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Plant Physiol, June 2001, Vol. 126, pp. 631-642
The Three-Dimensional Structure of Cystathionine
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
RESULTS
DISCUSSION
MATERIALS AND METHODS
LITERATURE CITED
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The pyridoxal 5'-phosphate-dependent enzyme cystathionine 
-lyase
(CBL) catalyzes the penultimate step in the de novo biosynthesis of Met
in microbes and plants. Absence of CBL in higher organisms makes it an
important target for the development of antibiotics and herbicides. The
three-dimensional structure of cystathionine -lyase from Arabidopsis
was determined by Patterson search techniques, using the structure of
tobacco (Nicotiana tabacum) cystathionine 
-synthase
as starting point. At a resolution of 2.3 Å, the model was refined to
a final crystallographic R-factor of 24.9%. The overall
structure is very similar to other pyridoxal 5'-phosphate-dependent enzymes of the -family. Exchange of a few critical residues within the active site causes the different substrate preferences between Escherichia coli and Arabidopsis CBL. Loss of
interactions at the 
-carboxyl site is the reason for the poorer
substrate binding of Arabidopsis CBL. In addition, the binding pocket
of Arabidopsis CBL is larger than that of E. coli CBL,
explaining the similar binding of L-cystathionine and
L-djenkolate in Arabidopsis CBL in contrast to E.
coli CBL, where the substrate binding site is optimized for the
natural substrate cystathionine.
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
RESULTS
DISCUSSION
MATERIALS AND METHODS
LITERATURE CITED
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The whole genome sequence of the
flowering plant Arabidopsis has been determined very recently
(Arabidopsis Genome Initiative, 2000
). This is the first known complete
plant genome sequence and it provides the foundation for detailed
functional characterization of plant genes. The function of a gene
product is closely related to its three-dimensional structure and can
be fully understood only when its three-dimensional structure is known.
Projects to determine the three-dimensional structures of the gene
products of whole genomes with high-throughput techniques have been
started and are designated as structural genomics projects (Burley,
2000).
Cystathionine -lyase (CBL) from Arabidopsis is an enzyme of the Met
biosynthesis pathway of this plant. Met is an essential amino acid that
can only be synthesized by microorganisms and plants.
In bacteria and higher plants, the synthesis of homo-Cys, the
direct precursor of Met, proceeds via a transsulfuration reaction involving two pyridoxal 5'-phosphate (PLP)-dependent enzymes. Cystathionine -synthase (CGS) first catalyzes the synthesis of cystathionine from Cys and activated homo-Ser. Cystathionine is then
cleaved by CBL to produce homo-Cys, pyruvate, and ammonia. Both enzymes
(CGS and CBL) belong to the -family of PLP-dependent enzymes
(Alexander et al., 1994). CGS is homologous to CBL in its amino acid
sequence (Belfaiza et al., 1986) and quaternary structure (Holbrook et
al., 1990; Kreft et al., 1994; Kaplan and Flavin, 1996). The native
recombinant Arabidopsis CBL is a tetramer composed of four identical
subunits of 46 kD, each being associated with one molecule of pyridoxal
5'-phosphate. A Schiff base is formed between the cofactor and the
holoenzyme at the 
-amino group of Lys-278 (Ravanel et al., 1996).
The amino acid sequence deduced from the sequence of Arabidopsis CBL
(aCBL) cDNA showed significant homology with the Escherichia
coli CBL (eCBL; 22% identity). Higher identities are obtained
with E. coli and Arabidopsis CGS (32% and 28%,
respectively; Ravanel et al., 1995) and tobacco (Nicotiana
tabacum) CGS (36.8% for a common stretch of 367 amino acid
residues). The structures of eCBL (Clausen et al., 1996), E. coli CGS (Clausen et al., 1998), and tobacco CGS (Steegborn et al., 1999) have been solved by x-ray crystallography at a resolution of 1.83, 1.5, and 2.9 Å, respectively.
Plant and bacterial CBLs are inhibited by
L-amino-ethoxyvinylglycine (AVG), an antimicrobial
amino acid (Martel et al., 1987; Droux et al., 1995; Ravanel et al.,
1996; Clausen et al., 1997; Turner et al., 1998), and rhizobitoxine, an
antibacterial and phytotoxic amino acid (Owens et al., 1968; Giovanelli
et al., 1972; Rando, 1975; Minamisawa et al., 1990).
