Synthesis of L-Cysteine and Its Analogues by Intact Cells Containing Cysteine Desulfhydrase1

Agric. Biol. Chem., 45 (1), 259-263, 1981 259

Synthesis of L-Cysteine and Its Analogues by Intact Cells Containing Cysteine Desulfhydrase1 Haruyuki Ohkishi, Daikichiro Nishikawa, Hidehiko Kumagai* and Hideaki Yamada** Central Research Laboratories, Mitsubishi Chemical Industries, Yokohama 227, Japan *Department ofFood Science and Technology, Kyoto University, Kyoto 606, Japan **Department of Agricultural Chemistry, Kyoto University, Kyoto 606, Japan Received August 8, 1980

Cysteine desulfhydrase catalyzes /^-replacement, the reverse reaction of a,/?-elimination, as well as a,^-elimination. These reactions were studied with intact cells of Aerobacter aerogenes 1-3-2 and Aerobacter cloacae IFO 12009 containing cysteine desulfhydrase. L-Cysteine and its analogues were synthesized by replacement and reverse reactions using intact cells. /?-Chloro-L-alanine, L-cysteine, S-methyl-L-cysteine, S-allyl-L-cysteine and L-serine were used as substrates together with hydrogen sulfide and methyl mercaptan to synthesize L-cysteine and S- methyl-L-cysteine via replacement reaction by intact cells. L-Cysteine synthesized from /?-chloro-L- alanine was confirmed to be entirely in L-form after isolation and identification of the product. The reverse reaction for synthesis of L-cysteine and S-methyl-L-cysteine from hydrogen sulfide or methyl mercaptan, pyruvate and ammonia was also catalyzed by intact cells. /?-Chloro-L-alanine was found to be the best substrate for synthesis of L-cysteine and S-methyl-L-cysteine by /^-replacement reaction.

Cysteine desulfhydrase catalyzes the degra- R; -SH, -SCH3, -OH, -Cl etc. dation of L-cysteine into pyruvate, ammonia R'; -SH, -SCH3 -SCH2CH3, ~S-<^) and hydrogen sulfide and requires pyridoxal phosphate as a co factor. Anapparently homo- etc. geneous preparation of the enzyme has been On the basis of these findings, we are prepared in our laboratory,1} and its properties examining practical application of this enzyme have been established in some detail. for preparation of L-cysteine and its deriv- Further investigation of the catalytic prop- atives. In the previous paper4) we reported the erties of the enzymehas revealed that besides distribution of cysteine desulfhydrase. In the a,/?-elimination (I), the enzyme catalyzes f$- present paper /^-replacement and the reverse replacement (II)2) and the reverse reaction reaction are studied with intact cells contain- (IH) 3) ing cysteine desulfhydrase. l-RCH2CHNH2COOH + H2O > CH3COCOOH + RH + NH3 (I) EXPERIMENTAL

l-C1CH2CHNH2COOH + R'H --» Chemicals. S-Ethyl-L-cysteine, S-propyl-L-cysteine and S-allyl-L-cysteine were prepared chemically according to l-R'CH2CHNH2COOH + HC1 (II) the method ofArmstrong et al.5) All other chemicals were commercial products. /?-Chloro-L-alanine and jS-chloro-D- RH + CH3COCOOH + NH3 > alanine were purchased from CyclochemicalCompany, RCH2CHNH2COOH + H2O (III) Ltd. Miroorganisms. Aerobacter cloacae IFO 12009 and f Microbiological Synthesis of L-Cysteine and Its Aerobacter aerogenes 1-3-2 were used as the enzyme Analogues. Part II. source. 260 H. Ohkishi, D. Nishikawa, H. Kumagai and H. Yamada

