THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 257. No. 22, Isue of November 25, pp. 13749-13756, 1982 Printed in U.S.A.

S-chloro-~-alanine Chloride-lyase (Deaminating) of Pseudomonas putida CR 1-1 PURIFICATION AND CHARACTERIZATION OF A NOVEL ENZYME OCCURRING IN ~-CHLORO-D-ALANINE- RESISTANT PSEUDOMONADS*

(Received for publication, January 13, 1982)

Tom Nagasawa, Haruyuki Ohkishi$, Bunsei Kawakami, Hironori Yamano, Hidekazu Hosono, Yoshiki Tad, and Hideaki Yamada From the Department of Agricultural Chemistry, Kyoto University, Kyoto, SOS, Japan and *Central Research Laboratory, Mitsubishi Chemical Industries, Yokohama, 227, Japan

A novel enzyme catalyzing cleavage of 3-chloroa- alanine to pyruvate, ammonia, and chloride ion is dis- tributed in some pseudomonads which have a resist- ance to high concentrations of 3-chloro-D-alanine. Pseu- domonas putida CR 1-1 (AKU 867) was found to have the highest activity of enzyme, which was inducibly formed by the addition of 3-chloro-~-alanine to the medium. The enzyme, tentatively called 3-chloro-~-aI- anine chloride-lyase, was purified from €? putida CR 1- 1 in seven steps. After the last step, the enzyme ap- peared to be homogeneous by the criteria of polyacryl- amide gel electrophoresis, analytical ultracentrifuge, and double diffusion in agarose. The enzyme has a molecular weight of about 76,000 and consists of two subunits identical in molecular weight (approximately 38,000). The enzyme exhibits absorption maxima at 278 nm and 418 nm, which are independent of the pH (6.0-9.0), and contains 2 mol of pyridoxal 5’-phosphate/ mol of the enzyme. The holoenzyme is resolved to the apoenzyme by incubation with phenylhydrazine and reconstituted by the addition of pyridoxal-P. The apoenzyme can be crystallized by adding ammonium sulfate. 3-Chloro-~-alanine chloride-lyase catalyzes an a,P-elimination reaction of 3-chlors-~-alanine and also, but to a lesser extent, D-cysteine and D-cystine. The enzyme also catalyzes a 8-replacement reaction of chlo- rine of 3-chloro-~-alanine with hydrosulfide to yield D- cysteine. The important role of this novel &lyase en- zyme in the detoxication of 3-chloro-~-alanine by P. putida CR 1-1 is also discussed.

3-Chloro-~-alanine has recently been shown to bind effi- ciently with transaminases and decarboxylases and to undergo a$-elimination reaction in situ (1-5). In some cases, during the course of these reactions, these enzymes were irreversively inactivated presumably by the reaction of an enzyme-bound aminoacrylic acid with some functional groups in the protein (1). Such self-destructive inactivation was also observed in the reaction of 3-chloro-D-alanine with amino acid racemase (6) , serine transhydroxymethylase (7), and D-amino acid trans- aminase (8). Thus, some of pyridoxal 5’-phosphate-dependent enzymes responsible for the metabolism. of amino acids were

* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dedicated to Professor Alexander E. Braunstein on the occasion of his 80th birthday.

inactivated by 3-chloroalanine. In search of the physiological effect of 3-chloroalanine on

the bacterial growth, Manning et al. (8,91) have demonstrated that 3-chloro-~-alanine is an effective antibacterial agent in vivo against Diplococcuspneumonia, Streptococcuspyogenes, Bacillus subtilis, and Escherichia coli. They indicated that the antibacterial action of this 3-chloro-])-alanine was due to the inactivation of both D-amino acid trarrsminase and alanine racemase (8,9), namely the preclusion of the biosynthesis of the peptidoglycan layer of the bacterial cell wall. Kaczorowski et al. (10, 11) explained that the greater efficiency of the D- isomer of chloroalanine compared with the L-isomer in inhi- bition of bacterial growth could be the result of a distinct path for enzymatic processing of each isomer by the membrane vesicles, 3-Chloro-~-alanine caused rapid inactivation of the dehydrogenase-coupled transport systems in the membrane vesicles. Unlike the D-isomer, 3-chloro-~-alanine did not in- activate the transport. These experimen:ts (12) showed that the oxidation of 3-chloro-D-alanine by a membrane-bound D- alanine dehydrogenase resulted in the inactivation of dehy- drogenase-coupled active transport systems in the membrane vesicles. The D-alanine dehydrogenase-catalyzed oxidation product was identified as chloropyruvate. They suggested this keto acid interfered with the ability of the energized mem- brane state to be used for the transport of most solutes.

These physiologically interesting effects of 3-chloro-o-ala- nine, as a suicide substrate or an inhibitor of active transport systems in membrane vesicles, prompted us to search for strains of bacteria resistant to this compound. When 3-chloro- D-danine was added to the culture medium at a concentration higher than 30 mM, the growth of most of the microorganisms was completely inhibited. Recently we isolated some bacteria belonging to the genus Pseudomonas which have a resistance to 3-chloro-~-alanine (13). These resistant pseudomonads grew well in the medium containing a high concentration of 3- chloro-D-alanine. In the crude cell extracts of these resistant strains, an enzyme activity, which degrades 3-chloro-~-ala- nine, was found. The enzyme was inducibly formed only by 3- chloro-malanine. We have attempted to obtain a homogene- ous enzyme preparation from Pseudomonas putida CR 1-1 (AKU 867). The purified enzyme catalyzed not only the a,P- elimination reaction of 3-ChlOrO-D-alanine to form pyruvate, ammonia, and chloride ion, but also the P-replacement reac- tion of 3-chloro-~-alanine in the presence of a high concentra- tion of sodium hydrosulfide to form D-cysteine. In this paper, we describe the purification and initial characterization of this novel enzyme which we have tentatively designated as “3- chloro-D-danine chloride-lyase (deaminating).”

