On the Metabolism of Organic Acids by Clostridium Acetobutylicum Part VII 307 Muscle and Was Named Crotonase5)

[Agr. Biol. Chem., Vol.25, No.4, p.306～313, 1961]

On the Metabolism of Organic Acids by Clostridium acetobutylicum

Part VII. Reduction of Crotonic Acid

By Hideo KATAGIRIand Tsunetake SUGIMORI Department of Agricultural Chemistry,Faculty of Agriculture, Kyoto University ReceivedSeptember 20, 1960

Molecular hydrogen was capable of generating the reduced coenzymes by the hydrogenase system of Clostridium acetobutylicum. It was pointed out that crotonic acid was activated and reduced quantitatively to butyric acid by intact cells and cell-free preparation of this organism in the presence of acetyl phosphate, 2, 3-dimercaptopropanol (BAL) and hydrogen gas.

INTRODUCTION tyric acid would involve one step of dehydra- In the fermentation of hexoses by acetone- tion and one step of reduction as described butanol fermentation bacteria, it is well below. known that pyruvic acid, which is derived or from hexose-diphosphate through the action of metalloaldolase1), is cleaved to acetyl-CoA and carbon dioxide2,3). It is with no doubt that two molecules of acetyl-CoA are con- Two types of unsaturated acids, namely both densed in forming acetoacetyl-CoA, and that vinylacetic and crotonic acids, are proposed as the latter is a common precursor of both the dehydrated intermediates. According to acetone and butyl compounds such as butyric the investigations on fatty acids metabolism acid and butyl alcohol. These butyl com- in animals, so-called Knoop's β-oxidation pounds are formed from acetoacetyl-CoA mechanism is shown as follows. through several steps of reduction. Gavard and Descourtieux4) found that the reduction R-CH2-CH2-CH2-CO-SCoA →R-CH2-CH=CH-CO-SCoA of acetoacetyl-CoA to β-hydroxybutyryl-CoA →R-CH2-CHOH-CH2-CO-SCoA was coupled with the dehydrogenation of →R-CH2-CO-CH2-CO-SCoA triose-phosphate in Embden-Meyerhof-Parnas →R-CH2-CO-SCoA+CH3-CO-SCoA→etc. pathway. In regard to the pathway from β- In this scheme -COSCoA means an acyl-CoA hydroxybutyryl-CoA to butyric acid, however, derivative. It was pointed out that each there has been no clear explanation with reaction in this scheme was reversible. These Clostridium acetobutylicum. As a matter of facts suggest that the pathway from β-hydroxy- course, it is expected that the pathway from butyryl-CoA to butyryl-CoA or butyric acid

β-hydroxybutyryl-CoA to butyryl-CoA or bu- in Cl. acetobutylicum might be analogous to

