©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
-Cystathionase from Bordetella avium
ROLE(S) OF LYSINE 214 AND CYSTEINE RESIDUES IN ACTIVITY AND CYTOTOXICITY (*)

(Received for publication, November 30, 1994; and in revised form, January 13, 1995)

Claudia R. Gentry-Weeks (§) Jennifer Spokes John Thompson

From the Laboratory of Microbial Ecology, NIDR, National Institutes of Health, Bethesda, Maryland 20892

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

beta-Cystathionase (EC 4.4.1.8) from Bordetella avium is a pyridoxal 5`-phosphate (PLP)-dependent enzyme that catalyzes the hydrolysis of L-cystine to yield pyruvic acid, NH(3), and thiocysteine. The latter compound is highly toxic toward MC3T3-E1 osteogenic cells, rat osteosarcoma cells, and other cell lines maintained in tissue culture (Gentry-Weeks, C. R., Keith, J. M., and Thompson, J.(1993) J. Biol. Chem. 268, 7298-7314). Site-directed mutagenesis has established that lysine 214 of the sequence TKYVGGHSD, is primarily responsible for internal aldimine binding of PLP in the holoenzyme. Translation of the DNA sequence of the beta-cystathionase gene (metC) from B. avium, reveals 4 cysteine residues/enzyme subunit (M(r) = 42,600), and spectrophotometric analysis with 4,4`-dithiodipyridine showed that there were no disulfide linkages in the native protein. beta-Cystathionase is inhibited by sulfhydryl-reactive agents, including N-ethylmaleimide (NEM). To elucidate the mechanism of NEM inhibition, each of the 4 cysteine residues at positions 88, 117, 279, and 309 was individually replaced by alanine or glycine. The mutant proteins C88A, C117G, C279G, and C309A were purified to homogeneity, and each was assayed for enzyme activity, PLP-binding, NEM sensitivity, and susceptibility to chymotrypsin digestion. The activities of mutant proteins C88A and C279G were comparable with that of the native enzyme, and since both forms were inhibited by NEM, neither cysteine 88 nor 279 are prerequisite for enzyme activity. By elimination, cysteine residues 117 and 309 must be the targets for alkylation, and resultant inactivation of beta-cystathionase, by the -SH reactive agent. Substitution of cysteine 117 and 309 with glycine and alanine, respectively, yielded the inactive proteins C117G and C309A. PLP was not detectable in these proteins, and their absorption spectra lacked the peak (at 420 nm) that is characteristic of internal PLP-Schiff base formation. Edman degradation revealed that C117G (M(r) 36,000) also lacked the first 63 amino acids comprising the N terminus of the native protein. The beta-cystathionase mutants C117G and C309A showed enhanced susceptibility to chymotrypsin digestion. Cysteine residues 117 and 309 may reside in conformationally sensitive environments, and in the native enzyme these amino acids most probably serve a structural function. Toxicity assays performed with the various mutant proteins obtained by site-directed mutagenesis established that only catalytically active forms of beta-cystathionase were cytotoxic for tissue culture cells.


INTRODUCTION

Recently we showed that a cell-free preparation of Bordetella avium (an avian pathogenic bacterium), was highly toxic toward a variety of eucaryotic cell lines maintained in tissue culture(1) . The purification, cloning, and sequencing of the gene encoding this cytotoxic protein revealed, surprisingly, that toxicity was caused by beta-cystathionase (EC 4.4.1.8). This pyridoxal 5`-phosphate (PLP)(^1)-dependent enzyme occurs in many bacterial species including B. avium, where, as a constituent of the biosynthetic pathway for methionine(2) , it catalyzes the cleavage of L-cystathionine to yield homocysteine, pyruvic acid, and NH(3)(3, 4, 5) . The enzyme may also utilize L-cystine as substrate, in which case the products of the beta-elimination reaction (5, 6, 7, 8) are pyruvic acid, NH(3), and the persulfide, thiocysteine(4, 5, 9) . L-Cystine is a component of the medium used for growth of tissue culture cells, and we proposed that cytotoxicity caused by beta-cystathionase was a consequence of two sequential steps. First, the enzyme catalyzed formation of thiocysteine from L-cystine present in the tissue culture medium. Second, the transfer of sulfane-sulfur (10, 11, 12) from the unstable compound thiocysteine to metabolically important and sulfane inhibitable enzymes within or at the surface of sensitive eucaryotic cells.

Interest in the cytotoxic potential of beta-cystathionase, and our sequencing of the gene (metC) from B. avium, prompted comparison with the beta-cystathionase(s) that have been described from Escherichia coli(13) and Salmonella typhimurium(14) . Analysis of the metC genes from the three bacterial species (Fig. 1) revealed considerable sequence similarity, particularly with respect to the conservation of the motif TKY(X)(X)GHSD. Previous studies by Martel et al.(15) involving reduction, carboxymethylation, and recovery of -pyridoxyllysine from beta-cystathionase of E. coli, provided evidence for participation of the lysine residue of this motif in Schiff-base formation with PLP in the holoenzyme. However, these chemical modification experiments did not establish this lysine residue (or, the equivalent lysine 214 of B. avium enzyme) as the sole or major determinant for PLP-binding in beta-cystathionase. Published reports suggest that neither the S. typhimurium and E. coli beta-cystathionase(s) are overtly sensitive to N-ethylmaleimide(16) . By contrast, the enzyme from B. avium is markedly inhibited by this -SH reactive agent. Our finding of 4 cysteine residues in this protein (Fig. 1), suggested that one or more of these residues fulfilled catalytic or structural functions in beta-cystathionase from B. avium.