13749

13750 a-chloro-~ -alanine Chloride-lyase (Deaminating) of P. putida EXPERIMENTAL PROCEDURES

Materials-3-Chloro-~~-alanine, 3-chloro-~-alanine, and 3-chloro- D-alanine were synthesized from DL-Serine, L-serine, and D-Serine, respectively, according to the method of Walsh et al. (14). DEAE- Sephacel, phenyl-CL-Sephmose, and a kit of low molecular weight standards were products obtained from Pharmacia. Pig heart lactate dehydrogenase (EC 1.1.1.27) was purchased from Oriental Yeast (Japan). Crystalline glutamate dehydrogenase (EC 1.4.1.2) from bo- vine liver, crystalline L-alanine dehydrogenase (EC 1.4.1.1) from B. subtilis, crystalline D-aIIIinO acid oxidase (EC 1.4.3.3) from hog kidney, catalase (EC 1.11.1.6) from bovine liver, D-cycloserine, D-penicilla-

pane-1-carboxylic acid were obtained from Sigma. Lyophilized alcohol mine, L-penicillamine, P-2- thienyb~~-alanie , and l-aminocyclopro-

dehydrogenase (EC 1.1.1.1) from yeast was purchased from Boehrin- ger. A membrane filter (Diaflo ultrafilter, UM 20) was obtained from Amicon. All other chemicals used were from commercial sources and of reagent grade quality. Ampholytes required for isoelectric focusing were the products of LKB-Produktor AB.

Microorganism and Conditions of Culture-P. putida CR 1-1 (AKU 867) was selected as a likely source of the enzyme for the purification. The basal medium for cultures consisted of 0.5 g of Polypepton (Daigo, Japan), 0.5 g of yeast extract (Oriental Yeast, Japan), 0.01 g of pyridoxine.HC1, and 0.1 g of NaC1/100 ml of tap water. The pH of the medium was adjusted to 7.0 by the addition of 4 M NaOH. P. putidu CR 1-1 was collected from an agar slant of the basal medium and inoculated into a subculture. The subculture (3 liters) was shaken reciprocally at 30 "C for 18 h, and, in turn, inocu- lated into a 100-liter jar fermentor containing 70 liters of the basal medium supplemented with 140 g of 3-chloro-~~-alanine.HCI. 3- Chloro-DL-alanine. HCI was sterilized by a Millipore filter (type HA, 0.45 pm). Incubation was carried out at 28 "C for 18 h with aeration (35 liter/min). The cells grown from 350 liters of the broth were harvested by a continuous flow centrifuge and washed with 0.15 M NaCl containing 0.1 mM EDTA. The yield of wet cells was approxi- mately 8.6 g/liter of the medium.

Enzyme Assay and Definition of Units-The enzymatic a$-elim- ination reaction was followed by routinely measuring the amount of pyruvate liberated from 3-chloro-~-alanine using a spectrophotomet- ric method with pig heart lactate dehydrogenase and NADH. The reaction was carried out at 30 "C in cuvettes containing 2 ml of 0.1 mmol of potassium phosphate (pH 8.0), 2.5 pmol of 3-chloro-~-alanine, 0.1 pmol of pyridoxal-€', 0.26 pmol of NADH, 10 units of pig heart lactate dehydrogenase, and an appropriate amount of the enzyme. The reaction was started by adding the substrate solution, and the decrease in absorption at 340 nm due to the consumption of NADH was monitored spectrophotometrically. The amount of pyruvate pro- duced was calculated from the molar extinction of NADH, 6220 "' cm" (Assay I).

In some cases, the reaction was carried out by omitting NADH and lactate dehydrogenase from the reaction mixture described above. The reaction was stopped by the addition of 0.25 ml of 30% trichlo- roacetic acid. The amount of pyruvate formed was determined by using a deproteinized filtrate according to the method of Friedemann and Haugen (15) (Asssy 11).

For the P-replacement reaction, the reaction system consisted of 10 pmol of 3-chloro-D-alanine, 0.1 mmol of sodium hydrosulfide, 0.1 p o l of pyridoxal-€', 0.1 mmol of potassium phosphate (pH 7.5), and an appropriate amount of the enzyme in a final volume of 1.0 ml. After incubation at 30 "C for 10 min, the reaction was stopped by the addition of 0.2 ml of 30% trichloroacetic acid. After centrifugation, cysteine in the supernatant solution was determined by Gaitonde's ninhydrin method (16) (Assay 111).

Protein was determined by its absorption at 280 nm. The absorp- tion coefficient of 0.489 mg" ml. cm", used throughout, was obtained by absorbance and dry weight determinations.

One unit of the enzyme is defined as the amount of the enzyme that catalyzes the formation of 1 pmol of pyruvate/min under the conditions of Assay I. The specific activity is expressed as units/mg of protein.

Other Enzyme Assays-Alanine racemase activity was determined as follows. L-Alanine (15 mM) was incubated with the purified 3- chloro-D-alanine chloride-lyase (4.2 to 21 pg) in the presence of 0.1 mM pyridoxal-P for 2 h at 37 "C in 0.1 M potassium phosphate (pH 8.0). The reaction was terminated by heating the solution for 2.5 min at 100 "C. After centrifugation for 10 min at 10,OOO X g for removal of precipitated protein, a portion of the reaction mixture was added to a test tube containing crystalline D-amino acid oxidase (0.3 mg), flavin adenine dinucleotide (0.1 mM), and catalase (400 units) in a final

volume of 2.4 ml of 60 mM potassium phosphate (pH 8.0). The D- alanine formed by the racemase was oxidized to pyruvate by D-aminO acid oxidase for 1 h at 37 "C, and the pyruvate was determined according to the method of Friedemann and Haugen (15).

The standard reaction mixture for the assay of D-amino acid transaminase consisted of 25 pmol of D-aminO acid, 80 pmol of potas- sium phosphate buffer (pH 8.0), 1 pmol of pyridoxal-€', 25 pmol of 2- ketoacid, and the purified 3-chloro-D-alanine chloride-lyase (4.2 to 21 pg) in a final volume of 1.0 ml. Enzyme was replaced by water in a blank. The reaction was initiated by addition of D-amino acid, and incubation was carried out at 37 "C for 30 min. 2-Ketoglutarate, pyruvate, and 2-ketobutyrate were examined as an amino acceptor, thereby the enzyme activity was examined by detecting glutamate, alanine, and 2-aminobutyrate formed with an automatic amino acid analyzer. The following amino acids were investigated as an amino donor: D-alanine, D-glutamate, D-glutamine, D-aspartate, D-threonine, D-methionine, D-nOrValine, D-norleucine, D-iSoleuCine, D-leuCine, D- tryptophan, D-serine, D-lysine, D-proline, and glycine. Furthermore, D-amino acid transaminase activity was examined by incubation of the purified enzyme (4.2 to 21 pg) with 10 pmol of D-glutamate, 5 pmol of pyruvate, and 1 pmol of pyridoxal-P in a final volume of 1.0 ml of 0.1 M potassium phosphate (pH 8.0) for 3 h at 37 "C. After the solution was heated at 100 "C for 2.5 min, the amount of 2-ketoglutarate formed was measured by addition of a portion of the solution to a cuvette containing 0.1 mM NADH and 60 mM potassium phosphate (pH 8.0) at 37 "C. When the absorbance at 340 nm was constant (usually after 1-2 min), glutamate dehydrogenase suspended in satu- rated ammonium sulfate (50 pl, 1 mg) was added to the cuvette. The decrease in the absorbance at 340 nm was a direct measure of the amount of 2-ketoglutarate present.