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; Burnell and Whatley, 1977; Gentry-Weeks et al., 1993;
Gentry-Weeks et al., 1995; Ravanel et al., 1996). The 50% inhibition
of initial activity values of 2.5 µM and 1.5 mM were determined for
N-ethylmaleimide and iodacetamide, respectively (Ravanel et
al., 1996). Therefore, it was suggested that at least one of the five
Arabidopsis CBL Cys residues is located in or near the active site of
the enzyme. The Arg-blocking reagent phenylglyoxal has no
inhibitory effect on the enzyme, leading the authors to the conclusion
that no Arg residue is involved in the catalytic function of CBL from
Arabidopsis (Ravanel et al., 1996).
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The mature enzyme from Arabidopsis is a poly-peptide of
approximately 46 kD. It is originally synthesized as a 50.4-kD
precursor that is processed within the chloroplast. The exact location
of the maturation site is not known; Gln44 appears to be a good
candidate for the cleavage of the precursor, leaving a protein of 45.7 kD (Ravanel et al., 1996).
Here, we report the crystal structure of Arabidopsis CBL at a resolution of 2.3Å. The size of the substrate binding pocket, as well as the exchange of several amino acids in the active site of E. coli and Arabidopsis CBL, underlie the differences in substrate affinity. As enzymes from anabolic pathways are important for plant growth they are attractive targets for the development of inhibitors used as herbicides.
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RESULTS |
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TOP
ABSTRACT
INTRODUCTION
RESULTS
DISCUSSION
MATERIALS AND METHODS
LITERATURE CITED
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Cloning, Expression, and Protein Purification
The cDNA coding for the putative mature aCBL was cloned into the
E. coli expression vector pASK75 (IBA, Institut für
Bioanalytik, Göttingen, Germany). The paCBL1 construct had
a C-terminal affinity-peptide Strep-I tag (Schmidt et al.,
1996), which after overexpression in E. coli strain
JM83/paCBL1 could be used during the purification on a Streptavidin
Sepharose column (IBA). About 10 mg of purified enzyme could be
obtained from 1 L of cell culture.
Structure Solution and Quality of the Final Model
The crystal structure of Arabidopsis CBL was solved by molecular
replacement at 2.3 Å resolution. The three-dimensional structure of
tobacco CGS was taken as search model because tobacco CGS exhibits the highest amount of identity (36.8%) to aCGL. Patterson
search techniques facilitate an x-ray crystal structure determination in such a way that one has to collect an x-ray data set from the native
crystal only. The preparation of heavy-metal or selenomethionyl derivatives is not necessary. The phase information is obtained by
searching the orientation and translation of the model of the related
structure in the unit cell of the other macromolecule by Patterson
search methods. In this study the program package AMoRe (Navaza, 1994)
was used, and the calculations revealed one prominent solution with a
correlation coefficient of 45.3 and a crystallographic R-value
of 54.1% for all diffraction data to 3.5 Å resolution.
The final model was made up of two identical monomers of 379 amino acid
and 253 water molecules. The N-terminal amino acids (Met57-Glu84) did
not appear in the electron density, indicating that this part of the
enzyme is highly flexible. In addition, residues 418-433 of the
C-terminal domain and the C-terminal Strep-tag are not
defined (Fig. 2). The final
crystallographic R-factor of R = 24.9% and
Rfree = 30.9% in a resolution range from 6.0 Å to 2.3 Å was rather high, due to the above-mentioned flexibilities. Properties of the final model are summarized in Table
I. A Ramachandran plot (Ramachandran and
Sasisekharan, 1968) shows 86.1% of the residues in the most favored
regions, 12.5% in the favored regions, 1.2% in the generously allowed
regions, and 0.3% in the disallowed region, as indicated by the
program Procheck (Laskowski et al., 1993). The residues located in the
disallowed region, Ser-258, Lys-278, and Ser-405, are well defined in
the electron density map. Lys-278 and Ser-405 are active site residues.
The 2Fo-Fc maps contoured
at 1.2 
showed continuous density for all main chain atoms except
for the N-terminal residues already mentioned and for residues
418-433, which are localized on the surface of the protein. All active
site side chains including the cofactor PLP are well defined; a
bicarbonate, which was already shown to be present in E. coli CBL (Clausen et al., 1996), was also found in the active site
of Arabidopsis CBL.
