S-Carboxymethylcysteine Synthase from Escherichia Coli

Agric. Biol. Chem., 53 (9), 2481 -2487, 1989 2481 S-Carboxymethylcysteine Synthase from Escherichia coli Hidehiko Kumagai,* Hideyuki Suzuki, Hiroki Shigematsu and Tatsurokuro Tochikura Department of Food Science and Technology, Faculty of Agriculture, Kyoto University, Kyoto 606, Japan Received April 14, 1989 An enzyme that catalyzes the synthesis of S-carboxymethyl-L-cysteine from 3-chloro-L-alanine (3-Cl-Ala) and thioglycolic acid was found in Escherichia coli W3110 and was designated as S- carboxymethyl-L-cysteine synthase. It was purified from the cell-free extract to electrophoretic homogeneity and was crystallized. The enzymehas a molecular weight of 84,000 and gave one band corresponding to a molecular weight of 37,000 on SDS-polyacrylamide gel electrophoresis. The purified enzyme catalyzed the ^-replacement reactions between 3-Cl-Ala and various thiol compounds. The apparent Kmvalues for 3-Cl-Ala and thioglycolic acid were 40mMand 15.4mM.The enzyme showedvery low activity as to the a,/^-elimination reaction with 3-Cl-Ala and L-serine. It was not inactivated on the incubation with 3-Cl-Ala. Theabsorption spectrum of the enzymeshows a maximum at 412nm, indicating that it contains pyridoxal phosphate as a co factor. The N-terminal amino acid sequence was determined and the corresponding sequence was detected in the protein sequence data bank, but no homogeneous sequence was found. 3-Chloro-L-alanine (3-Cl-Ala) acts as a so- called suicide substrate for some pyridoxal- Materials and Methods phosphate (PLP) dependent enzymes, such Reagents. Casamino acids and Tryptone were obtained as glutamate-oxaloacetic acid transminase,1} from Difco Laboratories, yeast extract from Oriental glutamate-pyruvic acid transaminase2) and Yeast Co., amido black 10B from E. Merck AGand phenylmethylsulfonyl fluoride from Calbiochem-Behring. amino acid racemase.3) On the other hand, Sephadex G-150 and G-100, and blue dextran 2000 and someother PLPenzymes that catalyze the a,/?- molecular weight markers were from Pharmacia. DEAE- elimination and /^-replacement reactions were Cellulofine AHand Cellulofine GC-700-mwere from found to be insensitive to 3-Cl-Ala and to Chisso Co. SC-Cys was from Tokyo Kasei Co., 3-Cl-Ala utilize it as a preferable substrate.4~7) Fur- was a generous gift from ShowalDenko Co., and PLP was thermore, the development of chemical syn- kindly provided by Dainippon Pharmaceutical Co. Other thesis techniques has madeit possible to pro- chemicals were purchased from commercial sources. duce 3-Cl-Ala economically on a large scale.8) Bacterial strain and culture. Escherichia coli W3110 was These observations prompted us to inves- obtained from the stock culture of the Laboratory of tigate and to develop an enzymatic method Industrial Microbiology, Department of Food Science and for the synthesis of useful amino acids from 3- Technology, Faculty of Agriculture, Kyoto University. Cl-Ala. Cells were picked up from an agar plate containing LB medium,10) inoculated into a tube containing 5ml of LB This paper reports the purification and prop- broth and then grown at 37°C overnight with reciprocal erties of the enzyme responsible for the syn- shaking. This preculture was transferred to a 2-1 Sakaguchi thesis of S-carboxymethyl-L-cysteine (SC-Cys) flask containing 500ml of modified Vogel and Bonner from 3-Cl-Ala and thioglycolic acid (TGA). mediumn) containing 0.5% glucose, 0.2% citric acid - H2O, SC-Cys exhibits mucolytic activity and is use- 0.35% NaNH4HPO4-4H2O, 1% K2HPO4, 0.02% Mg- SO4 7H2O and 0.05% Casamino acids, and then grown ful for loosening mucus in the trachea.9) at 28°C for 24hr with reciprocal shaking. Two such sub- * Corresponding author. 2482 H. Kumagai et al. cultures were inoculated into a 30-1 jar fermentor (type Dowex 1x2 (OH form) column (3.6 by 34cm) and MSJ-U 301; Marubishi Co.) containing 201 of the elution was carried out with 0.2n acetic acid solution. modified Vogel and Bonner medium. Cultivation was carried out at 30°C for 24hr with aeration (0.51/1 of HPLC. HPLCwas performed with an M6000Apump, a medium per min) and agitation (150 rpm). The grown cells U6Kinjector and an M440 UVdetector (wavelength fixed were harvested with a refrigerated continuous flow at 280nm) (Waters Assoc, Milford, MA, U.S.A.). centrifuge (type GLE; Carl Padberg GmbH). Paper chromatography. Some products of the enzyme Enzymeassay. SC-Cys synthase activity was determined reaction were detected and determined by paper chroma- with an automatic amino acid analyzer (type K-101-AS; tography. Ten fil of the reaction mixture was applied on a Kyowa Seimitsu Co.; with Kyowa Gel 62210-S) by filter pater (Toyo No. 51A), followed by development in measuring the SC-Cys synthesized. The assay solution the descending modewith a solvent system of rc-butanol- contained 50 /miol of 3-Cl-Ala, 50 /miol ofTGA, 0.1 mmol acetic acid-water (4 : 1 : 1). The development was repeated of potassium phosphate (pH 7.8), 20nmol of PLP, to obtain a good separation. The products were detected 4.0/rniol of EDTAand the enzyme in a final volume of by spraying a 0.3% ninhydrin solution in a mixture of 1.0ml. The reaction was started by the addition of 3-C1- acetone and ethanol (3: 1). For determination of the Ala, and incubation was carried out at 37°C and stopped product, the spot was cut out and extracted with 5ml ofa by the addition of0.5ml of0.3n HCI. 75%ethanol solution in water containing one drop of 5% O-Acetylserine sulfhydrylase activity was determined CuSO4. The amount of the product was estimated spec- according to Kredick and Becker.12) trophotometrically by measuring the absorbance at The enzymatic a,/?