OF VOl. 259, No. 16, of 25, PP. 10393-10403 1984 1% ... · The enzyme was purified from Streptomyces phaeo- chromogenes (IF0 3105) in nine steps. After the last steps, the enzyme

THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1% by The American Society of Biological Chemists, Inc

VOl. 259, No. 16, Issue of August 25, PP. 10393-10403 1984 Printed in C.S.A.

Cystathionine y-Lyase of Streptomyces phaeochromogenes THE OCCURRENCE OF CYSTATHIONINE y-LYASE IN FILAMENTOUS BACTERIA AND ITS PURIFICATION AND CHARACTERIZATION*

(Received for publication, December 21, 1983)

Toru Nagasawa, Hiroshi Kanzaki, and Hideaki Yamada From the Department of Agricultural Chemistry, Faculty of Agriculture, Kyoto University, Sakyo-Ku, Kyoto 606, Japan

Cystathionine y-lyase (EC 4.4.1.1) is widely distributed in actinomycetes, e.g. genera Streptomyces, Mi- cromonospora, Micropolyspora, Mycobacterium, No- cardia, Streptosporangium, and Streptouerticillium. The enzyme was purified from Streptomyces phaeochromogenes (IF0 3105) in nine steps. After the last steps, the enzyme appeared to be homogenous by the criteria of polyacrylamide gel electrophoresis, analytical centrifugation, and double diffusion in agarose. The enzyme crystallized in the apo form with the addition of ammonium sulfate. The enzyme has a molecular weight of about 166,000 and consists of four subunits identical in molecular weight. The enzyme exhibits absorption maxima at 278 and 421 nm and contains 4 mol of pyridoxal 5’-phosphate/mol of enzyme. L-Cystathionine, L-homoserine, DL-lanthionine, L-djenkolic acid, and L-cystine are cleaved as preferred substrates by the Streptomyces enzyme. The a,B-elimination reaction of L-cystathionine is also catalyzed by the enzyme at a ratio of about one-seventh of the a,y- elimination reaction.

Cystathionine &synthase (EC 4.2.1.22) and cystathionine y-synthase (EC 4.2.99.9) activities were also detected in crude extracts of S. phaeochromogenes, but cystathionine &lyase (EC 4.4.1.8) was not. Conse- quently, the reverse transsulfuration pathway in actinomycetes may be similar to that in yeast and molds.

Transsulfuration is the term used to describe the transfer of sulfur between L-cysteine and L-homocysteine. L-Cystathi- onine is an important intermediate in transsulfuration. There are two types of enzymes which catalyze L-cystathionine cleavage: cystathionine y-lyase (EC 4.4.1.1) and cystathionine @-lyase (EC 4.4.1.8). Cystathionine y-lyase catalyzes the cleavage of L-cystathionine to L-cysteine, a-ketobutyrate, and ammonia and was first found in rat liver extract by Carroll et al. (1). Matsuo and Greenberg (2) purified and crystallized the enzyme from rat liver. This enzyme is also found in Neurospora crassa (31, Aspergillus niduhns (4), Saccharomy- ces cereuisiae (3), and Sacchuromycopsts lipolyticu ( 5 ) . How- ever, the microbial enzyme has not been purified and its properties remained unclear. Cystathionine @-lyase catalyzes the a,@-cleavage of L-cystathionine to L-homocysteine, pyruvate, and ammonia and has been found widely in bacteria (3, 6-9). Previous studies have all indicated that procaryotic

* This work was supported in part by a grant-in-aid for scientific reserach from the Ministry of Education, Science, and Culture of Japan. 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.

organisms lack cystathionine y-lyase activity (3, 10). In Cephalosporium acremonium, L-cysteine is the direct

donor of sulfur for cephalosporin C; the addition of L-cysteine to the media does not stimulate synthesis of cephalosporin C. The sulfur atom of cephalosporin C is principally supplied by methionine via the reverse transsulfuration pathway (11). Treichler et al. (12, 13) have recently demonstrated that cephalosporin C formation depends on functional cystathionine y-lyase; mutants having impaired cystathionine y-lyase are also impaired in @-lactam antibiotic formation. They suggested that a cysteine precursor deriving from cystathionine, rather than cysteine itself, might be incorporated into the bicyclic molecule. In contrast to the numerous and exten- sive studies using Cephalosporium, the metabolism of cysteine and methionine by 8-lactam-producing actinomycetes is a topic which has been virtually ignored. Kern and Inamine (14) have recently examined for the first time the possible importance of reverse transsulfuration in the cephamycin C producer Streptomyces luctamdurans. Their studies with S. lactamdurans have demonstrated a bacterial cystathionine y- lyase for the first time time; however, they did not purify and characterize the y-lyase.

These interesting situations prompted us to study the actinomycetous cystathionine y-lyase. We first investigated the distribution of cystathionine and homoserine cleavage activities in bacteria and actinomycetes. Cystathionine y-lyase activity was not found in bacteria, but we found a wide distribution of the enzyme in actinomycetes. Cystathionine y-lyase occurred abundantly in Streptomyces phaeochromogenes (IF0 3105). S. phaeochromogenes was therefore chosen for purification of the enzyme. We were able to obtain a homogenous crystalline preparation of the enzyme. In the present paper, we described the purification of actinomycetous cystathionine y-lyase and investigate its physicochemical and enzymatic properties. Furthermore, we attempt to take the first step toward the elucidation of transsulfuration in actinomycetes.

EXPERIMENTAL PROCEDURES

Materials-L-Cystathionine, crystalline L-alanine dehydrogenase (EC 1.4.1.1) from Bacillm subtitis, L-amino acid oxidase (EC 1.4.3.2) from CrOtalW atr0.Z venom, D-cycloserine, D-penicillamine, L-penicillamine, @-2-thienyl-DL-alanine, L-aminocyclopropane-1-carboxylic acid, DL-propargylglycine, DL-vinylglycine, fi-cyano-L-alanine, DL- allylglycine, 0-acetyl-L-serine, and 0-succinyl-L-homoserine were obtained from Sigma. Hog kidney D-amino acid oxidase (EC 1.4.3.3) was from Boehringer Mannheim. DEAE-Sephacel, phenyl-Sepharose CL-IB, octyl-Sepharose CL-4B, Sephadex G-200, and a kit of low- molecular-weight standards were obtained from Pharmacia. Pig heart lactate dehydrogenase (EC 1.1.1.27), NAD, NADH, and marker proteins for molecular-weight determination on high-performance liquid chromatography were purchased from Oriental Yeast (Tokyo). DEAE-cellulose (DE52) was from Whatman. A membrane filter

10393

10394 Cystathionine y-Lyase of Streptomyces phaeochromogenes

(Diaflo Ultrafilter, YM-IO) was obtained from Amicon Corp. The ampholytes required for isoelectric focusing were made by LKB- Productor AB. 0-Chloro-L-alanine and P-chloro-D-alanine were syn- thesized from L-serine and D-serine, respectively, according to the method used by Walsh et al. (15). 0-Acetyl-L-homoserine was syn- thesized using the method of Nagai and Flavin (16). Hydroxylapaptite was prepared as described by Bernadi (17). All of the other chemicals used in this study were from commercial sources and of reagent grade.

Microorganisms and Culture Conditions for Studies on the Distri- bution of Microbial Cystathionine-cleaving Enzyme-Ninety-three bacterial strains belonging to 21 genera and 50 actinomycetous strains belonging to seven genera were used in the studies on the distribution of cystathionine y-lyase in bacteria and actinomycetes. The microorganisms came from stock cultures of the Institute of Fermentation, Osaka (IFO-type culture collection) and from our laboratory (AKU- type culture collection). The following bacterial genera were examined Achromobacter, Aerobacter, Agrobacterium, Alcaligenes, Arthro- bacter, Bacillus, Brevibacterium, Cellulomonas, Chromobacterium, Ci- torobacter, Corynebacterium, Erwinia, Escherichia, Micrococcus, Haf- nia, Proteus, Pseudomonas, Salmonella, Serratia, Staphylococcus, and Xanthmonas. The bacteria were cultivated, with shaking, at 28 "C for 24 h in a medium consisting of 0.5 g of Polypepton (Daigo, Osaka), 0.5 g of yeast extract (Oriental Yeast, Tokyo), and 0.1 g of NaC1/100 ml of tap water (pH 7.0) (Medium I). The cells were collected by centrifugation at 8500 X g at 4 "C for 20 min.