1) R. Gavard, Compt. rend., 238, 1620 (1954). the reversal of β-oxidation. The enzyme 2) R. S. Wolfe and D. J. O'Kane, J. Biol. Chem., 205, 755 (1953) . which catalyzes dehydration of β-hydroxy- 3) E. R. Stadtman, J. Biol. Chem., 196, 535 (1952). 4) R. Gavard and H. Descourtieux, Compt. rend., 239, 201 (1954). butyryl-CoA to crotonyl-CoA was isolated from On the Metabolism of Organic Acids by Clostridium acetobutylicum Part VII 307 muscle and was named crotonase5). But an MATERIALS AND METHODS enzyme corresponding to crotonase has not Enzyme Preparations been found in Clostridium bacteria. It is Cl. acetobutylicum grown in Speakman's culture presumable that the reduction reaction from medium was collected by centrifugation. After wash- butenoyl-compound to butyric acid would be ing with distilled water, the wet cells were suspended somehow coupled with dehydrogenation of in distilled water, 0.3 per cent sodium sulfide solution triose-phosphate or oxidative phosphoroclastic or 0.02M 2, 3-dimercaptopropanol (BAL) depending on the purpose of each experiment. reaction of pyruvate, because no other reac- For preparing dried cells, above mentioned cell-paste tion in which hydrogen atom is generated was spread as a film on the surface of a petri-dish and could be detected in this organism. From dried in vacuo on sulfuric acid. these points of view, the reduction of croto- Cell-free extract was prepared through sonic oscilla- nate by this organism was investigated. tion of forty ml of cell-suspension in 0.02M BAL It is well known that Clostridium bacteria containing about four grams of wet cells at 9KC for thirty minutes followed by centrifugation at 9,000rpm possess a hydrogenase system. Two types of hydrogenase are known in various bacteria6). for thirty minutes. One of them is found in Acetobacter, Azoto- Manometric Techniques for Hydrogen Uptake bacter and Hydrogenomonas, and is an irre- Warburg's manometers were used. Gas phase was exchanged by pure hydrogen, and the reaction was versible enzyme. This type of hydrogenase carried out at 30℃. activates molecular hydrogen in forming hydrogen atom. Another is the reversible Analysis of Volatile Acids Analysis of volatile acids such as crotonic and butyric one, which exists in Clostridium, Desulfo- acids was carried out by microdiffusion method and vibrio, anaerobic Micrococcus and Coli-aero- paper chromatography. Details of the methods were genes bacteria. The latter is not only capable similar to those described in Part VI8). of activating molecular hydrogen, but also Preparation of Acetyl-CoA Fraction liberating molecular hydrogen from reduced. According to the method by Peel and Barker9), coenzymes. It is with no doubt that the pro- acetyl phosphate and dried cells of Cl. acetobutylicum duction of hydrogen gas by Cl. acetobutylicum were treated as follows. Two ml of the reaction mix- is caused by this type of hydrogenase, since ture, which contained 10 micromoles of acetyl phos- the existence of hydrogenase in Cl. acetobutyli- phate, 100mg of dried cells, 0.5ml of 0.2M phosphate cum was demonstrated and its reversible func- buffer of pH 8.0 and 0.03 per cent sodium sulfide, was incubated at 30℃ for fifteen minutes. The mixture tion was also verified by Hongo7). In other was then adjusted to pH 3.5 by hydrochloric acid, and words, reduced coenzyme and molecular kept in boiling water bath for fifteen minutes. Un- hydrogen are mediated through reversible reacted acetyl phosphate is destroyed by this heat action of hydrogenase in this organism. It is treatment, but acetyl-CoA is not affected. After cool- expected, therefore, that molecular hydrogen ing, the mixture was neutralized by sodium hydroxide would be capable of generating the reduced and the solids were spun off. The clear supernatant coenzymes in the presence of active hydro- thus obtained was used as acetyl-CoA fraction. genase system. An attempt was made to show Miscellaneous Materials the utilization of gaseous hydrogen as the Crotonic acid, methylene blue, safranin T and BAL reducing agent in the reduction of crotonate were all of reagent grade. Hydrogen gas was generated to butyrate by this organism. through the reaction of zinc flour with dilute sulfuric acid and washed by acid, alkali, and water. Acetyl 5) J. R. Stern and A. del Campillo, J. Am. Chem. Soc., 75, 2277 phosphate was kindly supplied from Dr. I. Takeda. (1953). 6) H. Gest, Proc. Intern. Symp. Enz. Chem., 211 (1957). 8) H. Katagiri and T. Sugimori, This Journal, 25, 300 (1961). 7) M. Hongo, J. Agr. Chem. Soc. Japan, 32, 29 (1958). 9) J.L. Peel and H. A. Barker, Biochem. J., 62, 323 (1956). 308 Hideo KATAGIRI and Tsunetake SUGIMORI

Methyl-viologen was provided by courtesy of Dr. K. TABLE I. REDUCTION OF DPN AND FAD BY Ito. CELL-FREE EXTRACT

RESULTS AND DISCUSSION

Activation of Molecular Hydrogen by Cell-suspension and Cell-free Extract Washed cells suspended in distilled water

were capable of reducing methylene blue in vacuo in rather short period when the suspension was newly prepared. This activity, how-

ever, was rapidly depressed after storage for and incubated with sodium crotonate under several hours. Under atmospheric hydrogen, atmospheric hydrogen at pH 7.2 at 30℃. A these suspensions maintained such a reducing small amount of hydrogen was taken up. It activity for much longer periods. When sus- is presumable that the metabolic change of pended in 0.02M BAL, the reducing activity fatty acids would proceed in the intermediary for methylene blue was greatly stimulated and state of such acids, namely, energy-rich acyl maintained for about one week in ice-box. derivatives as compared with the mechanism Several dyestuffs having redox potentials lower found in animals as described previously. As than methylene blue, for instance safranin T acetyl-CoA is the precursor of butyl com- and methyl-viologen, were also readily reduced pounds formation of this organism, it is ex- by atmospheric hydrogen by cells of this organ- pected that intermediary energy-rich acyl ism suspended in BAL. These reduction was compounds might be acyl-CoA. It is proposed never detected when the cell-suspension was by Stadtman10) that CoA-linked reaction re-

