(Received for publication, November 30, 1994; and in revised form, January 13, 1995)
From the
-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
, 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
-cystathionase gene (metC) from B.
avium, reveals 4 cysteine residues/enzyme subunit (M
= 42,600), and spectrophotometric
analysis with 4,4`-dithiodipyridine showed that there were no disulfide
linkages in the native protein.
-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
-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
36,000)
also lacked the first 63 amino acids comprising the N terminus of the
native protein. The
-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
-cystathionase were cytotoxic for
tissue culture cells.
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 -cystathionase (EC 4.4.1.8). This pyridoxal
5`-phosphate (PLP)(
)-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, 4, 5) . The
enzyme may also utilize L-cystine as substrate, in which case
the products of the
-elimination reaction (5, 6, 7, 8) are pyruvic acid,
NH
, 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
-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 -cystathionase, and our sequencing of
the gene (metC) from B. avium, prompted comparison
with the
-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
-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
-cystathionase. Published
reports suggest that neither the S. typhimurium and E.
coli
-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
-cystathionase from B. avium.
Figure 1:
Comparison of the deduced
amino acid sequences of -cystathionase from B. avium (BABC), E. coli (ECBC) and S.
typhimurium(STBC). Numbering of the amino acids
of B. avium
-cystathionase is shown above the sequence
while the number of residues for the other
-cystathionases is
indicated at the end of the respective sequences. Boxes indicate identical amino acid residues between the
-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
-cystathionase from B. avium? Second, are the
contributions of the (4) cysteine residues to
-cystathionase activity of a conformational or catalytic nature?
Third, is catalytically inactive
-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.
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.
Figure 3:
SDS-polyacrylamide gel and Western
immunoblot of proteins of purified recombinant wild-type and mutant
-cystathionases. A, purified wild-type and mutant
-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
-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.
Figure 4:
Absorption spectra of wild-type and
mutant-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
-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 -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.
Figure 5:
Toxicity of wild-type and mutant
-cystathionases for MC3T3-E1 osteogenic cells. Purified
recombinant wild-type and mutant
-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
-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
-cystathionase and active C279G protein were identical to those
elicited by protein C88A, i.e. formation of detached,
spherical cells.
Figure 6:
SDS-polyacrylamide gel of
chymotrypsin-treated wild-type and mutant -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
-cystathionases. B,
chymotrypsin-digested
-cystathionases. Note that wild-type
-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.
Earlier studies in this laboratory demonstrated that
-cystathionase of B. avium was inhibited by
NEM(1) . In this context, it may be noted that
-cystathionase from Paracoccus denitrificans(24) is also sensitive to inhibition by NEM, whereas
neither the
-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
-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
-cystathionase from two
species but not others, we felt that the second possibility was the
most likely explanation for NEM-mediated inhibition of
-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
-cystathionases
are inaccessible to NEM.)
-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 -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
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
-cystathionase via formation of a
disulfide linkage(29) .
Throughout its purification,
-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
-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
-cystathionase from E. coli(15) showed that the
-NH
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
-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
-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
-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 -elimination reaction is a
question that has not yet been answered unequivocally for any of the
bacterial
-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
-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
-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
-cystathionase molecule with surface
components of the eucaryotic cells, is required for manifestation of
cytotoxicity.