Enzymepreparation. Intact cells containing cysteine the corresponding amino acids. Higher ac- desulfhydrase were used as enzyme. The preparation has tivity was found with jS-chloro-L-alanine than been described previously.40 L-cysteine. Analytical methods. L-Cysteine was determined by the acid ninhydrin method of Gaitonde.6) 5-Substituted-L- 2. Specificity of replacement reaction for cysteines were determined with an automatic aminoacid various aminoacids analyzer (Hitachi KLA-5), and by paper chromatography The replacement reaction between various with a solvent system of «-butanol-acetic acid-water amino acids and hydrogen sulfide or methyl (4: 1 : 1). Amino acids on paper chromatograms were mercaptan was studied with intact cells of detected by spraying ninhydrin reagent. Aerobacter cloacae IFO 12009. L-Cysteine and S-methyl-L-cysteine synthesized were deter- RESULTS mined by an automatic amino acid analyzer, as 1. Specificity of replacement reaction for shown in Table II. The replacement reaction mercaptans was observed with L-cysteine, S-allyl-L- The specificity of the replacement reaction cysteine, S-methyl-L-cysteine, L-serine and /?- for mercaptans was studied with intact cells of chloro-L-alanine, of which /?-chloro-L-alanine Aerobacter cloacae IFO 12009. The replace- showed highest productivity of both l- ment reaction wascarried out in the presence cysteine and S-methyl-L-cysteine. This result of various mercaptans and L-cysteine or /?- indicates that jS-chloro-L-alanine might be chloro-L-alanine. The amino acids synthesized usable as substrate for the preparation of l- were detected by paper chromatography and cysteine or 5-methyl-L-cysteine. identified by comparing their Rf values with those of authentic samples. As shownin Table 3. Reverse reaction I, all the mercaptans tested were converted to In the previous paper we reported that l- cysteine could be synthesized by the reverse Table I. Formation of l-Cysteine and reaction with intact cells containing cysteine ^-Substituted l-Cysteine from desulfhydrase.4) In this paper, synthesis of l- /?-Chloro-l-alanine or l- cysteine and 5-methyl-L-cysteine by the re- Cysteine and Mercaptans verse reaction from hydrogen sulfide or methyl by Intact Cells of Aerobacter cloacae IFO 12009 Table II. Replacement Reactions of The reaction was carried out at 30°C for 2hr in 10ml of Various Amino Acids a reaction mixture (pH 9.5) containing 2 mmol of The reaction was carried out at 30°C for 2hr. mercaptan, 2 mmolof /?-chloro-L-alanine or L-cysteine, 1 Conditions are described in Table I. mmol of NH4OH-NH4C1, 10 fimol of pyridoxal phosphate, 20 /rniol of EDTA, 5 mg of sodium dodecyl sulfate Substrate Amountof product (g/liter) and intact cells harvested from 50ml of the culture, l- Cysteine synthesized was determined according to the amino acid L-Cysteine S-Methyl-L-cysteine method of Gaitonde et al.6) Other amino acids synthesized were determined by paper chromatography. L-Cysteine 0.ll D-Cysteine Aminoacid formation S- Methyl- L-cysteine 0.19 Mercaptan S-Allyl-L-cysteine 0.03 0.07 jS-Cl-L-Alanine L-Cysteine L-Serine 0.003 0.01 £-Cl-L-Alanine 3.ll 1.47 ^-Cl-DL-Alanine 2.10 0.95 + CH^SH jS-Cl-D-Alanine + L-Methionine + L-Tyrosine + L-Tryptophan + CH, =CHCH,SH +++ L- Phenylalanine Microbiological Synthesis of L-Cysteine and Its Analogues 261

Table III. Properties of l-Cystine Isolated from Reaction Mixture 300 Optical activity Elemental analysis ] 20 H N [<(c=D JD 200 Product -199° 30.45 5.28 ll.38 Authentic -209° 29.75 5.17 ll.42 100 C alculated 29.99 5.03 ll.66