To detect amino acid decarboxylase activity, carbon dioxide was measured in a Gilson Differential Respiromater at 37 "C. To the main compartment of a two-armed Warburg vessel, in a total volume of 2.2 ml, were added: 1 ml of buffer I (50 mM potassium phosphate, 0.5 mM EDTA, 0.5 mM dithiothreitol, pH 7.0), 0.2 pmol of pyridoxal-P, and the purified 3-chloro-~-alanine chloride-lyase (8.4 to 42 pg). Amino acid (0.5 ml of a 50 mM solution) was placed in one side arm of the vessel and 0.3 ml of 3 M H2S04 in the other. After a 10-min equilibra- tion, substrate was tipped into the main compartment and carbon dioxide evolution was measured at 2-min intervals for 10 min. Acid from the other side arm was tipped into the reaction vessel to release dissolved carbon dioxide. Amino acid decarboxylase activity was examined by detecting the significant liberation of carbon dioxide under these conditions. The following D- and L-amino acids were examined as a substrate: alanine, glutamate, glutamine, aspartate, threonine, methionine, norvaline, isoleucine, leucine, tryptophan, ser- ine, lysine, proline, and glycine.

Serine transhydroxymethylase activity was conveniently measured by monitoring DL-threo-P-phenylserine cleavage (17). A typical 1-ml assay mixture contained 100 pmol of DL-threo-fi-phenylserine, 1 pmol of EDTA, 50 pmol of potassium phosphate (pH 7.3), and 4.2 to 21 pg of enzyme. Benzaldehyde production from DL-threo-P-phenylserine was measured at 279 nm employing a molar absorption value of 1400 M" cm" (18) for benzaldehyde. The cleavage of DL-threonine or L- allothreonine to form glycine and acetaldehyde was measured spec- trophotometrically at 30 "C with yeast alcohol dehydrogenase and NADH as described by Malkin and Greenberg (19) in the buffer containing 50 mM 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid, 1 mM EDTA, and 25 mM sodium sulfate (pH 7.5).

Other Assays-The amount of NHJ produced by the enzyme reaction was colorimetrically estimated by the phenol-hypochlorite method (20). The amount of chloride ion was determined by the method of Hattori (21).

Electrophoresis in Polyacrylamide Gels-Analytical electropho- resis in polyacrylamide gel was carried out in Tris/glycine buffer (pH 8.3) according to the method of Davis (22). Gels were stained for protein with Coomassie brilliant blue G-250 and destained in metha- nol/acetic acid/HzO (1:2:7). Sodium dodecyl sulfate-gel electropho- resis was performed in 10% of the polyacrylamide slab gels using the Tris/glycine buffer system described by King and Laemmli (23). Relative molecular mass of the subunit of the enzyme was obtained from the relative mobility of standard proteins.

determined as described by Winter and Karlson (24). The density Isoelectric Focusing-The isoelectric point of the enzyme was

gradient of the pH range 3-10 contained 2% ampholyte. Samples of the enzyme (about 1.5 mg) which had been exhaustively dialyzed against 0.13 M glycine were applied to the column after about one- fourth of the sucrose gradient had been formed. Electrofocusing was

3-Chloro-~ -alanine Chloride-lyase (Deaminating) of P. putida 13751

carried out a t 5 "C until there were no further changes in current (48 h), during which time the voltage was increased from 300 to 600 V. The column was then attached to a fraction collector and I-ml fractions were collected until the column was emptied. Absorption at 280 nm, pH, and 3-chloro-~-alanine chloride-lyase activity of the fractions were measured.

Gel Filtration on Calibrated Sephadex G-150"Analytical gel chromatography of the enzyme was performed in a column (2.0 X 110 cm) packed with Sephadex G-150. The gel bed was equilibrated with 10 mM potassium phosphate buffer (pH 7.0) containing 0.2 M KC1 and 1 mM 2-mercaptoethanol at 4 "C. A constant flow of the equilibrating buffer was maintained with the aid of a peristaltic pump (Tokyo Rikakikai MP-type). Small samples (1.0 ml) were chromatographed in all the experiments. The molecular weight of the enzyme was estimated according to Andrews (25). Proteins with the following molecular weights were employed as standards: horse heart cyto- chrome c, 12,400; chymotrypsinogen a, 25,700; hen egg ovalbumin, 45,000; bovine serum albumin, 68,000; yeast alcohol dehydrogenase, 150,000; rabbit muscle aldolase, 158,000; and bovine liver catalase, 248,000.

Ultracentrifugal Analysis-The purity of the purified enzyme and its sedimentation coefficient were determined by a Spinco model E ultracentrifuge equipped with a phase plate as a schlieren diaphragm. The molecular weight of the enzyme was determined by the ultracen- trifugal sedimentation equilibrium method according to the procedure of Van Holde and Baldwin (26). The experiments were carried out a Spinco model E ultracentrifu.ge equipped with Rayleigh interference optics. Multicell operations were employed in order to perform the experiments on five samples of different initial concentration ranging from 1.2 to 4.3 mg/ml with the use of an An-G rotor and double cells of different side-wedge angles. The rotor was centrifuged at 7447 rpm for 20 h at 20 "C. Interference patterns were photographed at intervals of 30 min to compare and make sure that the equilibrium was established. The relation between the concentration of the enzyme and the fringe shift was determined using the synthetic boundary cell.

Other Analytical Methods-Spectrophotometric measurements were made with a Hitachi 200-1-spectrophotometer with a 1.0-cm light path. Amino acids in the incubation mixture were identified by cwhromatography with authentic materials on a standard amino acid analyzer by the method of Spackman et al. (27).