The following genera of actinomycetes were examined: Micromo- nospora, Micropolyspora, Mycobacterium, Nocardia, Streptomyces, Streptosporangium, and Streptouerticillium. They were cultivated, with shaking, a t 28 "C for 48 h in the following media: 0.4 g of yeast extract, 1.0 g of malt extract (Difco), and 0.5 g of Polypepton/100 ml of tap water (pH 7.0) (Medium 11); 425 mg of Na L-glutamate, 1.0 g of glucose, 0.1 g of NH4CI, 20 mg of inositol, 0.2 g of K,HPO.,, 50 mg of NaCl, 50 mg of MgC12.6H20, 25 mg of CaC03, 2.5 mg of FeSO,. 7Hz0, 1 mg of ZnS04-7HZ0, 0.5 mg of MnS04.4-6H20, and 0.1 mg of p-aminobenzoate/100 ml of tap water (pH 7.0) (Medium 111); and 0.1 g of yeast extract, 0.1 g of meat extract (Mikuni, Tokyo), 0.1 g of cotton seed flour (Pharmamedia, Tokyo), 0.1 g of soybean hydroly- sate, and 1.0 g of glucose/100 ml of tap water (pH 7.0) (Medium IV). The cells were collected by centrifugation for 20 min at 8500 X g.

The cells were suspended in 0.1 M potassium phosphate buffer (pH 7.5) containing 20 p~ pyridoxal 5'-phosphate, 0.12 mM 2Na EDTA, and 0.2 mM dithiothreitol and disrupted with ultrasonic oscillation (19 kHz below 15 "C for 2-20 min). In some cases, the cells were opened with lysozyme, as described by Burgess and Jendrisak (18). The crude cell extract was centrifuged at 12,000 X g for 30 min, and the supernatant solution was dialyzed against the above buffer for 12 h and used as the cell-free extract.

S. phueochromogenes (IF0 3105) was selected as a likely source of enzyme for purification. The basal medium for cultivation consisted of 2 g of yeast extract, 0.3 g of glucose, 0.2 g of K2HP04, 0.1 g of MgClZ.2H20, 4 mg of CaC12.2HZ0, 2 mg of FeS0,-7Hz0, 2 mg of MnS04.4-6H20, 2 mg of CuS04.5Hz0, and 1 mg of ZnSO4.7H20/ 100 ml of tap water (Medium V). The pH of the medium was adjusted to 7.0 by the addition of 4 M NaOH. S. phueochromogenes was collected from an agar slant of the basal medium and inoculated into a subculture. The subculture (3.5 liters) was shaken reciprocally at 28 "C for 48 h and, in turn, inoculated into a 100-liter jar fermentor containing 70 liters of basal medium supplemented with 35 g of antifoam (AF-emulsion). Incubation was carried out a t 28 "C for 20 h with aeration (37 liters/min). The cells grown from 33.5 liters of broth were harvested by continuous-flow centrifuge and washed with 0.15 M NaCl containing 0.1 mM EDTA. The yield of wet cells was approximately 20 g/liter of medium.

Cystathionine y-Lyase Assay and Definition of Units-The enzymatic a,y-elimination reaction of L-homoserine was routinely followed by determination of a-ketobutyrate. The standard assay system contained 42 pmol of potassium phosphate buffer (pH 8.0), 50 pmol of L-homoserine, 40 nmol of pyridoxal-P, and the enzyme with a final volume of 1.0 ml. The enzyme was replaced by water in a control. Incubation was carried out at 30 "C for 30 min, and the reaction was terminated by the addition of 0.2 ml of 30% trichloroacetic acid. After centrifugation, a determination was made of a-ketobutyrate in the supernatant solution using the method described by Friedemann and Haugen (19) (Assay I). In some cases, 2.1 pmol of L-cystathionine were used as a substrate instead of L-bomoserine, with all other conditions remaining constant. The amount of L-cysteine formed was determined by Gaitonde's acidlninhydrin method (20) (Assay 11).

After the enzymatic a,B-elimination reaction, the amount of pyruvate liberated from a substrate was measured by spectrophotometry with pig heart lactate dehydrogenase and NADH. The reaction was carried out at 30 "C in cuvettes containing 2 ml of the following solution: 0.16 mmol of potassium phosphate buffer (pH 8.0), 0.15 pmol of pyridoxal-P, 0.26 pmol of NADH, 10 units of pig heart lactate dehydrogenase, the substrate, 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 that had been produced was calculated from the molar extinction of NADH, 6.220 M" .cm" (Assay 111).

Protein determination was carried out by the method of Lowry et al. (21) using crystalline bovine serum albumin as a standard. For cystathionine y-lyase from S. phaeochromogenes, protein was determined by its absorption at 280 cm. The absorption coefficient of 0.839 mg-'.ml-cm" was calculated using absorbance and dry weight determination and was constant throughout.

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

Other Assays-Cystathionine P-synthase (EC 4.2.1.22) activity was determined as follows. To block cystathionine y-lyase activity, the cell-free extract of S. phaeochromogenes was titrated with anti-cystathionine y-lyase serum (22). The reaction was carried out at 30 "C for 30 min in 1 ml of reaction mixture containing 0.1 mmol of HEPES' buffer (pH 8.6). 0.2 pmol of pyridoxal-P, 20 pmol of DL-homocysteine, 10 @mol of L-serine, and an appropriate amount of cell-free extract. The reaction was terminated by adding 0.2 ml of 30% trichloroacetic acid. After brief centrifugation, the supernatant was mixed with diethyl ether to extract the excess trichloroacetic acid, and the amount of cystathionine was determined with a standard amino acid analyzer.

Cystathionine y-synthase activity was measured by determining the formation of cystathionine from 0-acyl-L-homoserine (O-acetyl- or 0-succinyl-) and L-cysteine with an amino acid analyzer. The assay was carried out at 30 "C for 30 min in a reaction mixture (0.5 ml) consisting of 168 pmol of potassium phosphate buffer (pH 8.0), 32 nmol of pyridoxal-P, 12.5 pmol of 0-succinyl-L-homoserine (or 0- acetyl-L-homoserine), 5 pmol of L-cysteine, and an appropriate amount of antiserum-treated cell-free extract.

The enzymatic assay of 0-acetylserine sulfhydrylase (EC 4.2.99.8) was carried out at 30 "C for 30 min in 1.0 ml of the reaction mixture which consisted of 0.2 mmol of Tris/HCl buffer (pH 7.5), 1 pmol of EDTA, 0.1 mmol of 0-acetyl-L-serine, 80 pmol of sodium hydrosulfide, and an appropriate amount of cell-free extract. The reaction was terminated by adding 0.2 ml of 30% trichloroacetic acid. After brief centrifugation, the amount of L-cysteine in the supernatant solution was determined using Caitonde's acid/ninhydrin method (20).

0-Acylhomoserine sulfhydrylase (EC 4.2.99.10) activity was measured by determining the formation of homocysteine from O-succinyl- L-homoserine (or 0-acetyl-L-homoserine) and sodium hydrosulfide. One ml of reaction mixture contained 20 pmol of 0-succinyl-L- homoserine, 6 pmol of sodium hydrosulfide, 0.12 pmol of pyridoxal- P, 0.2 mmol of potassium phosphate buffer (pH 7.51, and an appropriate amount of cell-free extract. The reaction was run for 30 min at 30 "C and stopped by the addition of 0.2 ml of 30% trichloroacetic acid. After brief centrifugation, the supernatant solution was bubbled with nitrogen gas for 3 min to remove the remaining sulfide. The amount of homocysteine which had been found was determined using the method described by Ellman et al. (23).

Electrophoresis in Polyacrylamide-Sodium dodecyl sulfate-gel electrophoresis was performed in 10% of the polyacrylamide slab gels using the Tris/glycine buffer system described by King and Laemmli (24). The gels were stained for protein with Coomassie Brilliant Blue G-250 and destained in ethanol/acetic acid/H,O (31:6). The relative molecular mass of the enzyme subunit was obtained from the relative mobility of the standard proteins.

Isoelectric Focusing-The isoelectric point of the enzyme was determined using the method described by Winter and Karlson (25). The density gradient of pH range 3-10 contained 2% ampholyte. Samples of the enzyme (about 1.2 mg) were exhaustively dialyzed against 0.13 M glycine, then applied to the column after formation of about one-third of the sucrose gradient. Electrofocusing was carried

' The abbreviations used are: HEPES, N-2-hydroxyethylpipera- zine-N'-2-ethanesulfonic acid; DTT, dithiothreitol.