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Overall Fold
aCBL exists as an 4 tetramer in solution
(Ravanel et al., 1996) and crystalline state. Figure
3 shows ribbon plots of the active dimer
and the homotetramer. The asymmetric unit of the C2221 crystal form is built up by two monomers
that share active site residues, thus forming the active dimer. The two
monomers are related by a non-symmetric diad axis. Two active dimers,
related by 2-fold crystallographic symmetry, form the tetramer. The
dimensions of one monomer and the tetramer are 85 × 50 × 40 Å and 96 × 89 × 64 Å, respectively. Each monomer contains
one PLP cofactor and 379 amino acids with 34.3% -helical, 2.2%
310 helical, 14.1% -sheet conformation, and
28.9% turns, leaving 20.5% random coil structure. The cofactors of
the active dimers are separated by 22 Å. Like most PLP-dependent
enzymes of the -family, the monomer of aCBL comprises three
structurally and functionally distinct regions. The N-terminal domain
(residues 57-131) is only partially defined (residues 57-84 are
flexible) and interacts with the active site of the neighboring
monomer. The large PLP-binding domain (residues 132-325) carries most
of the catalytically important residues and is connected to the smaller
C-terminal domain (residues 326-464) by the long -helix 9 with a
kink near residue Ala-329. The N-terminal domain consists of one
-helix and an extended loop structure. A seven-stranded mainly
parallel -sheet (a, g, f, e, d, b, c, with g in antiparallel
orientation) that is bent around helix 3 is the central part of the
PLP-binding domain. It is sandwiched between helices 2, 5, 6, and 7, shielding it from the solvent accessible surface, and helices 3, 4, and
8, which are part of the interdomain surface. PLP, covalently attached to Lys-278 (Ravanel et al., 1996) is located near the C termini of
-sheets d, e, and f. The phosphate group binds near the N terminus
of helix 3. The major part of the C-terminal domain is a five-stranded,
almost antiparallel -sheet. Strands A, B, E, D, and C with
directions +, 
, +, , and are shielded from solvent by
helices 10-12.
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The active dimer-forming monomers associate tightly through hydrogen bonds, hydrophobic interactions and intermolecular active site interactions. The cofactor PLP is involved in several strong hydrogen bonds to amino acid residues of the adjacent subunit (Arg-129* and Tyr-127*). The extended loop of the N-terminal domain additionally stabilizes the dimer formation. Ala-119 and Gln-116 associate with Glu-395* and Ile-402* of the C-terminal domain. The helices 3, 4, 8, and 9 of the PLP-binding domain are in close contact with each other. Several hydrophobic residues of both symmetry mates (Leu-187, Val-191, Val-192, Leu-313, Ala-314, and Phe-316) build a large hydrophobic patch, which is further stabilized by hydrogen bonds between residues Gly-310(O)-Ser-156(OG), Ser-156(OG)-Ala-112(O), Asp-285(O)-Gln-110(NE2), and Arg-194(NH1)-His-166(O).
Upon association of the dimers AC and BD, a tetramer of overall 222 symmetry is formed. The most important elements for tetramer stabilization are the N-terminal half of helix 9 and the extended loop structure of the N-terminal domain.
Active Site Structure
Figure 4a shows the
2Fo-Fc electron density
map at 2.3-Å resolution of the aCBL active site region including
the PLP cofactor. Interatomic distances are indicated in Figure 4b. In
addition to the PLP-binding domain, parts of the C-terminal (C-terminal loop of strand C and strand D) and the neighboring N-terminal domain
(loop around Tyr-127*) are involved in building the substrate binding
pocket. The PLP cofactor is embedded by the N-terminal part of helix 3, the C termini of -strands d, e, and f, and their connecting loops.
Asp-253 plays a key role in stabilizing the positive charge of the
pyridine N1 nitrogen, thereby increasing the electrophilic character of
the cofactor. Its carboxylate OD2 forms a hydrogen bond (2.6 Å)
with the PLP nitrogen N1; two further interactions between OD1 and the
hydroxyl group (2.5 Å) as well as the amide nitrogen (2.8 Å) of
Ser-255 tether Asp-253 in an optimal position to interact with the
pyridine nitrogen. There are no specific interactions between the
methyl group at C2 and the apoprotein. The hydroxyl group at C3 of PLP
(presumably ionized) is placed in hydrogen bonding distance to a
well-defined water molecule (2.6 Å). The aldehyde function of PLP at
position 4 is observed as the internal aldimine resulting from the
reaction with the -amino group of Lys-278. UV absorbance of the
protein near 425 nm indicates protonation of this internal aldimin. The nitrogen NZ is involved in a weak hydrogen bond with the
hydroxyl group of Ser-275 (3.1 Å).