preheated at 100•Ž for ten minutes. It is quires reducing agents so as to make oxidized therefore evident that above mentioned reduc- CoA into active CoA-SH form. In this con- tion of several redox dyes is an enzymatic nection, several thiol compounds were ex- reaction. In other words, Cl. acetobutylicum amined. From the experiments on the effects is capable of activating molecular hydrogen of each 0.02 to 0.001M of several thiol com- and transporting activated hydrogen to pounds such as sodium sulfide, glutathione, methyl-viologen (E0=-0.446v), safranin T cysteine, thioglycollate and BAL, it was point- (E0=-0.289v) and methylene blue (E0= ed out that BAL was the most effective in +0.011v). stimulating the activity to take up hydrogen Employing a cell-free extract of the same with crotonate by cell-suspension. Table II organism, the enzymatic reduction of above shows the results obtained by use of dried described dyes was further verified under cells. atmospheric hydrogen. Moreover, it was con- It is evident from Table II that the hydro- firmed that the cell-free extract was able to gen uptake is enzymatic. The final molar reduce pyridine nucleotides and flavin nucleo- amount of volatile acids is equivalent to the tides readily with the aid of molecular hydro- initial within an experimental error. This gen. Table I indicates some experimental fact suggests that the carboxyl radical of results. crotonate is not affected through the uptake Reduction of Crotonate to Butyrate of hydrogen. Both crotonic and butyric acids Cells grown in Speakman's culture medium were detected by paper chromatography of

for twenty hours were suspended in water 10) E. R. Stadtman, J. Biol. Chem., 196, 527 (1952). On the Metabolism of Organic Acids by Clostridium acetobutylicum Part VII 309

TABLE II. REDUCTION OF CROTONATE TO BUTYRATE

acid distillate from the reaction mixture. Therefore, it is concluded that dried cells of Cl. acetobutylicum suspended in BAL did reduce crotonic acid to butyric acid utilizing molecular hydrogen as the hydrogen donor. It is assumed that the activation of thiol radical of CoA by BAL would be essential for the reduction of crotonate. It would suggest the participation of CoA-linked enzyme action. Effect of Acetyl Phosphate on the Reduction of Crotonate FIG. 1. Effect of acetyl phosphate on the reduction It was mentioned above that the metabolism of crotonate. of fatty acids by Cl. acetobutylicum is considered to resemble the fatty acid cycle in animals. Accordingly, it is presumable that each intermediary compound would be in the energy-rich acyl form, or acyl-CoA. With Cl. kluyveri, Stadtman11) pointed out the exis- crotonate. In this experiment, considerably tence of CoA transphorase, which transferred small amount of crotonate (6μmoles), was CoA from acetyl-CoA to propionic acid in used instead of the case in Table II (60μ forming propionyl-CoA and acetic acid. This moles), and the amount of hydrogen taken enzyme is also capable of transferring CoA to up was equal to the amount of crotonate various fatty acids. It is shown with Clostri- added. These results indicate that one mole- dium bacteria that acetyl-CoA is derived from cule of crotonate incorporated one molecule acetyl phosphate and CoA by phosphotrans- of hydrogen. Such a quantitative hydrogen acetylase action as follows. uptake was also observed with intact cells of

CH3-COOPO3H2+CoA-SH this organism as shown in Fig. 2. →CH3-CO-S-CoA+H3PO4 Consequently, it is believed that acetyl phos- From these points of view, acetyl phosphate phate plays a role in activating crotonate as follows. was added to the reaction mixture. As was shown in Fig. 1, acetyl phosphate revealed Acetyl phosphate+CoA→Acetyl-CoA+phosphate Phosphotransacetylase remarkable acceleration on the reduction of Acetyl-CoA+crotonate→Crotonyl-CoA+acetate 11) E. R. Stadtman, J. Biol. Chem., 203, 501 (1953). CoA transphorase 310 Hideo KATAGIRI and Tsunetake SUGIMORI

mechanism. If this is the case, it is easy to expect that acetyl-CoA would reveal predomi- nant effect on the activation and consecutive reduction of crotonate. Fig. 3 indicates the effect of acetyl-CoA fraction which was prepared from acetyl phosphate and dried cells of Cl. acetobutylicum as described above in Methods. On the whole, the reduction of crotonate by molecular hydrogen is illustrated as follows. Activation process:

(1) FIG. 2. Reduction of Crotonate by Intact Cells.