Cystathionine y-Lyase of Streptomyces phaeochromogenes 10395

out a t 5 "C until there were no further changes in current (44 h), during which time the voltage was increased from 300 to 600 V. The column was then attached to a fraction collector, and fractions (2.8 ml) were collected until the column was emptied. Absorption at 280 nm, pH, and enzyme activity of the fraction were measured.

Ultracentrifugal Analyses-The purity of the enzyme and its sedimentation coefficient were determined using a Spinco Model E ultracentrifuge equipped with a phase plate as a schlieren diaphragm and operating at 12,590 rpm with the boundary condition at the meniscus in the sector-shaped centrifuge cell. The molecular weight was calculated from the sedimentation and diffusion coefficients using the equation developed by Svedverg and Pederson (26).

The determination of molecular weight was carried out using the sedimentation equilibrium method as done by Van Holde and Baldwin (27). A Spinco Model E ultracentrifuge equipped with Rayleigh interference optics was used. Multicell operations were employed in order to perform the experiments on five samples of different initial concentration ranging from 0.51 to 3.37 mg/ml. An An-G rotor and double cells of different side-wedge angles were used. The rotor was centrifuged at 8225 and 9945 rpm for 13 h at 10 "C. Interference patterns were photographed at intervals of 30 min to ensure that equilibrium was established. The relation between the concentration of the enzyme and the fringe shift was determined using synthetic boundary cells.

High-performance Gel Permeation Liquid Chromatography-Six-k1 samples of the enzyme (Am = 8.0) were subjected to high-performance liquid chromatography (Hitachi 638-30, Tokyo) on a G-3000SW protein column (0.75 X 60 cm, Toyo Soda, Tokyo) at a flow rate of 0.3 ml/min and eluted with 50 mM potassium phosphate buffer (pH 7.5) containing 0.2 M NaCl at room temperature. The absorbance of the effluent was recorded at 280 nm. The molecular weight of the enzyme was then calculated from the mobility of the standard proteins.

Antiserum Preparation-Antibodies were elicited by injection of 3.2 mg of cystathionine y-lyase purified from S. phmochromogenes into a young white male rabbit. The antigen was dissolved in 0.9 ml of 10 mM potassium phosphate buffer (pH 7.5) containing 10 @M pyridoxal-P and 0.2 mM DTT, homogenized in an equal volume of complete Freund's adjuvant (Difco), and injected into a multiple subcutaneous site on the back. The animal received booster injections at 4 and 6 weeks. The booster injections were administered subcuta- neously on the neck with 1.1 mg of the antigen homogenized in an equal volume of incomplete Freund's adjuvant (Difco). On the 20th day following the final booster injection, blood was collected from the ear vein and artery and allowed to clot. The serum was centrifuged at 6000 X g for 10 min and the supernatant solution was stored at -80 "C.

Double diffusion was performed on microscope slides which were coated with 1% agarose (Difco) in 10 mM Tris/H,SO, (pH 8.0) containing 0.01% sodium azide following Ouchterlony's method (28). Antisera (5-20 pl) were added t u the center wells and antigens to the peripheral wells. Maximal precipitates developed within 30 h at room temperature, The plates were washed several times over 4 days with physiological saline and then stained with 0.5% Amido Black 10B in methanol/acetic acid (9:l) for 10 min. After a final washing with methanol/acetic acid (91), the plates were photographed.

Preparation of Cystathionine y- and @-Lyases for Serological Study-Rat liver cystathionine y-lyase was purified according to the method used by Matsuo and Greenberg (2). Four young male rats (180 g) were killed by decapitation, and the livers were excised from the well-bled carcasses and washed with cold 0.1 M potassium phosphate buffer (pH 7.5) containing 40 PM pyridoxal-P, 1 mM EDTA, and 1 mM DTT to remove the blood as completely as possible. The minced liver was suspended in the same buffer and homogenized. After centrifugation at 12,000 X g for 30 min, the supernatant solution was subjected to ammonium sulfate fractionation, heat treatment, and ethanol precipitation. The specific activity of the final preparation was 2.99 units/mg under standard conditions using L-homoserine was the substrate. The enzyme was preserved in 45% glycerol a t -20 "C.

Crude cystathionine y-lyase was prepared from Streptomyces oli- uochromgenes (IF0 3178). Streptomyces 1ydicu.s (AKU 2502) S. lactamdurans (IF0 13305), Streptouerticillium kentuchense (IF0 12880), Streptosporangium roseurn (IF0 3776), Micromonospora c h h a (IF0 121351, Micropolyspora angiospora (IF0 131551, Microellobosporia uiolucea (IF0 12517), Nocardia erythropolis (IF0 12539), Mycobacte- rium smegmatis (IF0 31541, Hansenulu jadinii (IF0 0987), Pinchia

wickerhumii (IF0 1278), and Candida utilis (IF0 0396). These strains were cultivated for 48 h at 28 "C in a 2-liter shaking flask containing 500 ml of Medium V (pH 7.0). The cells were harvested by centrifugation at 8500 x g a t 4 "C for 20 min and disrupted with an ultrasonic oscillator or a DYNO-MILL (W. A. Bachofen, Basel) in 0.1 M potassium phosphate buffer (pH 7.5) containing 0.12 mM EDTA, 20

pyridoxal-P, and 0.5 mM DTT. The cell debris was removed by centrifugation at 12,000 X g for 30 min and then the supernatant solution was dialyzed overnight against 10 mM potassium phosphate buffer (pH 7.5) at 5 "C. The precipitate that appeared during the dialysis was removed by centrifugation at 12,000 X g for 30 min, and the supernatant solution was used as the cell-free extract. This cell- free extract was fractionated with ammonium sulfate, and the 30- 60% saturated fraction was used for Ouchterlony's test.

Pseudomonas dachunhm (IF0 12048) was selected as a likely source of bacterial cystathionine @-lyase. This strain was cultivated in Medium I (pH 7.0) for 20 h. The cells were harvested from 2.5 liters of medium by centrifugation at 8,000 X g at 4 "C for 20 min, washed with 0.85% NaCl solution, and suspended in 0.1 M potassium phosphate buffer (pH 7.0) containing 0.1 mM pyridoxal-P and 0.1 mM DTT. The cells were disrupted with an ultrasonic oscillator, and the cell debris was removed by centrifugation at 12,000 X g for 30 min. After the dialysis of the supernatant against 10 mM potassium phosphate buffer (pH 7.0) containing 0.1 mM pyridoxal-P and 0.1 mM DTT, the cell-free extract was subjected to DEAE-Sephacel chromatography equilibrated with the same buffer. The active fractions were collected, concentrated by ultrafiltration, and preserved in 45% glycerol at -20 "C. The purified enzyme catalyzed the a.0- elimination of L-cystathionine at 9.7 x lo-' mol/min/mg of protein. Protein was determined by measuring the absorbance at 280 nm.

Other Analytical Methods-Spectrophotometric measurements were made with a Hitachi 220A spectrophotometer with a 1.0-cm light path. The amino acids in the incubation mixture were identified by co-chromatography with authentic materials on a standard amino acid analyzer (Kyowa Seimitsu K-101AS) using the method developed by Spackman et al. (29).

RESULTS

Distribution of Cystathionine y-lyase in Actinomycetes and Bacteria

Fifty actinomycetous strains from stock cultures of our laboratory (AKU-type culture collection) and IFO-type culture collection were tested for their ability to form the enzyme which catalyzes the a,y-elimination reaction of L-cystathionine. L-Homoserine and L-cystathionine were used as substrates, and the formation of a-keto acid and L-cysteine was measured (Table I). Gaitonde's acid/ninhydrin assay (20) was used to determine the amount of cysteine. This assay is highly specific and gives essentially no reaction with methionine or homocysteine. The lack of color development with homocysteine means that the assay is specific for y-lyase activity and does not measure any @-lyase activity (&lyase would generate homocysteine from cystathionine). The formation of a-keto acid, e.g. a-ketobutyrate or pyruvate, might result from the cleavage of L-cystathionine by cystathionine y-lyase or cystathionine p-lyase, respectively. However, the formation of L-cysteine is restricted to the enzymatic a,y-elimination reaction of L-cystathionine. Cystathionine y-lyase activity was found in all strains tested. All of the genera of actinomycetes were used in this part of the study. The strains belonging to the genus Streptomyces showed relatively strong cystathionine y-lyase activity. 5'. phaeochromogenes (IF0 3105) was chosen for the purpose of purification of the enzyme, as it produced abundant cystathionine y-lyase.