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The phosphate group is the main anchor of the cofactor within the
active site. The four phosphate oxygens are involved in a total of nine
interactions with the apoprotein. Gly-157 and Met-158 of the N terminus
of -helix 3 interact with OP1 and OP3, respectively. The
hydroxyl groups of Ser-275 and Thr-277 are within hydrogen bonding
distance to OP1 (2.8 and 2.7 Å); Ser-275 interacts additionally with
OP4 (2.9 Å) and the nitrogen NZ at position 4 (see above). All
other interactions originate from the neighboring subunit. Arg-129* and
Tyr-127* are involved in a total of four hydrogen bonds to OP2 and OP3,
thereby compensating the negative charge of the phosphate group.
Arg-129* additionally interacts with the phenolic hydroxyl group of
Tyr-181. The PLP pyridine ring is sandwiched between Ser-255 and
Ser-275 on the protein facing side and Tyr-181 on the opposite side of
the cofactor. The side chains of Ser-255 and Ser-275 lie within van der
Waals distance to the pyridine ring atoms, thereby minimizing any
movement of the cofactor in this direction. Furthermore, they can form stabilizing H-bonds with the 
-electron system of this ring. The phenolic ring of Tyr-181 is almost coplanar to the pyridine ring of
PLP. Stacking interactions between an aromatic side chain and the PLP
ring system are found in most PLP-dependent enzymes and are thought to
increase the electron sink character of the cofactor (Hayashi et al.,
1990; John, 1995). The positive charges of the guanidinium group of
Arg-129* and the protonated aldimine near Tyr-181 should lower the
pKa of the Tyr hydroxylic group, thereby enabling this
residue to play an essential role in catalysis by activating the
incoming substrate for transaldimation. This is similar to the
mechanism earlier proposed for eCBL (Clausen et al., 1996) and E. coli CGS (Clausen et al., 1998).
Comparison of aCBL with Other PLP-Dependent Enzymes
Overall Structure
As mentioned before, aCBL shows 22% identity to eCBL and 32% identity to E. coli CGS. A sequence alignment is shown in Figure 5a. PLP-dependent enzymes of the-family fold almost identically, despite moderate amino acid
identity. A least square superposition of the aCBL and eCBL dimers as
well as a superposition of their active site residues was carried out
using the program O (Jones et al., 1991). The
resulting overlays of the C traces and the active site residues are
shown in Figure 5, b and c, respectively.
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-helix 3 (Fig.
5b, region I) in eCBL corresponds to a gap in the amino acid sequence
of aCBL; the region around this gap is poorly defined in the aCBL
structure. The only conserved amino acids, Tyr-127* and Arg-129*, are
active site residues of the neighboring subunit. All secondary
structure elements of the PLP-binding and most of the C-terminal domain
superimpose almost perfectly. The C-terminal domain differs in
secondary structure elements and structural folds at two nearby surface
regions, marked IIa and IIb in Figure 5b. The aCBL amino acids 421 to
432 of region IIa were exchanged by poly-Ala because they were weakly
defined in the electron density map during refinement. Here, the
B-factors are approximately 120 Å2 (Fig. 2),
indicating a very flexible surface region. The corresponding region in
eCBL (residues 349-366) contains -helix 15 and a following loop
structure. Due to the flexibility of the aCBL fold, no information of
structural differences in this region was available. Region IIb
(382-396 in aCBL, 314-330 in eCBL) is situated near region IIa,
forming an extended loop, including -helix 11. Here, the B-factors
are only slightly above the average (around 60 Å2). The extended eCBL loop is exposed to the
solvent, in contrast to the corresponding loop structure of aCBL, which
is embedded by the C-terminal domain.
The PLP-binding and C-terminal domains contain several conserved
residues that are not directly involved in catalysis or binding of
cofactor and substrate. The small number of identical residues together
with the conservations in hydrophobicity pattern are obviously
sufficient to determine the common fold of the proteins. The central
-sheets contain only very few identical amino acids (all residue
numbers refer to aCBL amino acid sequence; Asp-253, Gly-289, Ser-381,
Phe-382, Ala-403, and Arg-440), whereas hydrophobic regions are
strictly conserved (hydrophobic: 152, 174, 175, 202, 220, 221, 222, 252, 271, 293, 355, 380, 383, 412, 413, 439, and 441; hydrophilic: 274, 354, 356, 401, and 442). Similar results are obtained for the
-helices. The loop and turn regions often show conserved Gly (131, 171, 268, 282, 310, 375, 407, and 444) or Pro (226 and 350) residues,
which allow tight turns or otherwise unfavorable backbone geometry.