Figure 1: Comparison of the deduced amino acid sequences of beta-cystathionase from B. avium (BABC), E. coli (ECBC) and S. typhimurium(STBC). Numbering of the amino acids of B. avium beta-cystathionase is shown above the sequence while the number of residues for the other beta-cystathionases is indicated at the end of the respective sequences. Boxes indicate identical amino acid residues between the beta-cystathionases. The asterisks designate the amino acid residues which were individually substituted by site-directed mutagenesis of the B. avium metC gene. The arrow designates the point of cleavage in the mutant protein C117G and the underlined residues are those which are lost during purification of this protein.



In this investigation we have posed the following questions. First, is lysine 214 the primary determinant for PLP binding by beta-cystathionase from B. avium? Second, are the contributions of the (4) cysteine residues to beta-cystathionase activity of a conformational or catalytic nature? Third, is catalytically inactive beta-cystathionase also toxic to tissue culture cells? To answer these queries, we have performed site-directed mutagenesis on the appropriate lysine and cysteine residues encoded by the metC gene from B. avium(1) . Lysine residue 214 and cysteine residues 88, 117, 279, and 309 have each been replaced by alanine (or glycine). The corresponding mutant proteins, designated K214A, C88A, C117G, C279G, and C309A have been purified to homogeneity, and each has been assayed for enzyme activity, PLP binding, and cytotoxicity.


EXPERIMENTAL PROCEDURES

Reagents

Reagents were purchased from the following suppliers: Gene Amp-DNA Amplification kit, Perkin/Elmer Cetus; reagents for synthesis of oligonucleotide primers, Applied Biosystems, Inc; Gene Clean II kit, BIO 101, Inc; restriction enzymes and molecular biology reagents and polyacrylamide sequencing gel solution (Gel Mix 8), Life Technologies, Inc.; Sequenase Version 2.0 DNA sequencing kit, United States Biochemical Corp.; DEAE-Sephacel, Blue-Sepharose CL-6B, phenyl-Sepharose CL-4B, Pharmacia LKB Biotechnology, Inc.; Ultrogel AcA 44, IBF Biotechnics, Inc.; BCA protein assay kit, Pierce; L-lactate dehydrogenase, Boehringer Mannheim; chemicals, including NADH, cystathionine, cystine, ampicillin, 4-chloro-1-naphthol, N-ethylmaleimide, and potassium phosphate, Sigma; 4,4`-dithiodipyridine (Aldrithiol), Aldrich; alpha-chymotrypsin, Millipore Corporation; 4-(2-aminoethyl)-benzenesulfonyl fluoride hydrochloride and goat anti-rabbit IgG horseradish peroxidase conjugate (IgG, H, and L chains, affinity purified), ICN Biochemicals; isopropyl beta-D- thiogalactopyranoside (IPTG), Calbiochem Corporation; electrophoresis materials and standards for SDS-PAGE and Western immunoblots, Bio-Rad; nitrocellulose filter paper, Schleicher and Schuell Inc.; tissue culture medium and supplements, Life Technologies, Inc. or BioWhittaker, Inc. Polyclonal rabbit sera against B. avium beta-cystathionase was prepared as described previously(1) .

Bacterial Strains, Plasmid Vectors, and Media

Escherichia coli DH5alpha (F, endA1, hsdR17 (r(k), m(k)), supE44, thi-1, , recA1, gyrA96, relA1, Delta(argF-lacZYA)U169, 80dlacZDeltaM15), competent cells were obtained from Life Technologies, Inc. E. coli SA2821 cells (an ompT Delta(degP::PstI-Km^r derivative of BL21(DE3)) (17) was provided by S. Adhya, National Cancer Institute, NIH. Plasmid pET-11d, a T7 expression vector containing a T7lac promoter and T7 terminator, was obtained from Novagen, Inc. E. coli DH5alpha and E. coli SA2821 were grown in Luria-Bertani (LB) medium supplemented with 150 µg of ampicillin/ml for plasmid maintenance. M9ZB (17) supplemented with ampicillin (150 µg/ml) and IPTG (0.25 mM) was the medium used for production of wild-type and mutant beta-cystathionase protein.

High Level Expression of the B. avium beta-Cystathionase Gene

A 6-kilobase DNA fragment containing the B. avium beta-cystathionase gene was previously cloned in pUC18 (generating pWDC372) and expressed in E. coli DH5alpha(1) . A 1.2-kilobase DNA fragment encoding only the beta-cystathionase gene was amplified by the polymerase chain reaction (Perkin/Elmer Cetus DNA Thermal Cycler) using the Gene Amp-DNA Amplification Reagent kit, template pWDC372 DNA, and primers: 5`-TCTGCCGCCTCTCCATGGTCTGACACCTCC-3` and 5`-GCAGCTGAGCGCGGATCCCTCAGGCGGCGCG-3`. The primers used for gene amplification generated NcoI and BamHI sites at the 5` and 3` ends of the beta-cystathionase gene, respectively. The amplified DNA fragment was electrophoresed through a 0.6% agarose gel, purified with reagents from the Gene Clean II kit, digested with NcoI and BamHI, ligated to NcoI-BamHI-digested pET-11d(17) , and transformed into the non-expression host E. coli DH5alpha. This construct, designated pWDC373, was confirmed by restriction enzyme analysis and was used as the template for site-directed mutagenesis of beta-cystathionase.