Ninety-three bacterial strains belonging to 21 genera were also tested for cystathionine y-lyase activity. Although most of the bacteria tested catalyzed the degradation of L-cystathionine to a-keto acid, no distinct formation of L-cysteine from L-cystathionine could not be detected. Cystathionine @-lyase may therefore be responsible for the formation of a-keto acid. Furthermore, Bacillus brevis, Corynebacterium aquaticum,

10396 Cystathionine y-Lyase of Streptomyces phueochromogenes

TABLE I Cystathionine y-lyase activity in actinomycetes

Each strain was grown in three kinds of media (11,111 and IV, 350 ml) for 48 h, as described under "Experimental Procedures." Keto acid and L-cysteine were measured using the methods described by Friedemann and Haugen (19) and Gaitonde (20), respectively. Protein was determined following Lowry et al. (21).

Cysteine formed Keto acid formed from cystathioine from cystathionine

Keto acid formed from homoserine

Strain Medium

activitp activity activity activity activity activity Total Specific Total Specific Total Specific

Micromonospora chulcea IF0 12135 IV 3.13 0.25 16.10 1.28 13.85

IF0 13504 coerulea IV 4.03 3.29 0.17 0.14 4.34 3.55 1.10

IF0 13155 Micropolyspora IV 3.34 0.47 0.16 0.02 4.34 0.61 angiosporo

Mycobacterium smegmatis IF0 3154 I1 4.36 0.28 0.29 8.76 phlei

4.63 AKU 2005 I1 3.49 0.37 8.67 0.93 4.80

0.56

Nocardia 0.51

asterodes IF0 3384 IV 5.63 0.61 13.47 1.46 11.31 1.22 IF0 3338 corallina I1 5.25 1.04 12.49 2.46 5.44 1.07 IF0 12539 erythropolis IV 9.71 2.14 8.02 1.77 47.30 IF0 13509 globerula

10.44 IV 9.72 1.80 17.41 3.22 9.43 1.74

Streptomyces lactamdurans IF0 13305 111 9.03 0.78 28.00 2.42 37.79 3.26

IF0 13306 tipmanii 111 9.55 1.36 64.97 9.27 63.96 9.13 AKU 2502 lydicw I11 12.52 1.09 27.83 2.74 33.06 2.89

IF0 3178 olivochromgenes 111 10.24 0.39 39.78 1.53 49.08 1.89 IF0 3105 phaeochromogenes I1 11.43 1.19 57.06 5.92 35.54 3.69 IF0 3226 rimosus I1 9.40 0.35 86.98 3.22 83.28 3.09 IF0 12827 virginiae 111 9.02 0.75 24.52 2.03 23.82 1.97 IF0 3776 Streptosporangium IV 5.08 0.71 7.25 1.02 10.30 1.45

roseum Streptoverticillium IF0 12880 111 13.11 4.23 23.91 10.93 21.68 6.99

kentuchense Total activity is expressed as micromoles/min.

* Specific activity is expressed as (micromoles/min/mg) X

Proteus morganii, Pseudomonas syringae, Pseudomonas mal- tophilia, and Xanthomonas campestris all exhibited the ability to cleave L-homoserine. In order to clearly characterize the enzyme, this enzyme was partially purified from P. morganii by ammonium sulfate fractionation and DEAE-Sephacel chromatography (data not shown). The substrate specificity of the enzyme was very similar to that of methionine y-lyase (EC 4.4.1.11) (30). The fact that the enzyme could not cleave L-djenkolic acid or L-cystine, which are good substrates of cystathionine y-lyase (31,32), implies that this enzyme might be methionine y-lyase.

Enzyme Purification

Steps 1-9 were carried out at 4 "C, and potassium phosphate buffer containing 20 FM pyridoxal-P, 0.12 mM EDTA, and 0.2 mM DTT was used, unless otherwise specified.

Step 1. Preparation of Cell-free Extract-Washed cells (670 g) were suspended in 2 liters of 0.1 M potassium phosphate buffer (pH 7.5) and disrupted with ultrasonic oscillation (19 kHz). The cell debris was removed by centrifugation at 12,000 X g for 30 min. The supernatant solution was then dialyzed overnight against 10 mM potassium phosphate buffer (pH 7.5) at 5 "C. The precipitate which appeared during the dialysis was removed by centrifugation at 12,000 X g for 30 min, and the resulting supernatant solution was used as the cell-free extract.

Step 2. First Ammonium Sulfate Fractionation-Solid ammonium sulfate was added to the cell-free extract to give 30% saturation (at 0 "C). The pH was maintained at 7.5 with ammonia solution. After stirring for 2 h or more, the precipitate was removed by centrifugation at 12,000 X g, and the supernatant solution was further saturated with ammonium

sulfate to give 60% saturation (at 0 "C). The suspension was then centrifuged at 12,000 X g, and the pellet was dissolved in 0.1 M potassium phosphate buffer (pH 7.5) and dialyzed for 36 h against three changes of 10 liters of 10 mM potassium phosphate buffer (pH 7.5).

Step 3. DEAE-cellulose Column Chromatography-The dialyzed enzyme solution was applied to a DEAE-cellulose column (3.5 X 60 cm) which had been equilibrated with 0.1 M potassium phosphate buffer (pH 7.5). The column was washed fully with 0.1 M potassium phosphate buffer (pH 7.5) containing 0.1 M KC1 until the absorbance of the effluent at 280 nm was reduced to 0.2 or less. The enzyme was then eluted with 0.1 M potassium phosphate buffer (pH 7.5) containing 0.2 M KC1. Fractions of 17 ml were collected, the active eluates were combined, and solid ammonium sulfate was added with stirring to bring the solution to 60% saturation. The precipitate was collected by centrifugation, dissolved in a small amount of 0.1 M potassium phosphate buffer (pH 7.5), and dialyzed against three changes of 10 liters of 10 mM potassium phosphate buffer (pH 7.5).

Step 4. Second Ammonium Sulfate Fractionation-The dialyzed enzyme preparation from Step 3 was then subjected to fine ammonium sulfate fractionation. The precipitate occur- ring between 50 and 60% saturation, the fraction having the highest specific activity, was dissolved in 0.1 M potassium phosphate buffer (pH 7.5) and dialyzed against three changes of 5 liters of 10 mM potassium phosphate buffer (pH 7.5).

Step 5. DEAE-Sephacel Column Chromatography-The enzyme solution from Step 4 was placed on a column (2.7 X 27 cm) containing DEAE-Sephacel which had been equilibrated with 0.1 M potassium phosphate buffer (pH 7.5). After the column was washed with the same buffer, the enzyme was

Cystathionine 7-Lyase of Streptomyces phaeochromogenes 10397

eluted with a linear gradient of KC1 (0-0.5 M, 400 ml in each container) in the same buffer at flow rate of 80 ml/h. Fractions of 3.3 ml were collected and the active fractions were pooled. Solid ammonium sulfate was added to the enzyme solution to give 60% saturation (at 0 "C). After centrifugation of the suspension at 12,000 x g, the precipitate was dissolved in 0.1 M potassium phosphate buffer (pH 7.5) and dialyzed against 5 liters of 0.01 M potassium phosphate buffer (pH 7.5).

Step 6. Hydroxylapatite Column Chromatography-The enzyme solution from Step 5 was placed on a column (2.8 x 14 cm) containing hydroxylapatite which had been previously equilibrated with 10 mM potassium phosphate buffer (pH 7.5). The column was washed fully with 10 mM potassium phosphate buffer (pH 7.5) until the absorbance of the effluent at 280 nm was reduced to 0.1 or less. The enzyme was then eluted with 200 ml of 50 mM potassium phosphate buffer (pH 7.5). Fractions of 3 ml were collected, and the active fractions were combined.