There are several other identical amino acids in both eCBL and aCBL
which are not involved in active site or secondary structure building.
The hydrophilic residues Glu-224, Asp-235, Asp-270, Glu-446, and
Asp-447 stabilize the termini of their corresponding secondary
structure elements, whereas the conserved amino acids around residue
283 form a loop in the active site region.
Active Site Structure
Detailed descriptions of the eCBL active site and the structure of the CBL inhibitor complex with AVG have been published (Clausen et al., 1996; Clausen et al., 1997). A superposition of the
active sites of aCBL and eCBL is shown in Figure 5c. The inhibitor AVG
was modeled into the structure by least square superposition of aCBL
and the eCBL-AVG inhibitor complex, using the program O. The
important catalytic residues Lys-278, Tyr-181, and Asp-253 align almost
perfectly. Similarly, the hydrogen bonding network around the
PLP-phosphate (Thr-277, Gly-157, Met-158, Arg-129*, and Tyr-127*) is
essentially conserved. There are two additional interactions with the
PLP-phosphate group in the aCBL structure, namely hydrogen bonds
between the hydroxyl group of Ser-275 and OP1 as
well as OP4. In addition, there are slight
differences in the vicinity of Asp-253. The equivalent Asp-185 from
eCBL interacts with the hydroxyl group of Thr-187 and the main chain
nitrogen of Asn-186. A water molecule connects Asp-185 and Glu-154,
which itself interacts with Ser-158. Thr-187 is exchanged to Ser-255 in
aCBL. The interaction with the hydroxyl group is conserved. Glu-224
(Glu-154 in eCBL) interacts with the main chain nitrogen of Tyr-181
instead of Asp-253.
Probably the most important amino acid exchanges involve the eCBL amino
acids Trp-340, Asp-116 and Tyr-238*, which are exchanged to Phe-406,
Arg-186 and Asn-307* in aCBL. These exchanges result in altered
positioning of the amino acid side chains. Furthermore, the quality of
aromatic interaction is changed (Trp versus Phe). More importantly,
there is an inversion of charges (Asp to Arg) as well as an exchange of
an aromatic residue by a polar, uncharged functionality (Tyr to Asn).
As a result, substrate binding in Arabidopsis should be altered, as
will be discussed below. The anion binding site Arg-440 (Arg-372 in
eCBL) and the bicarbonate are located further away from the cofactor
PLP, compared to the bacterial structure.
Enzyme Kinetics
The Km values for the substrates
cystathionine and djenkolate (structures see Fig. 1) are identical for
aCBL (0.22 mM in each case), while they differ by
a factor of nine for eCBL (0.04 and 0.36 mM,
respectively). eCBL binds the natural substrate cystathione much
stronger (0.04 mM, (Dwivedi et al., 1982)) than
aCBL (0.22 mM (Ravanel et al., 1996)).
Vmax values for
L-cystathionine and L-djenkolate are 5-fold higher with eCBL than
with aCBL, but each enzyme has similar Vmax
values for both substrates. Values of the kinetic constants for both
enzymes are summarized in Table II.
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
RESULTS
DISCUSSION
MATERIALS AND METHODS
LITERATURE CITED
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Substrate Binding and Inhibitory Studies
The substrate binding pocket of eCBL is smaller than that of aCBL.
This is due to the replacement of residues with larger side chains
lining the substrate binding site pocket by residues with smaller side
chains in the aCBL structure (Trp-340 to Phe-406, Tyr-338 to Val-404,
Phe-55* to Asp-126*, Glu-112 to Gly-182 and Tyr-238* to Asn-307*).
Several interactions which are present in the active site of eCBL are
lost or weakened in aCBL due to active site residue exchanges. Trp-340
of eCBL, which is positioned at the -carboxyl site of the substrate,
forms a hydrogen bond to OB2 of the bicarbonate in the native
structure and to O2B of L-aminoethoxyvinylglycine in
the complex, whereas the hydrophobic amino acid Phe-406 in aCBL lacks
this function. In addition, on the distal site of the pocket Tyr-238*,
which interacts indirectly with the amino group of the inhibitor, is
replaced by Asn-307*. The shorter side chain of Asn-307* is too far
away from the substrate binding pocket to interact directly or via an
active site water molecule with inhibitor or substrate. These
interactions that are involved in eCBL substrate binding are not
present in the aCBL active site.