Antiserum Preparation-Antibodies were elicited by the injection of 5.0 mg of 3-chloro-D-alanine chloride-lyase using two young, white, male rabbits. The antigen dissolved in 1.0 ml of 50 mM potassium phosphate buffer (pH 7.5) was homogenized in an equal volume of complete Freund's adjuvant (Difco) and injected into a multiple subcutaneous site on the back. After 4 weeks, the animal received a booster injection subcutaneously on the neck with 1 mg of the antigen homogenized in an equal volume of incomplete Freund's adjuvant (Difco). On the 7th and 14th day after the booster injection, blood was collected from the ear vein and allowed to clot. The serum was centrifuged at 6000 X g for 10 min and stored at -20 "C. Ouchterlony plates were made (28) using a 1% special Nobel agar in 10 mM Tris/ H$304 (pH 8.0) containing 0.01% sodium azide.

RESULTS

Bacterial Distribution of 3-Chloro-D -alanine Chloride-lyase

One hundred and twenty-one bacterial strains from the stock cultures of our laboratory (AKU type culture collection) were tested for their capability to grow in the nutrient medium containing d-chloro-~- or -D-alanine (Table I). The growth of most of the bacteria was completely inhibited by 3-chloro-~- alanine at a concentration of 15.6 mM. o d y 1 strain of the genus Arthrobacter, 2 strains of the genus Breuibacterium, and 6 strains of the genus Pseudomonas could grow under this condition, however, at a concentration of 31.2 mM 3- chloro-D-alanine, only these pseudomonads corresponding to P. putida (AKU 867,869, and 870), Pseudomonas fluorescens (AKU 868), Pseudomonas aureofaciens (AKU 836), and Pseudomonas aptata (AKU 849) had enough resistance to grow against a-chloro-~-alanine. 3-Chloro-~-alanine did not inhibit the bacterial growth as much as its D-enantiomer, about 50% of the tested bacteria could grow with a concentra- tion of 31.2 mM 3-chloro-L-alanine.

These resistant pseudomonads had the ability to form the

TABLE I Distribution of 3-chloro-~ - or D -alanine-resistant strains in

bacteria The bacterial strains were cultivated in the basal medium described

under "Experimental Procedures" containing 31.2 mM 3-chloro-~- alanine or 15.6 mM 3-chloro-D-alanine. After 24, 48, and 72 h of cultivation, the growth of each strain was examined. When the value of the absorbance at 610 nm was more than 1.0, the growth was judged to be positive.

No. of strains resistant to

Genera Tested strains 3-Chloro-L-ala- 3-Chloro-D-al- nine (31.2 ITIM) ani&(;5'6

Escherichia Aerobacter Erwinia Serratia Proteus Salmonella Alcaligenes Achromobacter Bacillus Agrobacterium Micrococcus staphylococcus Corynebacterium Arthrobacter Breuibacterium Bacterium Pseudomonas Xanthomonas

Total

7 4 2 2 2 1 2 2

22 6 5 3 5

10 7 1

38 2

121

7 4 2 2 1 1 2 2 7 5 1 0 3 4 3 1

20 0

65

0 0 0 0 0 0 0 0 0 0 0 0 0 1 2 0 6 0 9

enzyme catalyzing the degradation of 3-chloro-~-alanine in the cells, when they were grown in the culture medium sup- plemented with 3-ChlOrO-D-danine. P. put ida CR 1-1 (AKU 867) in which 3-chloro-~-alanine chloride-lyase occurred abundantly was chosen for the purpose of purification of the enzyme.

Enzyme Formation

The effect of various compounds on the induction of 3- chloro-D-alanine chloride-lyase was examined. P. put ida CR 1-1 was incubated in 500 ml of the basal medium containing 1 g of various tested compounds as an inducer. After cultiva- tion at 30 "C for 24 h with reciprocal shaking, the cells were collected by centrifugation at 10,000 X g and sonicated. The enzyme activity of the crude extract was measured using Assay I. The tested compounds were as follows: 3-ch loro-~- alanine, 3-chloro-~-alanine, D-alanine, L-alanine, D-cysteine, L-cysteine, D-cystine, L-cystine, D-Serine, L-serine, D-threo- nine, L-threonine, D-tryptophan, D-glUtamiC acid, D-aspara- gine, D-aspartic acid, and D-penicillamine. The enzyme was not formed in the absence of added 3-chloro-D-alanine. T h e enzyme was induced only by 3-chloro-~-danine among tested compounds.

For the large scale cult ivation, 3-chloro-~~-danine. HC1 was substituted for 3-chloro-D-alanine for economical reasons in nutrient broth as an inducer at the final concentration of 0.2%. The enzyme activity started to increase with the cell growth and the maximum activity was reached at an early stationary cell phase (Fig. 1). Rapid disappearance of the enzyme activity was observed immediately after the early stationary phase.

Enzyme Purification

Steps 1 t o 7 were carried out at 5 "C and the potassium phosphate buffers used for the following purification con- tained 10 (IM pyridoxal-P, 0.1 mM EDTA, and 1 mM 2-mercap- toethanol, unless otherwise specified.

Step 1. Preparat ion of Cell-free Extract-Washed cells

13752 3-chloro-D -alanine Chloride-lyase (Deaminating) of P. putida

FIG. 1. Induction of the enzyme during the bacterial growth. The bacteria were grown in a 100-liter jar fermentor under the conditions described under “Experimental Procedures.” The bacterial growth (0) was followed by absorption at 610 nm and the crude extracts of the cells harvested from 500 ml of culture broth were assayed for enzyme activity (0) under the conditions of Assay I.

(3,010 g) were suspended in 5 liters of 0.1 M potassium phos- phate buffer (pH 7.0) and disrupted with a DYNO-MILL (W. A. Bachofen, Switzerland). Cell debris was removed by cen- trifugation at 15,000 X g for 30 min.

Step 2. First Ammonium Sulfate Fractionation-Solid am- monium sulfate was added to the cell-free extract to give 30% saturation (at 0 “C). The pH was maintained at 7.0 with ammonia solution. After stirring for 12 h and removing the precipitate by centrifugation at 12,000 X g, the supernatant solution was further saturated with ammonium sulfate to give 50% saturation (at 0 “C). After 12 h, the suspension was centrifuged at 12,000 X g, and the pellet was dissolved in 50 mM potassium phosphate buffer (pH 7.0) and dialyzed against the same buffer.