Site-directed Mutagenesis of the beta-Cystathionase Gene

Alterations in single amino acids (18) were obtained by PCR amplification of pWDC373, using overlap extension and oligonucleotides altered in the target codon (Fig. 2). PCR was performed using the reagents and instructions of the Gene-Amp DNA amplification kit. Lysine residue 214 and cysteine residues at positions 88, 117, 279, and 309 were mutated to either alanine, glycine, arginine, or proline by substitution of the wild-type codon with the degenerate codon (G/C)(G/C)(G/C) in the oligonucleotide used for PCR amplification. The amplified, mutated beta-cystathionase gene was purified using reagents of the Gene Clean II kit, digested with NcoI and BamHI, ligated to NcoI, BamHI-digested pET-11d, and introduced into the expression host E. coli SA2821 (17) via electroporation. The mutations and fidelity of the PCR amplifications were confirmed by dideoxy DNA sequence analysis of the constructs using the Sequenase Version 2.0 DNA sequencing kit. The designations for these constructs indicate the wild-type amino acid, position, and mutant amino acid (R#R(m)) and are listed in Table 1.


Figure 2: Strategy for site-directed mutagenesis and recombinant PCR of the B. avium metC gene. For substitution of each amino acid, the 5` end of the B. avium metC was first mutated by PCR amplification using primer a and mutagenic primer c. In a separate PCR reaction, the 3` end of the metC gene was replicated by PCR amplification using primer b (which contains a region homologous to primer c) and primer d. The entire coding region of the metC gene, containing the altered codon was generated by recombinant PCR using primers a and d, and the PCR-generated 5` and 3` ends of the metC gene. Primers a and d are homologous to the 5` and 3` end of the metC gene and contain an NcoI site and BamHI site, respectively. The underlined codons of primers a and d designate the coding region of the amino terminus and carboxyl terminus of the metC gene, respectively, and the locations of the NcoI site and BamHI site are indicated above the primer. The primers used for mutagenesis of lysine 214 and cysteine residues 88, 117, 279, and 309 are designated b and c.





Purification of Wild-type and Mutant beta-Cystathionase(s)

For induction of expression of the recombinant wild-type and mutant beta-cystathionase gene, E. coli SA2821(pWDC373) was grown in 4 liters of M9ZB broth (17) containing ampicillin (150 µg/ml) at 30 °C to an optical density of 0.6-0.7 at 600 nm. IPTG and ampicillin were added to a final concentration of 0.4 mM and 150 µg/ml, respectively, and incubation was continued for 3 h at 30 °C. Cells were harvested by centrifugation at 10,000 times g for 30 min at 4 °C. Purification of recombinant wild-type and mutant proteins K214A, C88A, C117G, and C279G was performed as described previously(1) . At each step in the purification, column fractions which contained recombinant wild-type beta-cystathionase or active mutant protein were identified by the spectrophotometric assay for beta-cystathionase activity. During purification of inactive beta-cystathionase, the mutant protein was detected by reactivity of 20-µl samples of column fractions with antibody against B. avium beta-cystathionase in a Western immunoblot assay as described below. Mutant beta-cystathionase C309A formed insoluble inclusion bodies, and this protein was refractory to purification by our standard procedure. Cells containing mutant C309A protein were harvested by centrifugation at 13,000 times g for 10 min at 4 °C, suspended in 10 volumes of 10 mM potassium phosphate buffer, pH 7.1, and stored on ice for 16 h. During storage, the cells lysed spontaneously, and mutant C309A protein of a high degree of purity was recovered by centrifugation of the lysate at 1,450 times g for 10 min at 4 °C. Purified recombinant wild-type and mutant proteins were quantitated using instructions and reagents of the BCA Protein Assay kit.

SDS-PAGE and Western Immunoblot of Recombinant Wild-type and Mutant beta-Cystathionases

SDS-PAGE was performed with 10% SDS-polyacrylamide gels using a discontinuous buffer system and nitrocellulose filters containing protein were reacted with antibody against B. avium beta-cystathionase as described previously (1) .

Spectrophotometric Assay for beta-Cystathionase Activity and Inhibition of Enzyme Activity by NEM

Catalysis of L-cystine and L-cystathionine cleavage was determined spectrophotometrically by measuring the rate of NADH oxidation at 340 nm in the lactate dehydrogenase-NADH-coupled assay described previously(1) . To assay for inhibition by N-ethylmaleimide, purified mutant protein (0.5 µg) was incubated with NEM (final concentration, 1 mM) for 10 min at room temperature in 0.1 M Tris-HCl buffer, pH 8, prior to assay for enzyme activity.

NH(2)-Terminal Amino Acid Sequence of Mutant C117G beta-Cystathionase

The NH(2)-terminal amino acid sequence of mutant C117G enzyme was determined by automated Edman degradation in an Applied Biosystems model 470A gas-phase microsequencer as described in a previous article(1) .

Spectral Characterization of Pyridoxal 5`-Phosphate Cofactor

Absorption spectra of the purified proteins (300 µg/ml; dissolved in 0.1 M potassium phosphate buffer, pH 7.5) were obtained in a Beckman model DU-70 spectrophotometer. For determination of enzyme-associated PLP, purified recombinant wild-type and mutant beta-cystathionase were treated with 10 µl of 10 N NaOH, and the absorption spectrum at 388 nm was recorded. A molar extinction coefficient for pyridoxal 5`-phosphate (at pH 10, in 0.1 N NaOH) of 6,600 M cm(19) was used to calculate the concentration ratio of PLP/enzyme subunit.