Incubation time (hr) Fig. 1. Reverse Reaction by Intact Cells ofAerobacter aerogenes 1-3-2. The reaction was carried out at 30°C in 10ml of a 4. Identification of L-cysteine synthesized reaction mixture (pH 9.5) containing 2mmol of py- from P-chloro-L-alanine ruvate, 2mmol ofNaSH, 5 mmol ofNH4OH-NH4C1, 10 jS-Chloro-L-alanine was shown to be useful //mol ofpyridoxal phosphate, 20 /xmol ofEDTA, 5 mg of as substrate for the preparation of L-cysteine. sodium dodecylsulfate and intact cells harvested from The enzymatically synthesized L-cysteine was 100mlof the culture of Aerobacter aerogenes 1-3-2. S- Methyl-L-cysteine synthesized (#-à" ) was determined isolated from the large-scale incubation mix- with an automatic amino acid analyzer and L-cysteine ture as its oxidized form, L-cystine. Incubation synthesized (O-O) was determined by the method of Gaitonde et al.6) was carried out for 3hr in 40ml of a reaction mixture containing jS-chloro-L- ananine (1.28 g), Na2S (1.92g), sodium dodecyl sulfate (20mg), pyridoxal phosphate (1.1mg), Reaction mixture (hO ml) NH4C1-NH4OH buffer (20 mmol, pH 9.5) and

I - add 20 ml of 30% trichloroacetic acid intact cells of Aerobacter cloacae IFO 12009 SeparateSepa: harvested from 100ml of broth cultured for Dowex 50 X 8 (H+form 3x30 cm) |-wash with water (TOO ml) 16 hr. The yield of L-cysteine from /?-chloro-L- alanine and Na2S was 346 mg. L-Cysteine synthesized was isolated by the procedure L-Cy: shown in Fig. 2. The reaction was stopped by Con' vjteine|- elute fractionwith 2N adding 20 ml of 30% trichloroacetic acid, and icentrate in vacuo Precipitateipitate bacterial cells were removed by centrifugation. r- dissolve in water The supernatant was charged onto a Dowex Crystallize L-cystine 50x8 column (200-400 mesh, H+ form, 3 x 30cm), which was washed with 700ml of Fig. 2. Procedure for the Isolation of L-Cystine from Reaction Mixture. distilled water followed by 800ml of 2n- ammonia. The eluate with ammonia was evaporated to dryness at 50°C in vacuo. mercaptan and pyruvate and ammonia was During this procedure, L-cysteine was oxidized examined with intact cells of Aerobacter to L-cystine. L-Cystine was recrystallized from aerogenes 1-3-2. L-Cysteine synthesized was water in a yield of 253mg. It had same Rf determined by the method of Gaitonde et al.6) value as authentic L-cystine on the paper and S-methyl-L-cysteine synthesized was de- chromatogram. Elemental analysis and optical termined with the automatic amino acid ana- activity are shown in Table III. Its infrared lyzer. As shown in Fig. 1, the rate of synthesis absorption spectrum is shown in Fig. 3. These of 5-methyl-L-cysteine is higher than that of l- results confirm that the enzymatically synthe- cysteine. sized product is L-cysteine. 262 H. Ohkishi, D. Nishikawa, H. Kumagai and H. Yamada

U.O Wavelength (my ) 5.0 6.0 7-0 8.0 9-0 ll 13 15

n n Uooo 3600 3200 2800 2U00 2000 1800 1600 1U00 1200 1000 800 650 Wavenumber (cm~ )

Fig. 3. Infrared Absorption Spectra of Authentic (A) and Isolated L-Cystine (B).