(2)

The verification of enzymatic formation of (3) crotonyl-CoA by Cl. saccharobutylicum12) is also regarded as support for this activation

(4)

In activation process, acetyl phosphate plays a role in supplying energy-rich acyl bond, and acetyl-CoA is formed through the action of phosphotransacetylase. Active CoA, the ac- ceptor of acetyl radical, is probably present in the cells and is maintained in the active-SH form by BAL in the reaction mixture. Acetyl- CoA thus formed would activate crotonate by CoA transphorase action as shown in the equation (2). In a cyclic reduction process, no loss of acyl- FIG. 3. Effect of Acetyl-CoA Fraction on the Reduction CoA bond can be estimated. It is believed of Crotonate and Inhibitory Action of Acetate and that a small amount of crotonyl-CoA supplied, Arsenate. to the reaction system starts the cyclic reduction system, reducing crotonate to butyrate continuously. Inhibitory Action of Acetate and Arsenate As shown in Fig. 3, 0.01M of acetate or

12) J. Szulmajster, Compt. rend., 238, 2461 (1954). arsenate revealed severe inhibition on the re- On the Metabolism of Organic Acids by Clostridium acetobutylicum Part VII 311 duction of crotonate by cell-suspension of Cl. of crotonate. acetobutylicum. Experiments with Cell-free Extract It is considered that acetate accelerates the Cell-free extract was prepared as described reversal action of the above equation (2), and in Methods. Using this preparation, it was leads to the accumulation of acetyl-CoA. verified that the quantitative reduction of Moreover, acetate would replace crotonate in crotonate by hydrogen could occur in the the equation (4). This means decrease in crotonyl- and butyryl-CoA in the cyclic reduc- presence of both BAL and acetyl phosphate. The results are shown in Fig. 4. In this case, tion process, and the accumulation of acetyl- the lag period of about forty to fifty minutes CoA. Resultant acetyl-CoA would be con- was observed before the quantitative uptake verted to acetoacetyl-CoA by β-ketothiolase of hydrogen. It is assumed that the lag which would be subsequently reduced. This condensation reaction involves the net loss of period would be caused by the limiting rate of CoA transphorase. acyl-CoA bond, and therefore, it is assumed Summarizing the experimental results and that acyl-CoA bond would disappear from discussions, the mechanism of the reduction the reaction system when sufficient amount of free acetate was present. About the inhibitory action of arsenate, the following discussion would be available. It is known that phosphotransacetylase catalyzes cleavage of acetyl-CoA into CoA and free acetate in the presence of arsenate. Ac- cordingly, arsenate causes the liberation of acetate, and the latter forms further acetyl- CoA from other CoA derivatives in the reaction system through the action of CoA transphorase. These processes are similar to the case of acetate inhibition. In any way, the FIG. 4. Reduction of Crotonate by Cell-freeExtract. addition of acetate or arsenate leads to the net loss of acyl-CoA bond in the reaction system and consequent depression in the reduction 312 Hideo KATAGIRI and Tsunetake SUGIMORI of crotonate by hydrogen would be illustrated (1) schematically as follows. In this scheme, materials added to the reaction mixture are boldfaced. CoA is originally present in the cells. Many attempts to isolate the enzymes concerning the reduction of crotonate have been unsuccessful. Sensitivity of the enzymes of Clostridium bacteria to oxygen tension and temperature may be a reason why the investigations of the multi-enzyme system of this bacterium are still delayed. (2) Effect of FAD on the Reduction of Crotonate FAD revealed a coenzymic action in the dehydrogenation of butyrate by the cell-free extract of Cl. acetobutylicum as shown in Table III. Meanwhile, it is known in animal tissues that ethylene reductase, which catalyzes the redox reaction between crotonyl-CoA and butyryl-CoA, is a cuproflavoprotein13), and FIG. 5. Effect of FAD on the Reduction of Crotonate. contains FAD as the prosthetic group14). In this connection, the role of reduced FAD for the reduction of crotonate by the cell-free ex- this procedure, the yellow color of oxidized tract was investigated. FAD vanished. After transfusing the contents

TABLE III. DEHYDROGENATION OF BUTYRATE into the cuvette of spectrophotometer, each 0.2ml of sodium crotonate (20 μmoles) or water (for control test) was added and the increase in optical density at 450mμ for FAD or decrease at 340mμ for DPN was pursued.