Determination of -SH Groups

Sulfydryl groups in both native and urea-denatured beta-cystathionase were determined by the method of Grassetti and Murray(20) . The -SH reactive reagent 4,4`-dithiodipyridine was used in the assay, and reduced glutathione served as the standard. The absorbance of 4-thiopyridone ( = 19, 800 M cm) was measured at 324 nm, and a subunit M(r) 42,600 was assumed for beta-cystathionase in all calculations, except for mutant C117A (M(r) 36,000). The assays (1 ml) contained 0.1 M potassium phosphate buffer, pH 7.45, 1 mM 4,4`-dithiodipyridine; 0.05-0.2 mg of protein, and 5 M urea as denaturant. Absorbance readings were recorded after 30 min of incubation at room temperature.

Cytotoxicity Assay for MC3T3-E1 Osteogenic Cells

Assay for cytotoxicity for MC3T3-E1 mouse calvarial osteogenic clonal cells (21) was performed as described previously(1) . Purified recombinant wild-type and mutant beta-cystathionase proteins (K214A, C88A, C117G, C279G, and C309A) were added to individual wells to a final concentration of 3 µg/ml. After incubation for an additional 16 h, the cells were examined for morphological changes indicative of toxicity (spherical cells and detachment from the plate) with an Olympus model CK2 inverted microscope.

Sensitivity of beta-Cystathionase and Mutant beta-Cystathionases to Chymotrypsin

Chymotrypsin was used to compare the sensitivity of wild-type and mutant beta-cystathionases to proteolytic cleavage. Purified protein (3.5 µg) was digested at room temperature for 30 min with chymotrypsin (final concentration of 2 µg/ml) in a reaction volume of 20 µl containing 0.04 M Tris-HCl, pH 8, buffer and 0.05 M CaCl(2). The reaction was terminated by addition of AEBSF (final concentration 5 µg/ml) and the digested protein(s) were electrophoresed through a 10% SDS-polyacrylamide gel, and polypeptides were visualized by staining with Coomassie Brilliant Blue.


RESULTS

Substitution of Lysine Residue 214 and Cysteine Residues 88, 117, 279, and 309

Fig. 1indicates the similarity of B. avium beta-cystathionase to previously sequenced beta-cystathionases from E. coli(13) and Salmonella typhimurium(14) . Asterisks indicate the location of the lysine and 4 cysteine residues of interest in the B. avium enzyme. The lysine residue at position 214 and cysteine residues 88, 117, 279, and 309 were individually replaced with either alanine, glycine, arginine, or proline. Overlap extension PCR was used to synthesize the mutations using a degenerate oligonucleotide which was altered in the target codon by (G/C) (G/C) (G/C)(18) . Fig. 2illustrates the site-directed mutagenesis procedure and the oligonucleotides used to generate the desired mutations. All mutations and PCR fidelity were confirmed by DNA sequence analysis of the entire coding region of each mutated beta-cystathionase gene.

Expression and Purification of Mutant beta-Cystathionases

PCR-generated DNA fragments encoding the mutated metC gene were first cloned into the T7 RNA polymerase expression vector pET-11d and subsequently were transformed into E. coli SA2821 for gene expression. Mutant beta-cystathionase was produced upon induction of the E. coli host with IPTG, and cell extracts of two representative mutant proteins of each target residue were assayed for beta-cystathionase activity. All mutations of cysteine 88 (including C88A, C88R, C88G, C88P) yielded active enzyme, but the specific activity of C88P was only 10% that of other cysteine 88 mutant proteins. Cell extracts containing C279G were also active, but similarly prepared extracts containing mutant proteins C279A, C279R, and C279P failed to hydrolyze either L-cystine or L-cystathionine. beta-Cystathionase activity was not detectable in cell extracts containing mutant proteins derived by substitution at cysteine 117, cysteine 309, and lysine 214 (except K214A, which had a low level of activity). The active mutant proteins K214A, C88A, and C279G, and the inactive forms C117G and C309A, were chosen for further study, and the five proteins were purified to homogeneity (Fig. 3). Except for C309A (which was purified by a separate procedure), all mutant enzymes displayed chromatographic behavior similar to that of wild-type beta-cystathionase by their migration as dimers of (M(r) 70,000-85,000) during gel filtration chromatography. Mutant proteins C279G and C309A were prone to formation of inclusion bodies. With the exception of protein C117G, all purified mutant proteins co-migrated with the wild-type beta-cystathionase (M(r) 42,000) by SDS-PAGE. In freshly prepared extracts, C117G migrated as a 42-kDa protein; however, after purification to homogeneity, the apparent M(r) of C117G was 36,000 (Fig. 3A, lane 5). The NH(2)-terminal sequence of the protein was determined to ascertain whether this apparently low molecular weight represented anomalous migration of the protein during SDS-PAGE or partial hydrolysis of the protein during purification. Sequential Edman degradation revealed that the NH(2) terminus of the C117G protein was truncated with respect to the native enzyme and that cleavage occurred between residues Thr and Tyr of beta-cystathionase (see Fig. 1). Western immunoblots of the purified mutant and wild-type enzymes revealed that all mutant proteins reacted with polyclonal antibody raised against the native enzyme (Fig. 3B).