However, several substrate binding interactions are conserved. The
anion binding sites aCBL Arg-129* and eCBL Arg-58*, as well as aCBL
Tyr-181 and eCBL Tyr-111, are positioned almost identically in both
enzymes. Furthermore, an additional interaction, which is only relevant
in the Arabidopsis active site, results from the exchange of eCBL
Asp-116 to aCBL Arg-186. Asp-116 with a rather small side chain is
positioned too far from the binding pocket to interact with the
substrate, whereas the significantly longer side chain of Arg-186 in
aCBL, pointing toward the distal part of the substrate, further
interacts with the distal carboxyl group. The dimension of the eCBL
substrate binding pocket is optimized for the substrate cystathionine,
whereas the larger binding site of aCBL should accomodate cystathionine
and the longer substrate djenkolate with equal affinity. Without
considering the size of the active site cavity, substrate binding to
eCBL seems to occur with higher affinity, which may be due to the
stronger interaction at the -carboxyl site.
The influence of Arg- and thiol-blocking agents was examined earlier
(Ravanel et al., 1996). Although the Arg blocker phenylglyoxal had no
inhibitory effect on the enzyme, plant CBL was found to be
sensitive to the thiol-blocking inhibitors N-ethylmaleimide and
iodoacetamide, which in turn had no effect on the bacterial enzymes. It
was concluded that no Arg residue was involved in the catalysis of
aCBL, whereas at least one of the five aCBL Cys residues was postulated
to be located near the active site of the plant enzyme (Ravanel et al.,
1996). In contrast, our structure clearly showed several Arg, but no
Cys in the active site of the enzyme. The lack of inhibition by the Arg
blocker phenylglyoxal indicates that the interactions involving the
three active site Arg are strong enough to prevent the blocker from
binding. The guanidinium group of Arg-440 interacts with OB1 and OB2 of
the bicarbonate; the side chain of Arg-129* forms several hydrogen bonds to the PLP phosphate OP2, OP3, and the hydroxyl group of Tyr-181.
One of the guanidino nitrogens of Arg-186 lies within hydrogen bond
distance (3.2 Å) to the ND2 nitrogen of Asn-309*. The shape of
the active site in E. coli CBL may provide additional interactions favoring the binding of phenylglyoxal. The blocker alternatively could bind to an Arg outside the active site, thereby acting as a noncompetitive inhibitor.
Similarly, the inhibitory effects of N-ethylmaleimide can only be explained by allosteric effects because no Cys residue was identified in or near the active site.
Postulated Catalytic Mechanism
The catalytic mechanism of Arabidopsis CBL is similar to that
proposed for eCBL (Clausen et al., 1996). Clausen et al. already postulated an induced fit mechanism analogous to that of AAT
(Jansonius and Vincent, 1987). Further experiments to examine the
mechanism included the crystallization and structure determination of
ApoCBL from E. coli (Breitinger, 2000).
Attempts were made to solve the structure of the eCBL substrate complex by soaking the crystals with cystathionine. Under this treatment, the crystals cracked after several hours, indicating a conformational change of the protein upon substrate binding. The apo-eCBL crystals were unstable, but the structure could be solved to a resolution of 2.5 Å. It showed flexible regions in the N-terminal and PLP-binding domain. The loop, including residues Tyr56* and Arg58*, which interacts through several hydrogen bonds with the PLP phosphate in the native structure, is no longer defined in the apoprotein (Fig. 6). The region around Tyr-111 is also highly flexible. These flexibilities indicate that parts of the protein change their conformation during substrate binding, thereby shielding the active site region. Because Tyr-56* and Arg-58* are the only conserved amino acids in the specific region of the N-terminal domain, interaction directed to these residues seems to be the main driving force for this transition. The native structures of CBL, therefore, show the open conformation of the enzyme, where the active site is accessible to substrate.