Step 3. First DEAE-Sephacel Chromatography-The di- alyzed enzyme solution was applied to a DEAE-Sephacel column (5 x 80 cm) which had been equilibrated with 50 mM potassium phosphate buffer (pH 7.0). The column was washed with the same buffer until the absorbance at 280 nm of the effluent was reduced to 0.1 or less; then the enzyme was eluted with 0.1 M potassium phosphate buffer (pH 7.0). Fractions of 10 ml were collected, the active effluent fractions were com- bined and immediately precipitated with solid ammonium sulfate to a final concentration of 50%. The precipitate was collected by centrifugation, taken up in 50 mM potassium phosphate buffer (pH 7.0), and then dialyzed against the same buffer overnight.

Step 4. Second DEAE-Sephacel Chromatography-The enzyme from Step 3 was placed onto a column (3.5 x 58 cm) containing DEAE-Sephacel which had been equilibrated with 50 mM potassium phosphate buffer (pH 7.0). After washing the column with the same buffer, the enzyme was eluted with a linear gradient of KC1 (0 to 0.3 M, 1.3 liter in each container) in the same buffer at a flow rate of 50 ml/h. Fractions of 5 ml were collected and the active fractions were pooled.

Step 5 . Phenyl-Sepharose CL-4B Chromatography-The enzyme solution from Step 4 was cooled at 0 “C and ammo- nium sulfate was added in small portions with stirring to bring the solution to 5% saturation. The enzyme solution was placed on a column (2.5 x 36 cm) of phenyl-Sepharose CL-4B which had been equilibrated with 5ri saturated ammonium sulfate solution containing 10 mM potassium phosphate buffer (pH 7.0). It was eluted by lowering the ionic strength of ammonium sulfate (5 to Oc%, 300 ml in each container) in the same buffer. Fractions of 4 ml were collected; the active fractions were combined and precipitated with solid ammonium sulfate at a

final concentration of 50%. The precipitate was centrifuged at 12,000 X g and dissolved in 20 mM potassium phosphate buffer (pH 7.0), and then dialyzed against the same buffer overnight.

Step 6. Second Ammonium Sulfate Fractionation-The dialyzed enzyme preparation from Step 5 was then subjected to ammonium sulfate fractionation. The precipitate between 40 and 45% saturation was dissolved in 20 mM potassium phosphate buffer (pH 7.0) and dialyzed against the same buffer.

Step 7. Sephadex G-I50 Chromatography-The enzyme solution from the above step was placed onto a column (2.5 x 114 cm) containing Sephadex G-150 which had been previ- ously equilibrated with 20 mM potassium phosphate buffer (pH 7.0). The rate of sample loading and column elution was controlled at 3 ml/h by a peristaltic pump. The protein was eluted with the same buffer; fractions containing activity were combined and concentrated by ultrafiltration.

The overall purification achieved was approximately 9700- fold with a yield of 34.4%. The purified enzyme catalyzed the a,p-elimination of 3-chloro-~-alanine at 310 pmol/min/mg of protein under the standard conditions (Assay I). The result of the purification is summarized in Table 11.

Criteria for Purity

Polyacrylamide gel electrophoresis of the purified enzyme showed that it migrated as a single species as stained for protein on 7.5% gel. The purified enzyme showed only one band on sodium dodecyl sulfate-polyacrylamide slab gel elec- trophoresis (Fig. 2) and sedimented as a single sharp and symmetrical schlieren peak in the analytical ultracentrifuge in 20 mM potassium phosphate buffer (pH 7.0) containing 10 p~ pyridoxal-P and 0.1 mM 2-mercaptoethanol. Ampholyte electrofocusing also yielded only one absorption peak (pH 4.6) of protein and this peak coincided with 3-chloro-~-alanine chloride-lyase. The purity of this purified enzyme was exam-

TABLE I1 Purification of 3-chloro-~-alanine chloride-lyase (deaminating)

from P. putida CR 1-1 The enzyme activity was measured under the conditions of Assav

I.

Crude extract 57,000

DEAE-Sephacel (1st) 43,200 DEAE-Sephacel (2nd) 32.700 Phenyl-CL-Sepharose 31,000

SeDhadex G-150 19.600

(NH4)zSO.t (0.3-0.5) 52.500

(NH.8)ZSO.t (0.4-0.45) 23.800

W? 1.800,000

220.000 5.170 1,180

144 98.8 63.2

- units/rnp r;

0.032 100 0.239 92.1 8.36 75.8 27.7 57.4

215 54.4 24 1 41.8 310 34.4

- 1 2- 3 5 +

FIG. 2. Sodium dodecyl sulfate-slab gel electrophoresis o f% Chloro-D-alanine chloride-lyase. The conditions for sodium dode- cy1 sulfate-slab gel electrophoresis are given under “Experimental Procedures.“ A. marker proteins: I , phosphorylase h ( M , = 94,000); 2. bovine serum albumin (67.000); 3 , ovalhumin (43.000); 4, carbonic anhydrase (30,o(K)); 5, soybean trypsin inhibitor (20,000). B. the en- zyme, 27 p g . The direction of migration is from the cathode (left) to the anode.

S-Chloro-~-alanine Chloride- lyase (Deaminat ing) of P. pu t ida 13753

ined by a double diffusion in agarose. A single precipitin line was obtained with the crude cell extract of P. putida CR 1-1.

Crystallization and Reconstitution of the Apoenzyme The activity of the purified enzyme was found to be inde-

pendent of the added p-yridoxal-P. Furthermore, dialysis for 24 h against 50 mM potassium phosphate buffer (pH 7.0) containing 1 mM 2-mercaptoethanol failed to resolve the co- factor from the enzyme. The holoenzyme was converted to apoenzyme by dialyzing for 24 h against two changes of the following buffer: 300 volumes of 10 mM potassium phosphate buffer (pH 7.0) containing 50 mM phenylhydrazine and 1 mM dithiothreitol, then 800 volumes of 10 mM potassium phos- phate buffer (pH 7.0) containing 1 mM dithiothreitol. The enzyme thus treated had no detectable activity in the absence of added pyridoxal-P. The apoenzyme could be crystallized as small rods, by adding solid fine powdered ammonium sulfate to a solution of the enzyme (15 mg or more per ml) a t 0 "C while stirring gently with a glass rod (Fig. 3). The addition of ammonium sulfate was continued until the point where the induced turbidity ceased to disappear upon stirring. If dena- tured protein strands became visible, the solution was centri- fuged to remove them before the enzyme began to crystallize. Full activity was achieved a t a given concentration of pyri- doxal-P by the incubation for 4 h a t 25 "C. A saturation curve for pyridoxal-P is shown in Fig. 4, and the Michaelis constant was estimated as 0.023 PM for pyridoxal-P.