Step 7. Phenyl-Sepharose CL-4B Column Chromatogra- phy-The enzyme solution from Step 6 was cooled to 0 "C, and ammonium sulfate was added in small portions with stirring to bring the solution to 10% saturation. The enzyme solution was placed on a column (1.0 x 26 cm) of phenyl- Sepharose CL-4B which had been equilibrated with 50 mM potassium phosphate buffer (pH 7.5) containing 10% saturated ammonium suflate. The column was washed with 50 ml of 50 mM potassium phosphate buffer (pH 7.5) containing 10 and 5% ammonium sulfate. The enzyme was eluted by low- ering the ionic strength of ammonium sulfate (5 to 0%) in the same buffer. Fractions of 2.3 ml were collected and the active fractions were combined.

Step 8. Octyl-Sepharose CL-4B Column Chromatography- The enzyme solution from Step 7 was cooled, and ammonium sulfate was added in small portions with stirring to bring the solution to 10% saturation. The enzyme solution was placed on a column (0.8 X 10 cm) of octyl-Sepharose CL-4B which had been equilibrated with 50 mM potassium phosphate buffer (pH 7.5) containing 10% saturated ammonium sulfate. The enzyme passed through the column without any loss of activity, but some concomitant proteins were removed by this hydrophobic chromatography. The active fractions were combined and precipitated with solid ammonium suflate at a final concentration of 60%.

Step 9. Sephudex G-200 Column Chromatography-The precipitate from Step 8 was centrifuged at 12,000 X g and dissolved in 10 mM potassium phosphate buffer (pH 7.5). The enzyme solution was concentrated to about 2 ml by ultrafiltration and placed on a column (2.3 x 93 cm) containing Sephadex G-200 which had been equilibrated with 10 mM potassium phosphate buffer (pH 7.5) containing 0.1 M KCl. The rate of sample loading and column elution was kept at 4 ml/h by a peristaltic pump. The protein was eluted with the same buffer, and fractions containing cystathionine y-lyase activity were combined and concentrated by ultrafiltration and preserved in 45% glycerol at -20 "C.

Purification of approximately 3000-fold was achieved, with a yield of 28.9%. The purified enzyme catalyzed the a,y- elimination reaction of L-homoserine and L-cystathionine at 2.60 and 1.90 pmol/min/mg of protein, respectively, under the standard conditions. The purification is summarized in Table 11.

Criteria for Purity

The purified enzyme showed only one band on sodium dodecyl suflate-polyacrylamide slab gel electrophoresis (Fig. 1A). It sedimentated as a single sharp and symmetrical schlie-

TABLE I1 Purification of cystathionine y-lyase from S. phaeochromogenes Protein was determined by its absorption at 280 nm using the

absorption coefficient of 0.839 mg-I .ml .cm-l throughout. The reaction was carried out under standard conditions using L-homoserine as a substrate.

~ ~~ -~ ~ ~

s t ep Total Total Specific

protein activity activity

1. Cell-free extract

3. DEAE-cellulose

5. DEAE-Sephacel 6. Hydroxylapatite 7. Phenyl-Sepharose

8. Octyl-Sepharose

9. Sephadex (2-200

2. (NHI)*SO, (0.3-0.6)

4. (NH,),SO, (0.5-0.6)

CL-4B

CL-4B ~ "

ml: 85,800 48,500

1,970 582 279 114 21.3

units units/mg 73.3 0.000854 70.6 0.00154 69.7 0.0483 43.2 0.0741 41.2 0.148 32.1 0.383 22.3 1.05

% 100 96.3 95.1 58.9 56.2 43.8 30.4

9.54 21.8 2.28 29.7

8.15 21.2 2.60 28.9

2 3 4 5

I+

FIG. 1. Criteria for purity of cystathionine y-lyase. A, sodium dodecyl sulfate-slab gel electrophoresis. The conditions are given under "Experimental Procedures." Upper, marker proteins: I , phosphorylase b (M, = 94,000); 2, bovine serum albumin (67,000); 3, ovalbumin (43,000); 4, bovine erythrocyte carbonic anhydrase (30,000); 5, soybean trypsin inhibitor (20,100). Loruer, the purified cystathionine y-lyase from S. phaeochrornogenes, 28 pg. The direction of migration is from cathode (left) to anode. B, sedimentation pattern. This photomicrograph was taken at 64 min after reaching 59,780 rpm. The protein concentration was 6.0 mg/ml in 10 mM potassium phosphate buffer (pH 7.0) containing 20 pM pyridoxal-P, 0.12 mM EDTA, and 0.2 mM DTT.

ren peak in the analytical ultracentrifuge (Fig. 1B). Ampholyte electrofocusing also yielded only one absorption

peak of protein (PI 4.20). This peak is identical to that of cystathionine y-lyase.

The purity of the purified enzyme was tested by double diffusion in agarose. A single precipitin line was obtained with the crude cell-free extract of S. phaeochromogenes.

Absorption Spectra and Activity of H o b and Apoenzyme The activity of the purified enzyme was dependent on added

pyridoxal-P. The holoenzyme exhibited absorption maxima at 278 and 421 nm with a A27RAA421 ratio of approximately 3.97 (Fig. 2, curue A). No appreciable spectral shifts occurred by varying the pH (6.0-10.0). An absorption peak of 421 nm suggests that the formyl group of the bound pyridoxal-P forms an azomethine linkage with an amino group of the protein, as demonstrated in other studies of pyridoxal-P enzymes. Re-


FIG. 2. Absorption spectra of cystathionine ~-1yase. Curve A, holoenzyme (0.6 ml, 0.8 mg/ml) in 10 mM potassium phosphate buffer (pH 7.5); curve B, apoenzyme (0.6 ml, 0.8 mg/ml) prepared as described in the text; curue C, reconstituted enzyme (0.6 ml, 0.8 mg/ ml) preparaed by dialysis of the apoenzyme against 10 mM potassium phosphate buffer (pH 7.5) containing 1 mM DTT and 10 pM pyridoxal-P; curue D, the enzyme (0.6 ml, O.bmg/ml) after reduction with sodium borohydride following Matsuo and Greenberg (33). Each dialysis buffer was tested to act as a control.

duction of the enzyme with sodium borohydride by dialysis following Matsuo and Greenberg's method (33) affected both the activity and the absorption spectrum (Fig. 2, curue D). The reduced enzyme was catalytically inactive, and the addition of pyridoxal-P did not render it active. These results suggest that sodium borohydride reduces the aldimine linkage formed between the 4-formyl group of pyridoxal-P and an amino group of the protein. The holoenzyme was converted completely to apoenzyme by incubation for 3 h in 10 mM phneylhydrazine (pH 7.0), followed by dialysis against 4 liters of 10 mM potassium phosphate buffer (pH 7.5) containing 1 mM DTT. The apoenzyme could be reconstituted by overnight dialysis against 1 liter of 10 mM potassium phosphate buffer (pH 7.5) containing 10 p~ pyridoxal-P and 1 mM DTT. About 92% of the enzyme's initial activity could be restored a t a given concentration of pyridoxal-P. Spectra of the resolved and partially reconstituted enzyme are shown in Fig. 2, curves B and C, respectively. The Michaelis constant of the enzyme for pyridoxal-P was estimated to be 18 p~ after incubation for 2 h a t 30 "C.

CTystallization The purified enzyme solution was diluted with 10 mM

potassium phosphate buffer (pH 7.5) containing 20 p~ pyridoxal-P and 0.2 mM DTT to obtain a protein concentration of about 20 mg/ml. This solution was brought to 50% ammonium sulfate saturation by the addition of ammonium sulfate at 0 "C while stirring gently with a glass rod. The addition of ammonium sulfate was continued until the in- duced turbidity ceased to disappear upon stirring. The presence of crystallized material was evident from the silky sheen which appeared when the mixture was stirred. When dena-

tured protein strands became visible, the solution was centrifuged to remove them before the enzyme began to crystallize. Photomicrographs of the crystallized enzyme are shown in Fig. 3.

The crystallized enzyme was collected by centrifugation at 3000 X g for 15 min. The enzyme exhibited almost no activity in the absence of pyridoxal-]?; however, full activity was restored by the addition of 20 p~ pyridoxal-P to the reaction mixture. This confirms that the crystallized enzyme was in an apo form.

Molecular Weight and Structure of the Subunit

The sedimentation coefficient (s!~.,) and the diffusion coefficient (Dz0J of the enzyme were calculated to be 9.47 S (20 "C, 10 mM potassium phosphate buffer (pH 7.0) containing 20 p~ pyridoxal-P and 1 mM DTT) and 5.10 X loT7 cm2/ s, respectively. The molecular weight of the enzyme was estimated at 167,000 by the sedimentation velocity method (26). A molecular weight of 168,000 9,000 was obtained by the sedimentation equilibrium method (27), assuming a par- tial specific volume of 0.74. The molecular weight of the enzyme determined by high-performance gel-permeation liquid chromatography was 162,000 (Fig. 4).