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As shown in Figure 7, we
propose that the substrate enters the active site cleft from a position
above the conserved amino acids Arg-129* and Tyr-181. The substrate
will be oriented in proper position for transaldimation by interaction
with the general -carboxylate docking site Arg-440, the ionized PLP
hydroxyl group, Tyr-181, and Arg-129*. In the next step Tyr-181, a
conserved residue in all cystathionine-utilizing enzymes, abstracts a
proton from the -amino group of the bound substrate. After formation
of the external aldimine, the -carboxylate and the sulfur atom of
the substrate are fixed in a position, which allows the amino group of
Lys-278 to abstract the proton from C
. The PLP
cofactor delocalizes the negative charge of the developing carbanion
over its entire conjugated -system. Subsequently, Lys-278 transfers
the proton directly from C to
S
of the substrate. During this process, the
positively charged -amino group of Lys-278 is attracted by the
negative charge of the PLP phosphate group, directing it to hydrogen
distance position to S, thereby inducing
C
-S bond cleavage by protonation of
S, followed by the release of homo-Cys. The
remaining PLP derivative of aminoacrylate is converted to
iminopropionate by proton addition on C. After
release of homo-Cys, the transaldimation equilibrium between external
and internal aldimine should be shifted toward the latter, resulting in
release of iminopropionate, which is hydrolyzed to pyruvate and ammonia
outside the enzyme.
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Knowledge of the substrate binding site of CBL, coupled with detailed information of the differences in function, as well as three-dimensional structures of plant and bacterial CBLs should serve as a starting point for the development of specific enzyme inhibitors, herbicides, and antibiotics.
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MATERIALS AND METHODS |
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TOP
ABSTRACT
INTRODUCTION
RESULTS
DISCUSSION
MATERIALS AND METHODS
LITERATURE CITED
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Cloning Expression and Purification
The cDNA coding for the putative mature aCBL polypeptide,
starting with Met-57 of the respective coding sequence region of the GenBank/EMBL accession number L40511, was amplified from an
Arabidopsis cDNA library (Minet et al., 1992) by PCR with
Pfu DNA polymerase (Stratagene, La Jolla,
CA) according to the manufacturer's recommendations using
phosphorothioate-protected primers. The aCBL gene was amplified using
the oligonucleotides
5'-GCACTATCTAGATAACGAGGGCAAAACATGGAGAAAAGTGT-3' and
5'-GCTGATCCCGGGGAGAGGGAAGGTTTTGAAGG-3' as primers. The PCR product
was digested by XbaI/SmaI and cloned into
the XbaI/Eco47III-digested Escherichia coli expression vector pASK75 (IBA) yielding
the plasmid paCBL1, which codes for an aCBL polypeptide that is tagged
at its C terminus by the affinity peptide Strep-tag I
(Schmidt et al., 1996). The paCBL1 construct was used to transform
E. coli JM83 (Yanisch-Perron et al., 1985). For
overexpression, the E. coli strain JM83/paCBL1 was grown
at 26°C in Luria-Bertani medium containing 100 µg mL
1
ampicillin to a cell density of A550 = 0.5 and induced
with 200 µg L1 anhydrotetracycline (Skerra, 1994) for
16 h. Cells were harvested by centrifugation, resuspended in CP
buffer (100 mM Tris/HCl, pH 8.0, 1 mM EDTA, 10 µM pyridoxalphosphate) and disrupted by two passages
through a French pressure cell at 15,000 psi. The cell debris was
removed by centrifugation (18,000g for 30 min), and the
supernatant was subjected to 0% to 33% ammonium sulfate fractionation. The precipitate was resuspended in CP buffer and affinity purified on a Streptavidin Sepharose column (IBA). This protocol yielded about 10 mg of purified enzyme per liter cell culture.
Crystallization and Data Collection
Crystals were grown by sitting drop vapor diffusion
against a reservoir containing 100 mM
MES[2-(N-morpholino)-ethanesulfonic acid]·NaOH, pH
6.0, and 300 mM
(NH4)2SO4. Droplets were mixed from
4 µL of protein solution in 10 mM HEPES
[4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid], pH 7.0, 10 µM PLP, and 2 µL of reservoir. The crystals were grown
at room temperature to an approximate size of 200 µm × 100 µm × 40 µm within 1 week. The crystals belong to the
orthorhombic space group C2221 with cell dimensions of
a = 107.8 Å, b = 154.3 Å, c = 118.8 Å, and one
homodimer per asymmetric unit. Diffraction data were
collected at the Deutsche Elektronen Synchrotron (beamline BW7B,
Hamburg, Germany) at 100 K (
= 0.83 Å) on a 345-mm detector (MAR Research, Norderstedt, Germany). Before freezing, the crystals were incubated for a few seconds in 30% (v/v) PEG400, 50 mM HEPES (pH 7.0), 300 mM
(NH4)2SO4. A single crystal yielded
a data set complete to 2.3 Å resolution in a 90° 
-scan with a
1.0° frame width. Diffraction data were processed using the Denzo
program package (Otwinowski and Minor, 1996). The intensities were
scaled, reduced, and truncated to structure factors by the programs of the CCP4 program suite (Collaborative Computational Project, 1994). The
data collection statistics are summarized in Table I.