Figure 3: SDS-polyacrylamide gel and Western immunoblot of proteins of purified recombinant wild-type and mutant beta-cystathionases. A, purified wild-type and mutant beta-cystathionases (2.5 µg) were electrophoresed through a 10% SDS-polyacrylamide gel under reducing conditions and stained with Coomassie Brilliant Blue R250. B, in a duplicate gel, 0.5 µg of each protein was electrophoresed, transferred to nitrocellulose paper, and immunoreacted with antibody against B. avium beta-cystathionase (see ``Experimental Procedures''). The proteins applied to the gel are designated above the lanes. The molecular weight standards are indicated to the left of the first lane.



Enzyme Activity and PLP Binding by Mutant beta-Cystathionases

The activities of recombinant wild-type beta-cystathionase and mutant proteins K214A, C88A, C117G, C279G, and C309A were determined spectrophotometrically by their ability to generate pyruvate via hydrolysis of both cystathionine and cystine (Table 1) in the presence or absence of pyridoxal 5`-phosphate. Mutant proteins C88A and C279G displayed enzyme activities comparable to wild-type beta-cystathionase when either cystine or cystathionine were used as substrates. The fact that the activities of these forms were not significantly increased by addition of PLP to the reaction indicated the presence of bound PLP in these mutant proteins. By contrast, mutant proteins C117G and C309A showed no detectable beta-cystathionase activity, and the addition of PLP to the reaction mixture containing these proteins failed to restore catalytic activity. The absorption spectrum for each of the purified proteins was recorded over the range 200-600 nm. The spectra of the wild-type enzyme (Fig. 4A) and active mutant proteins C88A (Fig. 4B) and C279G (not shown) revealed a major absorbance peak ((max) = 423 nm), with a well-defined shoulder at (max) = 350-360 nm. The major peak is characteristic of PLP in the internal aldimine form(8) . However, the wavelength of maximum absorption of the smaller peak is considerably less than that ((max) 388-395 nm) usually ascribed to enzyme-associated PLP in the aldehyde form(22, 23) . The species at (max) 350-360 nm may represent a Schiff base intermediate in which the phenolic group at the 3 position of the pyridine ring of PLP has undergone deprotonation(8) . Neither free nor aldimine-linked PLP was detectable in the absorption spectra of any of the inactive proteins, including C117G (Fig. 4C) and C309A (not shown). The stoichiometric ratio of PLP/subunit of enzyme was estimated by measuring the absorption at 383 nm of the pyridoxal 5`-phosphate released upon exposure of the native enzyme and mutant proteins to alkaline conditions. Recombinant wild-type beta-cystathionase and mutant enzymes C88A and C279G contained 60-80% of PLP present in preparations of the native enzyme. Unexpectedly, the absorption spectrum of mutant K214A (Fig. 4D) showed a small but significant absorption peak ((max) = 423 nm) that is characteristic of PLP in the internal aldimine form. Extraction of the cofactor indicated that mutant K214A contained 10% of the PLP, and this mutant protein exhibited about 5% of the overall activity of the native beta-cystathionase (Table 1).


Figure 4: Absorption spectra of wild-type and mutantbeta-cystathionases. Purified protein (in 10 mM potassium phosphate buffer, pH 7.0) was placed in microcuvets (volume 300 µl and 1-cm light path), and spectra were obtained in a Beckman model DU-70 recording spectrophotometer. A, recombinant wild-type beta-cystathionase; 290 µg, B, mutant C88A protein; 430 µg, C, mutant C117G protein; 360 µg, and D, mutant K214A protein; 950 µg.



The ability of NEM to inhibit the activity of mutant proteins C88A and C279G was tested by treating these proteins with the -SH reactive agent prior to performing the spectrophotometric assay for beta-cystathionase activity. Treatment of the enzymatically active forms C88A and C279G with NEM resulted in almost complete loss (>90%) of enzymatic activity (Table 1), indicating that the NEM-susceptible sites were present and accessible in these mutant proteins.

Determination of the Number of Free -SH Groups in beta-Cystathionase

Spectrophotometric analyses using 4,4`-dithiodipyridine (20) revealed the theoretical four -SH groups in the urea-denatured protein (Table 2) that were predicted in the DNA sequence. These data are evidence of the lack of disulfide linkages in beta-cystathionase. Spectrophotometric determination of free -SH groups verified the deletion of a single cysteine residue in each of the cysteine-mutant proteins (Table 2).



Cytotoxicity of Mutant beta-Cystathionase(s) for MC3T3-E1 Osteogenic Cells

The availability of mutant proteins of beta-cystathionase raised a question as to whether these catalytically inactive species would also be toxic toward MC3T3-E1 cells. To this end, both active and inactive mutant beta-cystathionases were added separately to tissue culture cells, and after 16 h of incubation, the morphology of the cells was examined to assess cytotoxic effects. Mutant protein K214A displayed diminished capacity to kill the cells and addition of either of the inactive beta-cystathionases, C117G or C309A had no effect upon the morphology or growth of the cells in tissue culture (Fig. 5A). The two active mutant proteins C88A and C279G were toxic for MC3T3-E1 osteogenic cells, and visual inspection revealed that the tissue culture cells became spherical and detached from the tissue culture plates (Fig. 5B). Clearly, only catalytically active forms of beta-cystathionase are toxic toward MC3T3-E1 osteogenic cells.