DISCUSSION (c) CH +H 0 The formation of hydrogen sulfide from [E=N-C-C00H] 5-zflZ -H.,0 cysteine and cystine by bacteria has long been known. The enzyme responsible for this conversion was first demonstrated in cell (E,Enzyme; R.-H, -CH , -C£H -CH2CH=CH2 etc) extracts from Proteus vulgaris by Tarr7) and subsequently from Escherichia coll by Fig. 4. Schematic Representation of the Mechanism Fromageot and Desunuelle.8) The enzyme Discussed in the Text for Reactions Catalyzed by responsible for this conversion is called cys- Cysteine Desulfhydrase. teine desulftiydrase. Recently Yamada et al. studied the distri- methyl-L-cysteine, S-allyl-L-cysteine and l- bution of cysteine desulfhydrase9) and pre- serine could be used as substrates for the pared the enzyme in crystalline form from replacement reaction catalyzed by intact cells. Aerobacter aerogenes. The enzyme catalyzes /?- Of these amino acids /?-chloro-L-alanine was replacement2* and the reverse reaction of a,/?- shown to be the most useful for the prepara- elimination as well as a,j8-elimination.3) In the tion of L-cysteine and S-methyl-L-cysteine. For present investigation, the replacement and the reverse reaction, hydrogen sulfide and reverse reactions were studied with intact cells methyl mercaptan could be used as substrate of Aerobacter cloacae IFO 12009 and for formation of the corresponding amino Aerobacter aerogenes 1-3-2 containing high acids by intact cells. cysteine desulfhydrase activity. Hydrogen sul- Yamada et al. explained the catalysis by fide, methyl mercaptan, ethyl mercaptan, cysteine desulfhydrase2) by adapting the propyl mercaptan and ally! mercaptan could general mechanisms for the pyridoxal be used as substrates for the replacement phosphate-dependent reaction proposed by reaction to form the corresponding amino Braunstein and Shemyakin10) and by Metzler acids. Yamada et al. reported that /?-chloro-L- et al.n) From their studies with a crystalline alanine could be used as substrate for the preparation of cysteine desulfhydrase, replacement reaction catalyzed by crystalline Yamadaet al. postulated the mechanisms for cysteine desulfhydrase.2) We found that not the cysteine desulfhydrase catalyzed reaction only /?-chloro-L-alanine, but also L-cysteine, S- shownin Fig. 4. In the formation of puruvate Microbiological Synthesis of L-Cysteine and Its Analogues 263 from jS-chloro-L-alanine (a,/?-elimination re- REFERENCES action), the substrate interacts with the en- H. Kumagai, S. Sejima, Y. J. Choi, H. Tanaka and zyme to form enzyme-boundoc-amino acrylate H. Yamada, FEBS Lett., 52, 304 (1975). by elimination ofH+ and Cl~ (step a), and this H. Kumagai, H. Tanaka, S. Sejima and H. Yamada, is hydrolyzed to form pyruvate and ammonia. Agric. Biol. Chem., 41, 2071 (1977). In the presence of hydrogen sulfide (/?- H. Kumagai, Y. J. Choi, S. Sejima and H. Yamada, replacement reaction), the enzyme-bound a- Biochem. Biophys. Res. Commun., 59, 789 (1974). amino acrylate, rather than hydrolysis, can H. Ohkishi, D. Nishikawa, H. Kumagai and H. Yamada, Agric. Biol Chem., 45, 253 (1981). occur yielding L-cysteine by the reversal of the M. D. Armstrong and J. D. Lewis, /. Org. Chem., 16, formation of a-amino acrylate intermediate 749 (1951). (step b). More generally, the addition of M. K. Gaitonde, Biochem. J., 104, 627 (1967). mercaptans may also be catalyzed by the H. L. A. Tarr, Biochem. J., 27, 1869 (1933). C. Fromageot and P. Desnuelle, Enzymologia., 6, 80 enzyme to yield the corresponding S- (1939). substituted L-cysteines. In the case of intact H. Kumagai, Y. J. Choi, S. Sejima and H. Yamada, cells, the reactions mayproceed by the same Agric. Biol. Chem., 39, 387 (1975). mechanisms with L-cysteine, ^-substituted l- A. E. Braunstein and M. M. Shemyakin, Biokhimiya, cysteine or /?-chloro-L-alanine as substrate. 18, 393 (1953). D. E. Metzler, M. Ikawa and E. E. Snell, J. Am. Chem. Soc, 76, 648 (1954).