-elimination reaction was assayed by measuring the amount of pyruvate by a spectrophotometric method with lactate dehydrogenase and NADH.13) 500Spectrophotometric measurement.nm.Spectrophotometric One unit of an enzyme is defined as the amount of determination and measurement of absorption spectra enzyme that catalyzed the formation of 1 jiimol of the were carried out with a Hitachi 150-20 spectrophotometer. product under the assay conditions. Specific activity is expressed as units per mgprotein. lH-NMRspectrometric analysis. The ^-NMRspec- trum was measured in D2Owith a Hitachi Model R-22 Protein determination. Protein concentrations were de- (90 MHz)spectrometer and chemical shifts were expressed termined by the method of Lowry et al.l4) with bovine relative to DSSas an internal standard. serum albumin as a standard. Mass spectrometric analysis. The mass spectrum was Electrophoresis. Electrophoresis and protein staining measured with a Hitachi Model M-80 mass spectrometer were carried out according to the guide book from with electron impact ion at an ionizing voltage of 20eV. Pharmacia (Polyacrylamide Gel Electrophoresis-Labora- tory Techniques). Proteins on native gel electrophoresis Optical rotatory dispersion analysis. Optical rotatory were stained with amido black 10B and those on SDS dispersion was measured with a JASCO J-5 ORD re- polyacrylamide gel electrophoresis were stained with Co- corder. SC-Cys was dissolved in 1n HC1 at the con- omassie blue R-250. centration of 20 mg/ml. Molecular weight estimation. The molecular weight of Amino acid sequence analysis. Amino acid sequence the enzyme was determined by gel filtration on a high analysis was carried out by automated Edman degra- performance liquid chromatograph (HPLC) equipped dation with an Applied Biosystems gas phase sequencer with a TSK-GEL3,000 SWcolumn. The molecular weight (Model 470A) equipped online with a PTH-amino acid of a subunit was estimated by SDS polyacrylamide electro- analyzer (Model 120A). phoresis, using an LMWmolecular weight calibration kit purchased from Pharmacia. It was also determined by Purification ofSC-Cys synthasefrom E. coli W3110. All SDS gel filtration on a HPLC equipped with a TSK-GEL operation were carried out at 0 to 5°C unless otherwise 2,000 SWcolumn. stated. The pH of the enzyme solution was adjusted to 7.0 with a 2.5% ammoniumhydroxide solution after each Ion-exchange column chromatographies. Ion exchange addition of ammoniumsulfate. column chromatography was performed for the purification of SC-Cys produced through the enzyme reaction. Purification steps. Cation-exchange columnchromatography was performed (i) Preparation ofa cell extract. Cells harvested from 40 1 on a Dowex 50x8 (H+ form) column (3.6 by 25cm) and of culture (wet weight, 230g) were suspended in 1,150ml elution was carried out with a 0.25n NH4OHsolution. of 0.1 m potassium phosphate buffer, pH 7.8, containing Anion-exchange chromatography was performed on a 10mM2-mercaptoethanol, 20jjm PLP and 4.0mM EDTA E. coli S-Carboxymethylcysteine Synthase 2483 (this solution was namedBuffer A) and 1.0mMphenyl- eluted at the concentration of about 80mM. The active methyl sulfonyl fluoride, and disrupted with a Dyno-Mill fractions were combinedand concentrated by the addition (W. A. Bachofen, Maschinenfabrik, Basel, Switzerland) at of ammoniumsulfate to 90%saturation. the agitation rate of 3,000 rpm using glass beads of0.25 to (vii) Sephadex G-100 gel filtration. The enzyme solution 0.50mmin diameter. The supernatant solution was ob- was applied to a Sephadex G-100 column (2.0 by 102cm) tained by centrifugation. equilibrated with 50 mMBuffer A. The enzyme was eluted (ii) First ammoniumsulfate fractionation. Solid am- with the same buffer in 5-ml fractions. The active fractions moniumsulfate was addedto the enzymesolution to 35% were combined and concentrated by the addition of saturation. After standing for more than 1 hr, the super- ammoniumsulfate to 90%saturation. natant was collected by centrifugation and brought to 70% (viii) Sephadex G-150 gel filtration. The enzyme solution saturation in the same manner. After standing overnight, was applied to a Sephadex G-150 column (2.0 by 100cm) the precipitate was collected by centrifugation, dissolved equilibrated with 50 mMBuffer A.

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