Results are shown in Fig. 5. The increase in

optical density at 450mμ was verified, but no decrease in optical density at 340mμ for DPN was observed. These results indicate that FAD, not DPN, is coenzymic for the reduction

A quarter ml of cell-free extract, 0.5ml of of crotonate by this organism. 0.2M phosphate buffer of pH 7.0 and 0.042 Consequently, it is probable that hydrogen

μmole of FAD or 0.039 μmole of DPN were atoms activated by hydrogenase are transfer- inserted in a Thunberg-Borsook's tube Total red via FAD to the active crotonyl compound volume was 2.8ml. Head space of every tube in forming butyrate. was filled with hydrogen, and the tube was SUMMARY preincubated at 30℃ for sixty minutes. By It was confirmed that the cell-free extract 13) H. R. Mahler, J. Biol. Chem., 206, 13 (1954). 14) F. Lynen and S. Ochoa, Biochim. Biophys. Acta, 12, 299 (1953). of Cl. acetobutylicum was able to reduce pyri- Effect of Gamma-Ray upon Food Microorganisms. Part VIII 313 dine nucleotide and flavinenucleotide readily hibited by arsenate or acetate. It was pointed with the aid of molecular hydrogen. out that the reduction of crotonate was ac- The same preparation formed butyrate from celerated by FAD to some extent. crotonate utilizinghydrogen gas as reducing The mechanism of the reduction of croto- agents. This reduction proceeded quantita- nate to butyrate under atmospheric hydrogen tively by the addition of acetyl phosphate was considered to involve the activation of (or acetyl-CoA) and 2,3-dimercaptopropanol both crotonate and molecular hydrogen, and (BAL) at pH 7.2, while it was severelyin- the reduction of crotonyl-CoA to butyryl-CoA.

[Agr. Biol. Chem., Vol.25, No.4, p.313～318, 1961]

Effect of Gamma-Ray upon Food Microorganisms

Part VIII. Influence of the Addition of some Preservatives or Antibiotics

By Wataru WATANABE

Department of Agricultural Chemistry, Tokyo University of Education and the Niko Institute of Food,Niko co., Ltd. Received September 13, 1960

Possibility of enhancement in the lethal effect of gamma-ray by the aid of various sub- stances was investigated. This report deals with the survival-ratios of four strains of bacteria in the nutrient or the non-nutritious pure agar medium, to which various kinds of preservatives or antibiotics were added.

INTRODUCITION fore, during or after gamma-ray irradiation

The foodstuffs. irradiated with gamma-ray affecting the survival of bacteria, and has re- can be preserved for a longer period owing to ported that none of them enhanced the lethal the decreased number of microorganisms in effect of gamma-ray in nutritious state1～7). them. However, it is often observed that Heat processing or the addition of antibio- many foodstuffs undergo undesirable change tics or various preservatives are some of such in qualityby the irradiation.Therefore, it is methods. Shewan has conducted studies con- necessary to investigate methods that enhance cerning the preservation of fish meat by a the lethal effect of gamma-ray, by which the combined application of antibiotics and gam- foodstuffs can be preserved by means of irradi- 1) W. Watanabe, This Journal, 22, 68 (1958). ation at a dose causing no such undesirable 2) W. Watanabe, This Journal, 22, 255 (1958). change. 3) W. Watanabe, This Journal, 23, 73 (1958). 4) W. Watanabe, This Journal. 24, 75 (1960). The author has conducted a series of studies 5) W. Watanabe, This Journal. 24, 84 (1960). 6) W. Watanabe, This Journal. 24, 673 (1960). concerning the environmental conditions be- 7) W. Watanabe, This Journal. 24, 681 (1960).