When the enzyme was prepared with 1% sodium dodecyl sulfate and 50 mM 2-mercaptoethanol and electrophoresed on a gel containing 0.1% sodium dodecyl sulfate, a single band was observed (Fig. lA). The molecular weight corresponding to the band was estimated by comparing its mobility to that of reference proteins, and a molecular weight of 41,000 was obtained. The enzyme therefore probably consists of four subunits identical in molecular weight.

Pyridoxal-P Content

The amount of pyridoxal-P bound to tht! enzyme was analyzed, assuming that the molar extinction coefficient of pyridoxal-]? in 0.1 M NaOH is 66,000 at 388 nm (34) and that of the phenylhydrazone of pyridoxal-P is 24,500 at 410 nm (35). The enzyme underwent dialysis overnight against 10 mM potassium phosphate buffer (pH 7.0) containing 10 p M pyridoxal-P and 0.2 mM DTT and was then analyzed. An average pyridoxal-]? content of 4 mo1/168,000 g of the enzyme was obtained, indicating that 4 mol of pyridoxal-P are bound to 1 mol of the enzyme protein of the holoenzyme. Since the enzyme appears to be a homotetramer consisting of four identical subunits, each subunit may contain pyridoxal-P at a stoichiometric ratio of unity.

FIG. 3. Photomicrographs of crystalline apoenzyme of cystathionine y-lyase. Left, x 1000; right, X 200

Cystathionine 7-Lyase of Streptomyces phaeochromogenes 10399

I 50 60 70 80

Retention time (mid FIG. 4. Determination of the molecular weight of cystathi-

onine y-lyase by high-performance liquid chromatography. 0, cystathionine y-lyase purified from S. phaeochromogenes; 0, marker proteins: 1, glutamate dehydrogenase from yeast (M. = 290,000); 2, tyrosine phenol lyase from Citrobacter intermedius (195,000); 3, pig heart lactate dehydrogenase (142,000); 4, O-acetylserine sulfhydrylase from Bacillus sphaericw (68,000); 5, enolase from yeast (67,000); 6, adenylate kinase from yeast (32,000); 7, horse heart cytochrome c (12,400).

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

conditions using Assay I as described under "Experimental Procedures." The highly purified enzyme could be stored for 2 weeks without loss of activity in 10 mM potassium phosphate buffer (pH 7.5) containing 20 pM pyridoxal-P, 0.12 mM EDTA, and 0.2 mM DTT at 4 "C. The enzyme remained stable for 2 or more months at -20 "C in 45% glycerol solution containing 10 mM potassium phosphate buffer (pH 7.5), 20 pM pyridoxal- P, 0.12 mM EDTA, and 0.2 mM DTT. No loss of activity was observed when the enzyme was incubated in 42 mM potassium phosphate buffer (pH 8.0) containing 40 p~ pyridoxal-P at 30,35,40,45, and 50 "C for 10 min. Treatment at 55 "C caused approximately 15% loss of initial activity, while treatment at 60 "C resulted in close to 50% loss of initial activity.

Effect of p H and Temperature

The effect of pH on the activity of the enzyme was examined with L-cystathionine and L-homoserine as substrates. In two buffer systems, Tris/HCl and NH,Cl/NH,OH (final concentration 42 mM), the optima for the a,?-elimination reaction of L-cystathionine and L-homoserine were found to be approximately 9.0 (Fig. 5).

The effect of temperature on the activity of the enzyme was examined using L-homoserine as the substrate. When the potassium phosphate buffer (pH 8.0) was used, the optimal temperature for the a,y-elimination reaction was 50 "C.

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

cions of various amino acids was examined (Table 111). The reaction was carried out by modifying Assay I. In addition to L-cystathionine, several of its analogs, e.g., L-djenkolic acid, DL-lanthionine, L-cystine, and S-methyl-L-cysteine, served as preferred subtrates. L-Cysteine, D-cysteine, S-ethyl-L-cysteine, S-n-propyl-L-cysteine, S-n-butyl-L-cysteine, S-n- hexyl-L-cysteine, S-allyl-L-cysteine, N-acetyl-L-cysteine, and D-cystine were inert. L-Homoserine, DL-vinylglycine, and 0-

FIG. 5. Effect of pH on the activity of cystathionine y-lyase. The reactions were carried out for 30 min at 30 "C in the following buffers: potassium phosphate buffer (e), Tris/HCl buffer (O), NH.Cl/ NH,OH buffer (X), CH&OOK/CH3COOH buffer (H). A, cysteine formation from L-cystathionine; B, keto acid formation from L- homoserine.

TABLE 111 Michaelis constants (K,,,) and maximum reaction velocities (V& for

various substrates Substrate K , V,.

mM pmollminlmg

L-Cystathionine 0.204 1.37 L-Homoserine 12.5 2.37 8-Chloro-L-alanine 2.39 10.9 DL-Lanthionine 0.209" 3.76 L-Cystine 0.909 4.73 DL-Vinylglycine 5.18" 10.9 L-Djenkolic acid 1.82 S-Methyl-L-cysteine 1.09

a These values were calculated as the concentration of L-isomer. -

chloro-L-alanine were also effective substrates. These results suggest that the enzyme catalyzes both the a,& and a ,y - elimination reactions. There was no formation of pyruvate or a-ketobutyrate by the elimination reaction of DL-homocysteine, DL-homocystine, D-serine, L-serine, L-methionine, D-alanine, L-alanine, D-threonine, L-threonine, DL-alloth- reonine, 8-cyano-L-alanine, DL-ethionine, L-tryptophan, L- tyrosine, DL-allylglycine, /3-chloro-D-alanine, DL-diaminopropionic acid, 0-acetyl-L-serine, /3-2-thienyl-~~-alanine, or 1- aminocyclopropane-1-carboxylic acid, even when a large amount of the enzyme was added.

The kinetic studies were performed by modifying Assay I and Assay 111. Michaelis constants (K,) and maximum reaction velocities ( Vmax) for L-cystathionine, L-homoserine, 8- chloro-L-alanine, DL-lanthionine, L-cystine, DL-vinylglycine, L-djenkolic acid, and S-methyl-L-cysteine were calculated from the double-reciprocal plots (Table 111). L-Cystathionine showed the highest affinity for cystathionine y-lyase.

Inhibitors Various compounds were investigated for their inhibitory

effects on the enzyme activity using L-homoserine as a substrate. The enzyme was strongly inhibited after a 2-h incubation in hydroxylamine, D- and L-penicillamine, semicarba- zide, phenylhydrazine, 3-methyl-2-benzothiazolinone hydra- zone, and D-cycloserine (inhibition at 1 mM was 94.2, 83.8, 87.3, 94.8,95.9, 91.3, and 75.1%, respectively).

The Streptomyces cystathionine y-lyase displaced a high sensitivity to some thiol reagents (1 mM), e.g. p-chloromer-


curibenzoate, HgCL, AgN03, and ZnC12, showing from 60 to 100% inhibition after a 30-min incubation at 30 "C in each reagent. Sodium cyanide caused 82% inhibition a t 1 mM. DL- Propargylglycine and @-cyano-L-alanine, which inhibit mam- malian cystathionine y-lyase (36), strongly inhibited the activity of this enzyme (80-100% inhibition at 1 mM). ~ - C y s - teine, DL-homocysteine, L-methionine, and S-adenosyl-L-methionine also showed inhibitory effects on the &,?-cleavage activity of L-homoserine (inhibition at 5 mM was 89, 100, 14, and 57%, respectively).

Reaction Products and Stoichiometry of the Enzymatic Cleavage of L-Cystathionine and L-Homoserine

Next, the products and stoichiometry of the enzymatic cleavage of L-cystathionine were studied. The reaction mixture contained 0.21 mmol of potassium phosphate buffer (pH 8.0), 18.75 pmol of L-cystathionine, 20 pmol of pyridoxal-P, 20 pmol of DTT, and the enzyme in a final volume of 5.0 ml and was incubated a t 37 "C in an air-tight tube under a gentle nitrogen gas stream. After a 1-h incubation, 2.0 ml of the reaction mixture were taken up, and the amount of cysteine, total free SH groups, and ammonia in a reaction mixture was determined. Ammonia was measured colorimetrically using phenol hypochloride (37). The amount of cysteine was determined using Gaitonde's acid/ninhydrin method (20). The number of free SH groups was determined using a 5,5'- dithiobis-(2-nitrobenzoic acid) reagent as described by Ellman et al. (23) after the DTT in the reaction mixture had been completely removed by extracting with diethyl ether.