Figure 5: Toxicity of wild-type and mutant beta-cystathionases for MC3T3-E1 osteogenic cells. Purified recombinant wild-type and mutant beta-cystathionases were applied to separate wells containing MC3T3-E1 osteogenic cells. Following 16 h of incubation, the cells were observed for morphological changes associated with beta-cystathionase toxicity (i.e. spherical appearance and detachment of cells from the plate). A, cells treated with inactive mutant C117G protein (final concentration 3 µg/ml) showed no toxic effects. Equivalent amounts of inactive proteins K214A and C309A were similarly without effect upon the cells. B, cells treated with catalytically active mutant C88A protein (final concentration 3 µg/ml). The effects of wild-type beta-cystathionase and active C279G protein were identical to those elicited by protein C88A, i.e. formation of detached, spherical cells.



Sensitivity of Wild-type and Mutant beta-Cystathionases to Chymotrypsin

Improperly folded or denatured proteins are generally more susceptible to cleavage by proteases (trypsin, chymotrypsin, etc.) than the same proteins in their native state. Chymotrypsin cleaves at the COOH-terminal side of aromatic residues (tryptophan, tyrosine, and phenylalanine), and examination of the primary amino acid sequence of beta-cystathionase revealed 26 potential chymotrypsin cleavage sites. It was felt that chymotrypsin treatment might allow comparative analysis of the conformational status of native and mutant beta-cystathionase(s). To this end, wild-type and mutant forms of beta-cystathionases were incubated with chymotrypsin, and the resultant polypeptide profiles were examined by SDS-PAGE (Fig. 6). Chymotrypsin-digested preparations of the wild-type and mutated K214A and C88A beta-cystathionases exhibited similar protein digestion patterns, i.e. there were approximately seven polypeptide bands detectable by SDS-PAGE (Fig. 6B, lanes 2 and 3, respectively). By contrast, C117G, C279G, and C309A beta-cystathionases were almost completely degraded by chymotrypsin to yield small fragments barely detectable by SDS-PAGE (Fig. 6B, lanes 4-6). The marked susceptibility of the mutant proteins to proteolytic digestion suggests an increase in either the number or accessibility of chymotrypsin digestion sites, and by inference, of considerable conformational change in these proteins.


Figure 6: SDS-polyacrylamide gel of chymotrypsin-treated wild-type and mutant beta-cystathionase(s). Purified proteins (3.5 µg) were incubated for 30 min at room temperature with chymotrypsin (final concentration; 2 µg/ml). The digests were electrophoresed through a 10% SDS-polyacrylamide gel and polypeptides were stained with Coomassie Brilliant Blue R250. A, untreated beta-cystathionases. B, chymotrypsin-digested beta-cystathionases. Note that wild-type beta-cystathionase and mutants K214A and C88A are moderately resistant to chymotrypsin treatment while C117G, C279G, and C309A are highly sensitive to cleavage by the protease. The molecular weight standards are indicated at the left of the gel.




DISCUSSION

Earlier studies in this laboratory demonstrated that beta-cystathionase of B. avium was inhibited by NEM(1) . In this context, it may be noted that beta-cystathionase from Paracoccus denitrificans(24) is also sensitive to inhibition by NEM, whereas neither the beta-cystathionase(s) from S. typhimurium nor that of E. coli is inactivated by this compound(16) . These findings suggested to us that for B. avium beta-cystathionase, NEM interacts either with catalytically functional cysteine residues, or that alkylation of -SH groups and incorporation of the bulky NEM moiety into the protein elicits gross conformational change(s) and attendant loss of enzyme activity. Since it is unlikely that cysteine residues would be prerequisite for catalysis by beta-cystathionase from two species but not others, we felt that the second possibility was the most likely explanation for NEM-mediated inhibition of beta-cystathionase from B. avium and P. denitrificans. (A facile explanation for the differing sensitivities to the -SH reagent may be the fact that cysteine residues of S. typhimurium and E. coli beta-cystathionases are inaccessible to NEM.)

beta-Cystathionase of B. avium contains 4 cysteine residues at positions 88, 117, 279, and 309 of the protein sequence. To define their role(s) in enzyme function, each of these residues was individually replaced with either alanine, glycine, proline, or arginine, by means of oligonucleotide-directed, site-specific mutagenesis. Cysteine residues were substituted with alanine since this amino acid does not alter the main chain conformation, nor does the incorporation of this amino acid introduce charge or steric effects within the protein(25) . Furthermore, alanine is the most abundant amino acid in proteins and is found in both buried and exposed positions and in all varieties of secondary structure(26, 27) . Glycine was also chosen to replace cysteine residues since this amino acid does not introduce a charge or a bulky side chain into the polypeptide, and the rotational flexibility of this residue also allows the protein to assume variable conformations(28) . The use of a primer with the degenerate codon (GC) (GC) (GC) for generation of mutant proteins allowed the substitution of proline and arginine for the cysteine residues; both represent a nonconservative change in the target amino acid.