Molecular Replacement and Structure Refinement
Structure solution by Patterson search techniques was carried
out using the program package AMoRe (Navaza, 1994). The coordinates of
the tobacco (Nicotiana tabacum) CGS dimer (Steegborn et
al., 1999) were used as a search model. Residues were mutated to Ala or
Gly, and the PLP cofactor was omitted. The AMoRe calculations revealed
one prominent solution with a correlation coefficient of 45.3 and a
crystallographic R-factor of 54.1% using diffraction data to a
resolution of 3.5 Å.
The quality of the electron density maps for model building was
improved by 2-fold real space averaging using the program AVE (Jones,
1992). The non-crystallographic symmetry (NCS) operators were
determined using program LSQMAN (Kleywegt and Jones, 1994). Model
building was done with program O (Jones et al., 1991)
and refinement with the CNS software (Brünger et al., 1998) using the parameters of Engh and Huber (1991). No NCS restraints were used
due to the sufficient good resolution. The minimize protocol with
an overall anisotropic B-factor was applied. B-factors were refined
isotropically, yielding a final root mean square deviation value
for bonded Bs of 3.45. The crystallographic R-factor was used for
monitoring the stage of the refinement procedure, omitting 5% of the
structure factors in order to calculate
Rfree values. The initial averaged
2Fo-Fc electron density map showed well-defined electron density for the PLP cofactor. At the end of the refinement model, building was done in unaveraged electron density maps
independently in both monomers. Finally, 253 water and two bicarbonate
molecules were incorporated.
Rebuilding and refinement cycles converged at
Rcryst and Rfree
values of 24.9% and 30.9%, respectively. Three stretches of the
polypeptide chain were invisible in the electron density maps; the
N-terminal part, i.e. residues 57 to 84; the region between residues
418 to 433; and the 11 amino acids of the C-terminal Strep-tag (Fig. 2). These parts are invisible most
probably due to their high flexibility. This feature of the structure
accounts for the relatively high final R values. The final model
contained 5,671 protein atoms, 30 cofactor atoms, and 266 solvent
atoms. Further details are given in Table I. The stereochemistry of the
model was analyzed with programs CNS and PROCHECK (Laskowski et al.,
1993). For the comparison of NCS-related molecules, the program LSQMAN
was used.
Coordinates
The coordinates of the refined model of aCBL have been deposited in the Research Collaboratory for Structural Bioinformatics Protein data bank (accession code 1IBJ).
| |
FOOTNOTES |
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Received February 12, 2001; returned for revision March 5, 2001; accepted March 22, 2001.
1 Present address: Institute for Biochemistry, Friedrich-Alexander-University of Erlangen-Nürnberg Fahrstr. 17, D-91054 Erlangen, Germany.
* Corresponding author; e-mail: messersc{at}biochem.mpg.de; fax 49-89-8578-3516.
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LITERATURE CITED |
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TOP
ABSTRACT
INTRODUCTION
RESULTS
DISCUSSION
MATERIALS AND METHODS
LITERATURE CITED
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, and -family.
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Biochemistry
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Biochim Biophys Acta
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Plant Physiol
104: 1215-1220[Abstract]-synthase and -cystathionase have common peptide sequences of the active site and are inhibited similarly by aminoethoxyvinylglycine.
In
TK Korpela, P Christen, eds, Biochemistry of Vitamin B6. Birkhäuser Verlag, Basel, pp 341-344-cystathionase in Salmonella typhimurium.
Biochim Biophys Acta
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Biochem J
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Plant Mol Biol
29: 875-882[CrossRef][Web of Science][Medline]-synthase from Nicotiana tabacum reveals its substrate and reaction specificity.
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47: 189-196[CrossRef]This article has been cited by other articles:
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-Lyase
from Arabidopsis and Its Substrate Specificity