The reaction mixture was then divided in half. One-half was combined with an equal volume of 30% hydrogen peroxide to oxidize cysteine and homocysteine to cysteinic acid and homocysteinic acid, respectively (38). The mixture was run through an amino acid analyzer, and the elution peaks coincided with those of authentic L-cysteinic acid and DL-homocysteinic acid. In addition, to confirm this identification, the formed cysteine and homocysteine were oxidized to cystine and homocystine, respectively, with the addition of FeC13 (38). After brief centrifugation, the supernatant solution underwent amino acid analysis; the elution peaks coincided with those of authentic L-cystine and L-homocystine.

In order to capture and identify the found cysteine, sodium iodoacetate was added to the remaining reaction mixture in a slightly higher concentration than that of the free SH groups. To confirm the configuration of the product, the captured S- carboxymethylcysteine (1.0 pmol) was incubated at 30 "C for 1 h with shaking in 2.0 ml of the reaction mixture containing 1.65 units of L-amino acid oxidase, 0.3 pmol of FAD, 500 units of catalase, and 0.2 mmol of potassium phosphate buffer (pH 8.0). When the incubated mixture was passed through an amino acid analyzer, it was found that the peak corresponding to formed S-caraboxymethylcysteine had disappeared. Iden- tical treatment with D-aminO acid oxidase did not provoke the disappearance of the peak. The enzymatically formed cysteine is thus shown to be the L-isomer. The amount of formed L-cysteine, total free SH groups, and ammonia was determined to be 12.73, 14.49, and 14.39 pmol, respectively. The amount of L-homocysteine (1.76 pmol) was calculated by subtracting the amount of L-cysteine from the amount of free SH groups.

To determine the amount of a-keto acids, the reaction was stopped by adding 6 ml of 99% ethanol to the remaining half of the reaction mixture (3.0 ml). The identification and the determination of formed a-keto acid were carried out with L- alanine dehydrogenase (39). As we found that L-cysteine inhibited L-alanine dehydrogenase activity, L-cysteine was

transformed to S-carboxymethyl-t-cysteine by adding sodium iodoacetate. The above reaction mixture was evaporated to dryness under reduced pressure. To the residue were added 0.15 mmol of NH4C1/NH40H buffer (pH 9.0), 0.1 mmol of NADH, and 7.0 units of L-alanine dehydrogenase. After incubation at 37 "C for 1 h, the solution was diluted and subjected to amino acid analysis. The formed amino acids were identified as L-alanine and L-a-aminobutyrate and determined to be 1.94 and 12.80 pmol, respectively. The remaining L-cystathionine in the reaction mixture was measured with an amino acid analyzer and found to be 3.80 pmol.

The amount of formed L-cysteine (12.73 pmol) and L- homocysteine (1.76 pmol) closely agreed with formed a-ketobutyrate (12.83 pmol) and pyruvate (1.94 pmol), respectively. The amount of formed ammonia, total free SH groups, and total a-keto acids agreed closely with the amount of consumed L-cystathionine (14.95 pmol). Consequently, it can be said that Streptomyces cystathionine y-lyase catalyzes the heter- ogeneous decomposition of L-cystathionine. The a,y-elimination reaction forms L-cysteine, a-ketobutyrate, and ammonia, and the a,p-elimination forms L-homocysteine, pyruvate, and ammonia. The a,8-elimination reaction proceeds at about one-seventh the ratio of the a,y-elimination reaction.

The stoichiometry of L-homoserine was carried out as described above, except that 86.67 pmol of L-homoserine were used as a substrate. After a 1-h incubation, 8.83 pmol of L- homoserine were degraded and resulted in the formation of 8.81 pmol of a-ketobutyrate and 8.98 pmol of ammonia. The results indicate that a-ketobutyrate and ammonia are formed stoichiometrically by a a,y-elimination reaction in accordance with the consumption of L-homoserine.

Serological Properties Immunodiffusion techniques (28) were used to test the

ability of antiserum prepared against the purified cystathionine y-lyase of S. phaeochromogenes to cross-react with cystathionine y-lyase from actinomycetes, yeasts, and rat liver. Precipitin bands were formed with cystathionine y-lyase de- rived from s. oliuochromogenes (IF0 3178), s. lydicus (AKU 2502), S. lactamdurans (IF0 133051, S. bn tucheme (IF0 12880), S. reseum (IF0 3776), M. chalcea (IF0 12135), M . angiospora (IF0 13155), and M. uiolacea (IF0 12517), and spurs were formed when the enzymes from these actinomycetes were placed in neighboring wells (Fig. 6). This indicates that cystathionine y-lyase from S. phaeochromogenes is only partially identical to that from the actinomycetous microorganisms described above. In contrast, cystathionine r-lyase from N. erythropolis (IF0 12539) and M. smegmatis (IF0 3154), which are also actinomycetes, formed no precipitin bands under the same conditions.

Cystathionine y-lyase from rat liver and yeasts, e.g. P. wickerhumii (IF0 1278). H. jadinii (IF0 0987), and C. utilis (IF 0396), cystathionine 8-lyase from P. dachunhae (IF0 12048), and methionine y-lyase from Pseudomonas putida (IF0 3738) did not form precipitin bands (Fig. 6). These enzymes did not share any antigenic determinants with the S. phaeochromogenes enzyme.

Other Enzymes Related to the Biosynthesis of Sulfur- containing Amino Acids

We next attempted to clarify the transsulfuration process in S. phaeochromogenes by examining six kinds of enzyme activity in the crude extracts of S. phueochromogenes grown on a synthetic medium (Medium VI) and a nutrient medium (Medium V) (Medium VI is described in Table IV). The following enzymes were studied cystathionine y-lyase (EC


FIG. 6. Double-immunodiffusion analysis of cystathionine y-lyase from S. phaeochromogenes. A, the purified cystathionine y-lyase (4.6 X unit) from S. phaeochromogenes; 1 , rat liver cystathionine y-lyase (0.20 unit); 2, cystathionine 8-lyase (9.0 X unit) from P. dacunhae, 3, methionine y-lyase (2.3 X unit) from P. putida; 4, crude cystathionine y-lyase from P. wickerhumii (2.8 X

unit); 5, C. utilis (1.9 X lo-' unit); 6, H. jadinii (2.0 X unit); 7, S. oliuochromogenes (2.3 X unit); 8, S. kentuchense (2.1 X unit); 9, S. lydicus (1.4 X unit); 10, M. uiolacea (3.0 X 10" unit); 11, M. chulcea (4.9 X 10" unit); 12, S. phaeochromogenes (4.9 X unit).

TABLE IV Enzyme actiuities related to the transsulfuration in S.

phaeochromogenes S. phaeochromogenes was grown at 28 "C in Media V and VI for 48

and 240 h, respectively. Medium VI is composed of 1 g of glycerol, 0.2 g of NaN03, and 0.1 g of KIHPOl in 100 ml of tap water (pH 7.0). Cystathionine y-lyase activity is shown as the formation of a-ketobutyrate from L-homoserine. Protein was determined using the method described by Lowry et al. (21). " ~~ ~.

Enzyme Enzyme activity

Medium V Medium VI pmol/min/mg protein

Cystathionine y-lyase 4.07 X 10-~ 3.53 X 10-~ Cystathionine 8-lyase ND" ND Cystathionine y-synthase

Ab 2.97 X 10-~ 0.82 X 10-~ B' 5.65 X 10-~ 2.08 X 10-~

Cystathionine &synthase 0.92 X 10-3 0.49 X 10-3 O-Acetylserine sulfhydrylase 0.88 X 10-~ 0.63 X 10-3 0-Succinylhomoserine sulfhydrylase 1.1 1 X 1.48 X 0-Acetylhomoserine sulfhydrylase ND ND "

ND, not detected.

cystathionine y-synthase.

cystathionine y-synthase.