Absorption Spectra and Activity of Holo and Apoenzyme The holoenzyme exhibits absorption maxima a t 278 and 418

nm with a A L i H ~ 4 1 H ratio of approximately 3.44. The spectrum was independent of pH between 6.0 and 9.0. The occurrence of the absorption peak a t 418 nm suggests that the formyl group of the bound pyridoxal-P forms an azomethine linkage with an amino group of the protein, as in other pyridoxal-P enzymes studied so far. Reduction of the enzyme with sodium borohydride by the dialysis method of Matsuo and Greenberg (29) affected both the activity and the absorbance spectrum (Fig. 5). The reduced enzyme was catalytically inactive and the addition of pyridoxal-P did not reverse the inactivation. These results suggest that sodium borohydride reduces the aldimine linkage formed between the 4-formyl group of pyri- doxal-P and an amino group of the protein. Spectra of the resolved and reconstituted enzymes are also shown in Fig. 5.

Molecular Weight and Structure of Subunit The sedimentation coefficient (s.,,. , r ) of the enzyme is 6.6 S.

The molecular weight of the enzyme was estimated a t 73,000 on a calibrated column of Sephadex G-150. A molecular weight

FIG. 3. Photomicrograph of crystalline apoenzyme of 3- chloro-D-alanine chloride-lyase (x 1450).

, K p ~ p = 0.023UM

L-P W R l W X A

FIG. 4. Activity of 3-chloro-~-alanine chloride-lyase as a function of pyridoxal-P concentration. Apoenzyme (0.48 pg; spe- cific activity, 310) was incubated in assay medium with the indicated concentration of pyridoxal-I' for 4 h at 25 "C, then 20 min at 30 "C before initiation of the reaction by the addition of 3-chloro-D-alanine. The enzyme activity was determined in the usual way (Assay I ) . The concentration of pyridoxal-I' that gives half-maximum activity ( K I W = 0.023 p ~ ) was obtained from an Eadie-Hofstee plot of these data.

0.3

W u 2

0.2

0 v)

a

m

0.1

0 300 350 400 450 5

WAVELENGTH (nm) 10

FIG. 5. Absorption spectra of 3-chloro-~-alanine chloride-ly- ase. Curve A, holoenzyme (0.7 mi, 2.1 1 mg/ml) in 10 mM potassium phosphate buffer (pH 7.0); Curve B, holoenzyme reduced with sodium borohydride by the method of Matsuo and Greenberg (29) and dialyzed against the same buffer; Curve C, apoenzyme (0.7 ml, 2.11 mg/ml) prepared as described in the text. The reconstituted enzyme (0.7 ml, 2.1 1 mg/ml) (Curve D) was prepared by dialyzing the apoen- zyme against 10 mM potassium phosphate buffer (pH 7.0) containing 1 mM dithiothreitol and 0.3 mM pyridoxal-1'. The reference cuvette contained the respective dialysis buffer.

of 76,000 f 2,000 was also obtained by the sedimentation equilibrium method (26), assuming a partial specific volume of 0.74. When the enzyme was pretreated with 1% sodium dodecyl sulfate and 50 mM dithiothreitol and electrophoresed on a gel containing 0.1% sodium dodecyl sulfate, a single band was observed. The molecular weight corresponding to the band was estimated to be 38,000 based on its mobility relative to those of the reference proteins (Fig. 2). The enzyme appears to consist of two identical subunits.

Pyridoxal-P Content The enzyme was dialyzed overnight against 20 mM potas-

sium phosphate buffer (pH 6.5 or 7.0) and the amount of pyridoxal-P was analyzed, assuming that the molar extinction coefficient of pyridoxal-P in 0.1 M NaOH is 66,000 at 388 nm

13754 3-Chloro-D -alanine Chloride-lyase (Deaminating) of P. putida

(30) and that of the phenylhydrazine of pyridoxal-P is 24,500 at 410 nm (31). An average pyridoxal-P content of 2 mol/ 76,000 g of protein was obtained, indicating that 2 mol of pyridoxal-P are bound to 1 mol of the enzyme protein in the holoenzyme. Since the enzyme appears to be a homodimer consisting of two identical subunits, each subunit may contain pyridoxal-P a t a stoichiometric ratio of unity.

Stability of the Enzyme The stability of the enzyme was examined under various

conditions using the method of Assay I. The highly purified enzyme could be stored in 10 mM potassium phosphate buffer (pH 7.0) containing 0.1 mM pyridoxal-P and 1 mM 2-mercap- toethanol at 4 "C for 4 weeks without loss of activity, The enzyme remained stable in 45% glycerol solution containing 10 mM potassium phosphate (pH 7.0) and 1 mM 2-mercapto- ethanol for 4 or more months in a deep freezer (at -20 "C). When the enzyme was incubated in 10 mM potassium phos- phate buffer (pH 7.0) containing 1 mM 2-mercaptoethanol and 0.1 m~ pyridoxal-P a t 30, 40, and 45 "C for 15 min, no loss of activity was observed. The heat treatment at 50,55, and 60 "C for 15 min caused about 4756, 80%, and 100% loss of initial activity, respectively. The enzyme was stable in the pH range of 6.0-8.0 when the enzyme solution (0.1 mg/ml) was heated a t 50 "C for 15 min in the following buffers (a final concentra- tion, 0.1 M); CH3COOK/CHaCOOH, pH 4.0-5.5, K2HP04/ KH2P04, pH 6.0-8.0; Tris/HCl, pH 7.0-9.5; NH4C1/NH40H, pH 8.5-10.5.