Inspection of Fig. 1would suggest cysteine 88 of B. avium beta-cystathionase as the functionally most important cysteine residue because this residue occurs at the same position in the highly conserved motif ELEGG(X)(X)C in all three bacterial enzymes. Surprisingly, PLP remained tightly bound, and catalytic activity was retained when cysteine 88 (and cysteine 279) were replaced by alanine or glycine, respectively. Furthermore, since both of these mutant proteins remained sensitive to inhibition by NEM, the data implicated cysteine 117 or cysteine 309 as the NEM-sensitive target(s) in the native holoenzyme. The following observations provide evidence that the latter cysteine residues occupy conformationally important positions: First, site-directed substitution of these residues yielded inactive enzymes (C117G and C309A) in both cases. Second, although the two mutated proteins contain lysine at position 214, neither protein contained PLP and the absorption spectra lacked the peak at 420 nm that is characteristic of the formation of the lysine 214-PLP Schiff base. Third, both C117G and C309A are demonstrably more susceptible to proteolytic digestion by chymotrypsin than either the native or catalytically active mutant enzymes. Fourth, automated Edman degradation revealed that truncation of the first 63 amino acids from the NH(2) terminus had occurred during purification of mutant protein C117G. In summation, these results suggest that cysteine 117 and cysteine 309 are conformationally important residues which, in the native enzyme, facilitate the formation of the internal aldimine between the C-4 formyl moiety of the cofactor and the -amine of lysine 214. Furthermore, spectrophotometric analysis established that the 4 cysteine residues are in the reduced state, and the presence of reducing agent dithiothreitol had no inhibitory effect upon enzyme activity. Cysteine 117 and 309 cannot therefore contribute to the structural or conformational stability of beta-cystathionase via formation of a disulfide linkage(29) .

Throughout its purification, beta-cystathionase from B. avium tenaciously retains the PLP cofactor, and the holoenzyme exhibits a stoichiometric ratio PLP/subunit of 1:1(1) . To date, three bacterial beta-cystathionases have been cloned and sequenced, and all contain a highly conserved motif of nine amino acids: TKY(X)(X)GHSD. Previous chemical modification studies involving borohydride reduction of beta-cystathionase from E. coli(15) showed that the -NH(2) of this conserved lysine residue formed an internal aldimine with the C-4 formyl moiety of PLP. Our site-directed substitutions of lysine 214 in the B. avium enzyme (to yield mutant proteins K214A and K214R) confirm, and amplify the role postulated for this conserved lysine residue. Certainly, lysine 214 is a functionally important residue in the beta-cystathionase from B. avium, and even conservative replacement of this basic amino acid with arginine produced a PLP-deficient catalytically inactive protein. Somewhat surprisingly, substitution of lysine 214 with alanine did not entirely abolish either cofactor binding or enzymatic activity. Indeed, the purified mutant protein (K214A) retained about 5% of the PLP (in aldimine form) and exhibited 10% of the catalytic activity of the wild-type beta-cystathionase. It seems reasonable that lysine 214 is primarily responsible for PLP binding, but it is also evident that other ligands must also contribute to retention of the cofactor by the holoenzyme from B. avium. Although novel with respect to cofactor binding by beta-cystathionase, our findings are not without precedent in the literature pertaining to PLP-dependent enzymes. For example, lysine residues 87 and 145 participate in internal aldimine formation with the PLP of tryptophan synthase (23, 30) and D-amino acid aminotransferase(22, 31) , respectively. However, site-directed substitution of the aforementioned lysyl residues (to yield K88T, and K145A or K145Q, respectively) does not eliminate binding of cofactor by these mutant proteins.

Whether the lysine residue of the TKY(X)(X)GHSD motif also serves a catalytic role in the beta-elimination reaction is a question that has not yet been answered unequivocally for any of the bacterial beta-cystathionases. Should this prove to be the case for the B. avium enzyme, then for the mutant protein K214A one must assume that a second lysine residue (in proximity to the active site) can partially fulfill the catalytic function normally assumed by K214. Since the primary sequence contains only 7 lysine residues (including K214), judicious choice or sequential site-directed mutagenesis of these amino acids, may permit identification of the ``alternate'' active-site lysine residue.

The preparation, by site-directed mutagenesis, of both active and inactive forms of beta-cystathionase from B. avium has provided further insight to the toxicity of this enzyme toward osteogenic and other cell lines in tissue culture. Inactive or mutant proteins of low activity such as K214A, C117G, and C309A had no discernible effect on MC3T3-E1 osteogenic cells, whereas the catalytically active mutant proteins C88A and C279G, mimicked the cytotoxicity of wild-type beta-cystathionase. Mutant K214A had only 5% of wild-type activity and therefore was unable to produce the concentration of thiocysteine required to elicit gross morphological changes in the tissue culture cells. These observations show conclusively that enzyme activity, and not simply interaction of the beta-cystathionase molecule with surface components of the eucaryotic cells, is required for manifestation of cytotoxicity.


FOOTNOTES

*
A portion of this work was presented at the 93rd General Meeting of the American Society for Microbiology, May 23-27, 1994, Las Vegas, Nevada (Gentry-Weeks, C., Spokes, J., and Thompson, J.(1994) Abstr. B-181, p. 60). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence and reprint requests should be addressed: Lab. of Microbial Ecology, Bldg. 30, Rm. 532, 30 Convent Dr., MSC 4350, NIDR, NIH, Bethesda, MD 20892. Tel.: 301-496-6060; Fax: 301-402-0396.

(^1)
The abbreviations used are: PLP, pyridoxal 5`-phosphate; NEM, N-ethylmaleimide; PAGE, polyacrylamide gel electrophoresis; AEBSF, 4-(2-aminoethyl)-benzenesulfonyl fluoride hydrochloride; IPTG, isopropyl beta-D-thiogalactopyranoside; PCR, polymerase chain reaction.


ACKNOWLEDGEMENTS

We acknowledge the generosity of our colleagues at the National Institute of Dental Research: Jerry Keith for his support and interest throughout our investigations and Edith Wolff and Dwayne Lunsford for critical review of the manuscript. We thank Nga Y. Nguyen for providing the N-terminal amino acid sequence data.