' 0-Acetyl-L-homoserine was used as a substrate for the assay of

0-Succinyl-L-homoserine was used as a substrate for the assay of

4.4.1.1), cystathionine @-lyase (EC 4.4.1.8). cystathionine y- synthase (0-acylhomoserine thiol lyase, EC 4.2.99.9), cystathionine @-synthase (serine sulfhydrylase, EC 4.2.1.22), 0- acetylserine sulfhydrylase (cysteine synthase, EC 4.2.99.8), and 0-acylhomoserine sulfhydrylase (homocysteine synthase, EC 4.2.99.10). In order to ensure precise measurement of the activities of cystathionine y-synthase, cystathionine @-synthase, and cystathionine @-lyase, concomitant cystathionine y-lyase was removed by treating the crude extract with anti- cystathionine y-lyase serum. The activity of the enzymes differed little in the two media (Table IV).

Cystathionine @-lyase is universally distributed in bacteria,

0 10 20 30 40 50 14 Antis- (PO

FIG. 7. Immunotitration of crude extract of S. phaeochromogenes with the anti-cystathionine y-lyase serum. The crude extract containing 0.12 unit of cystathionine y-lyase was incubated with various concentrations of anti-cystathionine y-lyase serum a t 37 "C for 30 min and then stored at 5 "C overnight. After centrifugation at 10,000 X g for 30 min, the remaining activity of each supernatant was measured using L-homoserine and L-cystathionine as substrates. 0, cysteine formation from L-cystathionine, 0, keto acid formation from L-cystathionine; X, keto acid formation from L- homoserine.

but was not detected in our samples. To confirm the absence of cystathionine @-lyase, the crude extract was incubated with various concentrations of anti-cystathionine y-lyse serum at 37 "C for 30 min and stored at 5 "C overnight. After centrifugation at 10,000 x g for 30 min, the remaining activity of each supernatant was measured using L-homoserine and L- cystathionine as substrates. Fig 7 shows the activities of the cells cultivated in Medium V. Similar results were obtained in Medium VI. The cysteine- and keto acid-forming activities from L-cystathionine were also checked. There was no formation of keto acid (pyruvate) after removal of cystathionine y-lyase, which corresponded to cystathionine @-lyase activity. Cystathionine @-lyase was therefore assumed to be lacking in the cell-free extracts.

Cystathionine y-synthase activity which catalyzes the synthesis of cystathionine from L-cysteine and 0-acetyl-L-homoserine or 0-succinyl-L-homoserine, respectively, was found. 0-acetylhomoserine sulfhydrylase activity was not detected.

Two reverse transsulfuration reactions were found to occur in the crude extracts: cystathionine y-lyase and cystathionine @-synthase. These findings constitute an important exception to the classic description of sulfur metabolism in procaryotes and eucaryotes and suggest that the metabolism of sulfur by actinomycetes may differ significantly from that in other bacteria.

DISCUSSION

The studies described here deal with the enzymological and physicochemical characterization of the Streptomyces enzyme. The enzyme was purified and crystallized from S. phueochromogenes, as cystathionine y-lyase occurs most abundantly in this species. The physicochemical properties of the Streptomyces enzyme resembled those of cystathionine y- lyase from rat liver; the molecular weight, sedimentation, and diffusion coeffients (Streptomyces enzyme, 1.62-1.68 X lo5, 9.47 S, 5.10 X cm2/s, respectively; rat liver enzyme, 1.9


X lo5, 8.90 S, 4.10 X cm2/s, respectively), subunit structure (four subunits), pyridoxal-P content (4 mol of pyridoxal- P/mol of the enzyme), and absorption spectrum (Streptomyces enzyme, A,,, = 421 nm; rat liver enzyme, A,,, = 427 nm,) are all similar. The isoelectric point of the Streptomyces enzyme (PI 4.20) is much lower than that of the rat liver enzyme (PI 6.05). (2). K,,, values for pyridoxal-P of Streptomyces enzyme and rat liver enzyme are different (1.8 x lo-' and 4.3 x mM, respectively); rat liver enzyme binds pyridoxal-P more tightly than does Streptomyces enzyme. There are also some differences with respect to the catalytic properties of the enzymes. The relative activity exhibited by the Streptomyces enzyme on different substrates was found to decrease in the order lanthionine > homoserine > djenkolic acid > cystathionine, while the order for rat liver enzyme was homoserine > cystathionine > djenkolic acid > lanthionine. The Strepto- myces enzyme showed a higher affinity for L-cystathionine (K, = 0.204 mM) than did rat liver enzyme (K, = 3 mM). The calculated K,,, values for the Streptomyces enzyme and the rat liver enzyme are 12.5 and 2 mM, respectively, for L-homoserine. Maximum velocities were also determined, and from these figures and the molecular weight (166,000), turnover numbers were calculated. The rates for the Streptomyces enzyme were: L-cystathionine, 227.4 mol/min/mol at 30 "C; L-homoserine, 393.4 mol/min/mol. Higher turnover numbers are reported for the rat liver enzyme (2): L-cystathionine, 2340 mol/min/ mol at 37 "C; L-homoserine, 6400 mol/min/mol at 37 "C. Furthermore, rat liver cystathionine y-lyase catalyzed the a,@-cleavge of ~-2,3-diaminopropionic acid (40), while the Streptomyces enzyme could not degrade this compound. Al- though cystathionine y-lyase has been shown to be present in Neurospora (3) as well as Streptomyces, it has not been purified to homogenity, and the physicochemical properties of the Neurospora enzyme remain unclear. The catalytic properties of the partially purified Neurospora enzyme were studied by Flavin and Slaughter. (41). A comparison of the Streptomyces enzyme with the Neurospora enzyme reveals that L-cystine and DL-lanthionine are preferred substrates for both enzymes, while L-homocystine is inert. The optimum pH differs for the a,y-elimination of L-cystathionine catalyzed by rat liver, Neurospora enzyme, and Streptomyces enzyme (8.0, 7.4, and 9.0, respectively).

There is almost no information in the existing literature on the metabolism of sulfur-containing amino acids by filamentous bacteria. The experiments reported here confirm the universal distribution of cystathionine y-lyase in seven genera of actinomycetes: Micromonospora, Micropolyspora, Mycobac- terium, Nocardia, Streptomyces, Streptosporangium, and Streptouerticillium. y-Lyase may in fact be common among the actinomycetes. In addition, as cystathionine P-synthase was thought to occur only among eucaryotes, the presence of the enzyme in S. phaeochromogenes strongly suggests that the transsulfuration metabolism of S. phaeochromognes resembles that of the fungi and differs from that of the other bacteria. As cystathionine y-lyase activity was almost nonexistent, it might be concluded that transsulfuration of S. phaeochromogenes proceeds as follows: homocysteine + (cystathionine) + cysteine. How, then, is homocysteine supplied? Streptomyces y-lyase also catalyzes the a,&elimination reaction at about one-seventh the ratio of the a,y-elimination reaction and produces homocysteine. Does this have a physiological signif- icance? Direct synthesis of homocysteine also occurs when 0- succinyl-L-homoserine and sodium hydrosulfide combine with extracts from S. phaeochromogenes. The Salmonella enzyme which catalyzes this reaction is identical to cystathionine y- synthase (42). It remains to be determined whether cystathi- mine y-synthase activity is identical to the enzyme that

catalyzes the sulfhydrylation of 0-succinyl-L-homoserine in S. phaeochromogenes or not. The similar properties of the purified Streptomyces y-lyase and the Neurospora enzyme and the similar transsulfuration mechanism exhibited by these microorganisms could have implications for our knowl- edge of the evolution of the sulfur metabolism in microorganisms. The production of cephamycin C by S. lactamdurans was strongly inhibited by propargylglycine, a mechanism- based inhibitor (14). Furthermore, some Streptomyces sp. produce sulfur-containing P-lactam antibiotics, as Penicillium sp. produce penicillin N and Cephalosporium sp. produce cephalosporins. I t might therefore be possible that the occurrence of cystathionine y-lyase is a necessary precondition for the production of such P-lactam antibiotics.

Acknowledgments-We thank Dr. K. Soda, professor at Kyoto University, for sample for methionine y-lyase from P. putida. We also wish to thank Dr. J. Imanishi, professor at Kyoto Prefectural University of Medicine, for the preparation of antibodies and his technical assistance in this work.

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OF VOl. 259, No. 16, of 25, PP. 10393-10403 1984 1% ... · The enzyme was purified from Streptomyces phaeo- chromogenes (IF0 3105) in nine steps. After the last steps, the enzyme