Substrate Specificity and Kinetic Properties The ability of the enzyme to catalyze elimination reaction

of various amino acids was examined. The reaction was carried out by modifying Assay 11. The enzyme was specific for 3- chloro-palanine; 3-chloro-~-alanine (2 mM) was not degraded. In addition to 3-chioro-~-alanine (V,,,,,, 310 pmol/min/mg) which was the preferred substrate, D-cysteine served as an effective substrate (V,,,,,, 106 pmol/min/mg). The formation of pyruvate from D-cystine was also catalyzed, but to a lesser extent, by the enzyme (27 pmol/min/mg). The a,p-elimination of D-serine could barely be detected (0.03 pmol/min/mg). The formation of pyruvate or 2-ketobutyrate by the elimination reaction of L-cysteine, L-cystine, D-tryptophan, L-tryptophan, L-serine, D-threonine, L-threonine, D-alanine, L-alanine, D-ho- moserine, L-homoserine, S-methyl-L-cysteine, DL-homocys- teine,P-2-thienyl-~~-alanine, and l-aminocyclopropane-l-car- boxylic acid was not detected, even though a large amount of enzyme was added. The kinetic studies were performed by

I "1.. ....,__ 2 I ...., ~ ; 2 1 0

Ifs x 10-4 11s x 10-4

FIG. 6. Effect of substrate concentration on 3-chloro-~-ala- nine chloride-lyase activity. The reactions were carried out at 30 "C in the reaction mixture containing 225 ng of the enzyme (specific activity, 310), 0.1 pmol of pyridoxal-P, 0.2 pmol of NADH, 10 units of pig heart lactate dehydrogenase, 0.1 mmol of potassium phosphate buffer (pH 8.0), and variable amount of 3-chloro-~-alanine (a) or D- cysteine ( b ) as indicated, in a total volume of 2.89 ml. Velocity ( u ) was expressed as micromoles of pyruvate formed/min and substrate concentration ( S ) as moles/liter.

modifying Assay 1. The K,,, value for 3-chloro-~-danine was 61 p~ at pH 8.0 (Fig. 6a) . Lineweaver-Burk kinetic data of D-cysteine were linear over a range of concentration between 35 pM and 0.35 mM, and K , value for D-cysteine was 0.18 mM at pH 8.0; however, a prominent substrate inhibition of D- cysteine was observed at a higher concentration than 0.35 mM (Fig. 6b).

Effect of pH For these experiments, Assay I1 was used to measure 3-

chloro-D-alanine chloride-lyase activity. The effect of pH on the activity of the purified enzyme was examined with 3- chloro-D-alanine as the substrate. In both Tris/HCl and NH4C1/NH40H buffer (a final concentration, 50 mM) used, the optimum for the a$-elimination reaction of 3-chloro-~- alanine was found at pH 9.0.

Inhibitors Various compounds were investigated for their inhibitory

effects on enzyme activity. The enzyme was hardly inhibited after a 20-min incubation a t 30 "C by hydroxylamine .HCl, L-penicillamine, semicarbazide. HCI, and cysteamine. 2 HC1 (a final concentration, 10 mM), which were inhibitory to most pyridoxal-P-requiring enzymes, whereas phenylhydrazine and the D-enantiomers of penicillamine and cycloserine were stronger inhibitors (inhibition at 10 mM: 77%, 30%, and 22%. respectively). The enzyme showed relatively high resistance to the carbonyl reagents. 3-Chloro-~-alanine chloride-lyase displayed a high sensitivity t o some of the thiol reagents (1 mM), iodoacetate, HgC12, AgNOz, CuS04, which showed 75-100% inhibition. Potassium cyanide inhibited the enzyme almost completely at 1 mM.

Reaction Products and Stoichiometry The stoichiometry of the enzymatic a,p-elimination of 3-

chloro-D-alanine was examined with a reaction mixture con- taining 0.2 mmol of potassium phosphate buffer (pH 7.0), 5 pmol of 3-chloro-D-alanine, 0.2 pmol of pyridoxal-P, and the enzyme in a final volume of 4.0 ml. The reaction was carried out at 30 "C for 10 min and terminated by the addition of 1 ml of 1 M HzS04. The denatured protein was removed by centrifugation. The amounts of pyruvate, ammonia, and chlo- ride ion were determined by the method described under "Experimental Procedures." Formed ammonia and chloride ion were determined as 1.33 and 1.34 pmol, respectively. Formation of 1.37 pmol of pyruvate was also observed by the method of Assay 11. The results indicated that pyruvate, ammonia, and chloride ion were formed stoichiometrically with a consumption of 3-chloro-D-alanine. The identity of pyruvate was further confirmed with alanine dehydrogenase. The deproteinized solution was evaporated under reduced pressure. To the residue were added 0.5 mmol of N H d X NH40H buffer (pH 9.0), 0.1 pmol of NADH, and 10 units of purified alanine dehydrogenase to make a final volume of 1 ml. After incubation at 37 "C for 60 min, the solution was subjected to an amino acid analyzer. The amino acid formed was identified as alanine and amount determined was 1.34 pmol.

P-Replacement Reaction Catalyzed by 3-Chloro-~ -alanine Chloride-lyase

In the previous paper (13), we stated that 3-chloro-~-alanine chloride-lyase catalyzed the P-replacement react,ion in the presence of a high concentration of sodium hydrosulfide. The product of the P-replacement reaction catalyzed by the en- zyme was identified as D-cysteine by physicochemical means. The ratio of the a,p-elimination and the ,&replacement reac-

3-Chloro-D -alanine Chloride-lyase (Deaminating) of P. putida 13755

tions were controlled by the concentration of sodium hydro- sulfide in the reaction mixture (Fig. 7). At the concentration of 0.1 M sodium hydrosulfide, the pyruvate formation (a$- elimination reaction) was almost completely depressed. Under this condition, when the P-replacement reaction dominated, about 90% of added 3-chloro-~-alanine was converted to D- cysteine. According to the method of Lineweaver and Burk, the apparent K , values were 8.1 X M for 3-chloro-~- alanine and 4.5 X lo-' M for sodium hydrosulfide in the p- replacement reaction. The maximum velocity of the synthesis of D-cysteine was calculated to be 1620 pmol/min/mg. It has been known that some pyridoxal-P-dependent enzymes have multiple catalytic functions, e.g., tryptophanase (32), tyrosine phenol-lyase (33), and cysteine desulfhydrase (34). The mech- anism of a,p-elimination and /?-replacement reactions by 3- chloro-D-alanine chloride-lyase probably includes the forma- tion of a-aminoacrylate as an intermediate as reviewed by Snell and Di Mari (35) and Davis and Metzler (36). Among multifunctional lyases requiring pyridoxal-P, 3-ChlOrO-D-ala- nine chloride-lyase is the first enzyme which is specific to D- amino acid.

