    0.4). Treatment of Factor I1 with phosphodiesterase or alkaline phosphatase did not alter its retention on HPLC, suggesting that Factor I1 contains neither a phosphodiester bond nor a terminal phosphate, as expected for riboflavin. To obtain further evidence that Factor I1 is riboflavin, both were treated with KIO,. After treatment with KI04, both the product from Factor I1 and that derived from riboflavin co-chromatographed on HPLC. The UV-visible spectrum of each was identical (A,,, = 442, 370, and 266 nm), and they co-migrated on TLC systems A (RF = 0.61 (major), 0.71 (minor)) and B ( R F = 0.65). These results establish that Factor I1 is riboflavin. Furthermore, it was found that under the conditions used to isolate Factor 11, FMN and FAD are stable. Therefore, Factor 11 is not derived from FMN or FAD during the course of isolation.
Factor I from MeOH-precipitated E I1 had the same retention time as FMN on HPLC3 Factor I has a UV-visible spectrum identical to FMN (A-FMN = 266, 373, and 445 nm; A,, Factor I = 266,372, and 444 nm) and co-chromatographed with FMN on TLC systems A and C. When Factor I was treated with alkaline phosphatase, the product co-chromatographed with riboflavin in HPLC and on TLC systems A and C, consistent with the identification of Factor I as FMN. Alkaline phosphatase-treated Factor I and riboflavin were incubated with KIO,. The product derived from Factor I co-chromatographed with the product obtained from riboflavin in HPLC and on TLC systems A and C. These results establish that Factor I is FMN.
The amount of FMN was equal to that of riboflavin as determined by comparing peak areas after HPLC. The absolute amount of FMN on E I1 was also estimated by HPLC.
There were 0.5 mol of FMN (and, therefore, 0.5 mol of riboflavin) per mol of E 11. This number represents a lower limit on the amount of flavin on E I1 because 1) O2 causes release of flavin from E I1 and E I1 is exposed to significant quantities of 0, during purification and 2) not all of the flavins may actually be liberated from E 11.
Identification of Lactyl-CoA-Lactate, a product of acrylyl-CoA hydration by E I and E 11, is measured colorimetrically.
In this method, the lactate is first oxidatively decarboxylated to acetaldehyde under strongly acidic conditions and then the amount of acetaldehyde determined. Treatment of the assay products with base sufficient to hydrolyze any thioesters did not increase the amount of lactate detected colorimetrically. It is likely that under the assay conditions, lactyl-CoA can be When E I1 was precipitated with HClO,, the retention time of Factor I was different from any of the standards and varied from 16 to 29 min. This material was not further investigated.
hydrolyzed to lactate so that the colorimetric assay does not distinguish between lactyl-CoA and lactate. Therefore, the product resulting from acrylyl-CoA hydration was examined further. The products of acrylyl-CoA hydration assays (see Table IV, Complete Reaction Mixture) were chromatographed on the PRP HPLC column. The eluent was collected, neutralized, and dried and then assayed for lactate colorimetrically. Two peaks that gave a positive reaction in the colorimetric assay for lactate were observed. The first, which cochromatographed with lactic acid, had a k' = 0.3 and contained 11% of the apparent lactate. The second had a k' = 1.2 and contained the remaining material that gave a positive result in the colorimetric assay. This second peak was repurified, again using the PRP column. The NMR spectrum of this material represents the sum of the spectra of lactate and CoA. No sulfhydryl groups were detected with 5,5'-dithiobis(2-nitrobenzoic acid) in the purified product, whereas after base hydrolysis, the concentration of sulfhydryl groups measured by 5,5'-dithiobis(2-nitrobenzoic acid) was equal to the concentration of lactate measured colorimetrically. The UV spectrum of the purified material was very similar to the spectrum of acetyl-coA. Thus, the product of acrylyl-CoA hydration is lactyl-CoA, and the colorimetric assay for lactate will measure lactyl-CoA and lactate equally well.

DISCUSSION
Cell-free extracts of C. propwnicum catalyze the conversion of lactate to propionate. Our results indicate that this conversion proceeds through CoA thioesters and that acrylyl-CoA is probably an intermediate (see equation below).
The requirement for CoA and acetyl phosphate suggests the presence of a CoA transferase system involved in the formation of lactyl-CoA. A CoA transferase from C. propionicum that transfers CoA from acetyl-coA to lactate, acrylate, or propionate was recently isolated (22). When [3-'H3]lactate was used, an isotope effect was found in the conversion of lactate to propionate, indicating that abstraction of a 8hydrogen is partially rate-limiting. This is consistent with intermediate formation of acrylyl-CoA and would not be expected if lactyl-CoA was reduced directly to propionyl-CoA. The existence of an acrylyl-CoA reductase was demonstrated, and it was shown that inactivation of this enzyme abolishes the reduction of lactate to propionate but not the hydration of acrylate to lactate. Finally, we have purified two enzymes which together catalyze the conversion of acrylyl-CoA to lactyl-CoA.
The enzyme system which converts lactyl-CoA to acrylyl-CoA is surprisingly complex. It consists of two proteins which we designated E I and E 11. E I1 consists of two nonidentical subunits, and E I consists of one subunit. The reaction requires ATP in substoichiometric amounts. While nonhydrolyzable ATP analogues cannot replace ATP, GTP can. Thus, it is likely that ATP hydrolysis is required. The mechanism by which ATP activates the system is unknown, and we were unable to demonstrate a time dependence of this activation (data not shown). Our results differ from those reported earlier (22) that ATP inhibited lactate reduction. This discrepancy may be due to the greater purity of our preparations. Furthermore, two flavins, riboflavin and FMN, are associated with E 11. This is the first enzyme we know of that uses riboflavin.
The mechanism of the hydration is, for reasons pointed out in the Introduction, of considerable interest, and the possible role of the flavins and ATP is intriguing. It was proposed that 2-hydroxygiutarate dehydration involves an enzyme-bound hydroxyl radical as shown in Scheme 3a (23). While the involvement of a radical during dehydration of 2-hydroxycarboxylic acids is attractive, we think an enzyme-bound hydroxyl radical is highly unlikely. Instead, we propose the mechanism shown in Scheme 3b. Initially, a P-hydrogen radical is abstracted. The resultant carbon radical combines with a group M, which could be a highly reduced metal or a flavin. The group M donates one electron and hydroxide is eliminated. M is reduced to its original state by the initially abstracted hydrogen. While we have no direct evidence for this mechanism, a number of workers have demonstrated that