REFERENCES

  1. Gentry-Weeks, C. R., Keith, J. M., and Thompson, J. (1993) J. Biol. Chem. 268, 7298-7314 [Abstract/Free Full Text]
  2. Cohen, G. N., and Saint-Girons, I. (1987) in Escherichia coli and Salmonella typhimurium: Cellular and Molecular Biology (Ingraham, J. L., Low, K. B., Magasanik, B., Schaecter, M., and Umbarger, H. E., eds) pp. 429-444, American Society for Microbiology, Washington, D. C. _
  3. Cavallini, D., De Marco, C., Mondovi, B., and Mori, B. G. (1960) Enzymologia 22, 161-173
  4. Delavier-Klutchko, C., and Flavin, M. (1965) Biochim. Biophys. Acta 99, 375-377 [Medline] [Order article via Infotrieve]
  5. Flavin, M. (1962) J. Biol. Chem. 237, 768-777 [Free Full Text]
  6. Davis, L., and Metzler, D. E. (1972) in The Enzymes (Boyer, P. D., ed) Vol. VII, pp. 33-74, Academic Press, Inc., New York
  7. Miles, E. W. (1986) in Vitamin B6: Chemical, Biochemical, and Medical Aspects (Dolphin, D., Poulson, R., and Avramovic, O., eds) Vol. I, pp. 253-310, John Wiley and Sons, New York
  8. Snell, E. E., and Di Mari, S. J. (1970) in The Enzymes (Boyer, P. D., ed) pp. 335-370, Academic Press, Inc., New York
  9. Abdolrasulnia, R., and Wood, J. L. (1980) Bioorgan. Chem. 9, 253-260
  10. Toohey, J. I. (1989) Biochem J. 264, 625-632 [Medline] [Order article via Infotrieve]
  11. Wood, J. L. (1982) Adv. Exp. Med. Biol. 148, 327-341 [Medline] [Order article via Infotrieve]
  12. Wood, J. L. (1987) Methods Enzymol. 143, 25-29 [Medline] [Order article via Infotrieve]
  13. Belfaiza, J., Parsot, C., Martel, A., Bouthier de la Tour, C., Margarita, D., Cohen, G. N., and Saint-Girons, I. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 867-871 [Abstract]
  14. Park, Y. M., and Stauffer, G. V. (1989) Mol. & Gen. Genet. 216, 164-169
  15. Martel, A., Bouthier de la Tour, C., and Le Goffic, F. (1987) Biochem. Biophys. Res. Commun. 147, 565-571 [Medline] [Order article via Infotrieve]
  16. Delavier-Klutchko, C., and Flavin, M. (1965) J. Biol. Chem. 240, 2537-2549 [Free Full Text]
  17. Studier, F. W., Rosenberg, A. H., Dunn, J. J., and Dubendorff, J. W. (1990) Methods Enzymol. 185, 60-89 [Medline] [Order article via Infotrieve]
  18. Horton, R. M., Cai, Z., Ho, S. N., and Pease, L. R. (1990) BioTechniques 8, 528-535 [Medline] [Order article via Infotrieve]
  19. Peterson, E. A., and Sober, H. A. (1954) J. Am. Chem. Soc. 76, 169-175
  20. Grassetti, D. R., and Murray, J. F., Jr. (1967) Arch. Biochem. Biophys. 119, 41-49 [Medline] [Order article via Infotrieve]
  21. Sudo, H., Kodama, H.-A., Amagai, Y., Yamamoto, S., and Kasai, S. (1983) J. Cell Biol. 96, 191-198 [Abstract]
  22. Futaki, S., Ueno, H., Martinez del Pozo, A., Pospischil, M. A., Manning, J. M., Ringe, D., Stoddard, B., Tanizawa, K., Yoshimura, T., and Soda, K. (1990) J. Biol. Chem. 265, 22306-22312 [Abstract/Free Full Text]
  23. Miles, E. W., Kawasaki, H., Ahmed, S. A., Morita, H., Morita, H., and Nagata, S. (1989) J. Biol. Chem. 264, 6280-6287 [Abstract/Free Full Text]
  24. Burnell, J. N., and Whatley, F. R. (1977) Biochim. Biophys. Acta 481, 246-265 [Medline] [Order article via Infotrieve]
  25. Kasturi, S., Kihara, A., FitzGerald, D., and Pastan, I. (1992) J. Biol. Chem. 267, 23427-23433 [Abstract/Free Full Text]
  26. Ashkena, A., Presta, L. G., Marsters, S. A., Camerato, T. R., Rosenthal, K. A., Fendly, B. M., and Capon, D. J. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 7150-7154 [Abstract]
  27. Bordo, D., and Argos, P. (1991) J. Mol. Biol. 217, 721-729 [Medline] [Order article via Infotrieve]
  28. Branden, C., and Tooze, J. (1991) Introduction to Protein Structure , pp. 247-268, Garland Publishing Inc., New York
  29. Bewley, T. A., and Li, C. H. (1969) Int. J. Protein Research. I, 117-124
  30. Lu, Z., Nagata, S., McPhie, P., and Miles, E. W. (1993) J. Biol. Chem. 268, 8727-8734 [Abstract/Free Full Text]
  31. Nishimura, K., Tanizawa, K., Yoshimura, T., Esaki, N., Futaki, S., Manning, J. M., and Soda, K. (1991) Biochemistry 30, 4072-4077 [Medline] [Order article via Infotrieve]

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