Further Characterization of 3-Chloro-D -alanine Chloride- lyase

It is known that amino acid racemase (6), serine transhy- droxymethylase (7), and D-amino acid transaminase (8) cata- lyze a$-elimination of 3-chloro-~-alanine to pyruvate, am- monia, and chloride ion; however, during the course of these reactions, these enzymes were concomitantly inactivated. T o make clear that the lyase activity of the Pseudomonas enzyme with 3-chloro-D-alanine as substrate is not simply due to an already known characterized pyridoxal-P-dependent enzyme such as amino acid racemase, D-amino acid transaminase, amino acid decarboxylase, or serine transhydroxymethylase, we have sought to examine whether the Pseudomonas enzyme exhibits those catalytic activities or not. These activities were deliberately checked by adding an adequate amount of the enzyme to each reaction mixture under the reaction conditions as described under "Experimental Procedures." For the assay of D-amino acid transaminase, three representative keto acids (2-ketoglutarate, pyruvate, and 2-ketobutyrate) were used as

5l 10 mM

0 30 50 90

FIG. 7. Formation of D-cysteine by the P-replacement reac- tion catalyzed by 3-chloro-~-alanine chloride-lyase. The incu- bation was carried out at 30 "C with a reaction mixture (5 m l ) containing 50 pmol of 3-chloro-~-alanine, 0.5 pmol of pyridoxal-P, 0.5 mmol of potassium phosphate (pH 7.5), various concentrations of sodium hydrosulfide, and 1.21 pg of the enzyme. Aliquots of the reaction mixture were withdrawn at intervals to follow the reaction. The formed pyruvate (0) and D-cysteine (0) were determined hy using Assay I1 and Assay 111, respectively, as described under "Experimental Procedures."

an amino acceptor and various kinds of D-amino acids were examined. Decarboxylase activity of various D- or L-amino acids was also tested. To detect serine transhydroxymethylase activity, C,Cp-aldol cleavage activity of DL-threonine, L ~ O - threonine, or DL-threo-P-phenyiserine was measured instead of using serine as a substrate. In conclusion, the Pseudomonas enzyme did not exhibit those catalytic activities.

DISCUSSION

P. putida CR 1-1, which is resistant to 3-chloro-~-alanine, has been found to produce an enzyme which catalyzes a&- elimination reaction of 3-chloro-~-alanine. The general equa- tion for the action of the enzyme is shown in Reaction I.

D-C~CH~CH(NH~)COOH + HzO (1)

+ HCI + CHaCOCOOH + NHJ

The a,p-elimination reaction of 3-chloro-~-alanine has been demonstrated with amino acid racemases (6), serine transhy- droxymethylase (71, and D-amino acid transaminase (8); how- ever, as the reaction proceeded, these enzymes were inacti- vated. Such self-destructive inactivation by 3-chloro-~-alanine was not observed in the reaction with the Pseudomonas enzyme. It is therefore conceivable that the enzyme from Pseudomonas would catalyze a similar reaction without suf- fering inactivation. Thus, this enzyme could be a variant type that permits turnover but is refractory to alkylation. There- fore, we have deliberately sought whether the enzyme that we have isolated can catalyze racemization, decarboxylation, or transamination by designing the assays approximately so that it would be possible to detect such enzymes, if present. However, the purified enzyme lacked those catalytic activities. Next we suspected that Reaction I might have been provoked by certain enzymes, such as D-serine dehydratase or D-cysteine desulfhydrase which were not "specific for 3-chloro-~-ala- nine." These enzymes may possibly make 3-chloro-D-alanine labile by the formation of Schiffs base and subsequent re- moval of the a-proton. The enzyme barely attacked D-serine, whereas it catalyzed the degradation of D-cysteine (Reaction 11).

D-HSCH~CH(NH~)COOH + Hz0 (11)

+ HzS + CRCOCOOH + NHs

Previously it was reported that D-cysteine desulfhydrase ac- tivity was detected in E. coli (37), but the details of the enzyme were unknown. The Pseudomonas enzyme was in- ducibly formed by 3-chloro-~-alanine. The inducibility was enhanced in accordance with the increase of the concentration of 3-chloro-D-alanine up to 15.2 mM.' No enzyme activity was detected in the cells cultivated in the ordinary nutrient broth and the supplements of D-cysteine, D-cystine, D-alanine, and D-serine were entirely inert as an inducer. Hence the enzyme appears to be directly related to the metabolism of 3-chloro- D-alanine in itself, not with D-cysteine. The induction of this enzyme was insensitive to catabolite repression by a carbon source such glucose, succinate, glycerol, or citric acid.' The enzyme was inducibly formed as long as 3-chloro-~-alanine was in the medium. Furthermore, all pseudomonads which have resistance to high concentrations of 3-chloro-~-alanine definitely possessed the ability to produce this enzyme. These results suggest that the physiological role of the enzyme might be to detoxify 3-chloro-~-alanine. The enzyme also catalyzed the ,&replacement reaction to form D-cysteine from 3-chloro- D-alanine and sodium hydrosulfide (Reaction 111).

' T. Nagasawa, H. Yamano, H. Hosono, and H. Yamada, unpuh- lished experiments.

13756 3-Chloro-D -alanine Chloride-lyase (Deaminating) of P. putida

D-C~CH~CH(NH~)COOH + NaHS (111)

+ NaCl + D-HSCH~CH(NH~)COOH

The ,&replacement reaction proceeded a t a higher rate than the elimination reaction of 3-ChlOrO-D-alanine. Recently Sakai et al. have found that cyanide-resistant strain Enterobacter sp. 10-1 forms the enzyme which converts sodium cyanide with o-acetyl-L-serine to 3-cyano-~-alanine (38). Accordingly, it might be possible that 3-chloro-D-alanine is converted to another nontoxic compound by the P-replacement reaction of the enzyme.

Kaczorowski et aZ. (11, 12) have proposed that the bacteri- cidal property of 3-chloro-~-alanine is attributed to the dam- age of the energized activity transport system in membrane vesicles by chloropyruvate, which was metabolically formed from 3-chloro-~-alanine. From the view point of Kaczorowski et aZ., it seems reasonable that the enzyme should localize in the membrane fraction or the culture filtrate in order to decompose 3-chloro-~-alanine or to convert it to another compound. However, our preliminary experiments revealed that the enzyme localized in the soluble cytoplasm fraction of the 24-h cultured cells.' This fact indicates that the antimi- crobial activity of 3-chloro-~-alanine is not due to the mem- brane enzyme. This argument is reinforced by the finding that the growth inhibition by 3-chloro-D-alanine can be reversed by D-amino acids that are substrates for amino acid racemase or D-amino acid transaminase (8, 9). Furthermore, transport of several amino acids was not blocked.

How does the enzyme take part in the detoxication of 3- ChlOrO-D-ahine? Further investigation to elucidate the de- tailed mechanism is currently in progress.

1.

2.

3.

4.

5. 6.

7.

8.

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