the quinonoid intermediate from the opposite side of the PLP-ring. By site-directed
mutagenesis, kinetic and computational studies, it has been demonstrated that alanine
racemase catalyzes the racemization by a two-base mechanism in which Tyr 265 is the base
abstracting the Cα-proton from L-alanine while Lys 39 is the base abstracting the Cα-proton
from D-alanine [20-23]. Evidence has been also provided that alanine racemase catalyzes the
transamination of both enantiomers of alanine as a side reaction and that: 1) the α-hydrogen
of L-alanine is transferred suprafacially to the C4’ of PLP by Tyr 265; 2) Lys 39 plays the
role of a counterpart for Tyr 265 and is specific for D-alanine [24]. It should be noted that
alanine racemase, together with PLP-dependent amino acid racemases of broad substrate
specificity [25], represents the first class of PLP-enzymes catalyzing the hydrogen removal
on both sides of the plane of a substrate-cofactor complex during transamination.
and alanine racemase. The complex between alanine racemase and alanine phosphonate (PDB 1BD0;
represented as green sticks), and the complex between cystalysin and aminoethoxyvynilglycine (PDB
1C7O; represented as yellow sticks) are shown. Oxygen atoms are colored orange, nitrogen atoms black
and phosphorus atoms red .The position of the water molecule W733H is also shown. Figure was
obtained using pyMol software.
The structural similarities between cystalysin and alanine racemase led to analyze the
interaction of cystalysin with alanine, a compound which does not contain a suitable leaving
group and cannot be substrate of an α,β-elimination reaction.
As reported in Table 2, cystalysin is able to catalyze the racemization of both
enantiomers of alanine [26]. Considering that racemization is a side reaction for the enzyme,
it takes place with a remarkable kcat value (about 1 s-1), which is only about 10-fold lower
than that of the main reaction at the same pH. This raises the question of a possible
110 Barbara Cellini, Riccardo Montioli and Carla Borri Voltattorni
physiological meaning for T.denticola in the synthesis of the bacterial cell walls. However,
considering the high Km for alanine, it cannot be excluded that the racemase activity could be
a mere corollary of the chemical properties of the enzyme. Spectroscopic analyses of the
interaction of cystalysin with L-and D-alanine have indicated that, along with the racemase
activity, the enzyme catalyzes the half-transamination of both enantiomers of alanine with
turnover times measured in minutes (Table 2). Moreover, apo-cystalysin, in the presence of
PMP, catalyzes the reverse transamination of pyruvate. Thus cystalysin is able to perform
transamination in both direction: from PLP to PMP and from PMP to PLP [26].
Table 2. Steady-state kinetic parameters for the alanine racemase and transaminase
catalytic activities in 20 mM potassium phosphate buffer pH 7.4 at 25°C
kcat (s-1) 1.05 ± 0.03 1.4 ± 0.1
10-4 x kcat (s-1) 4.50 ± 0.05 1.0 ± 0.1
Cystalysin: An Example of the Catalytic Versatility… 111
blue and phosphorus purple. Hydrogen bonds are shown in cyan. The * denotes a residue that belongs
to the neighbouring subunit. This Figure was obtained using pyMOL.
According to a generally accepted mechanism, it has been postulated that the
racemization of alanine catalyzed by cystalysin proceeds as follows (Figure 9): (i)
transaldimination between Lys 238 bound with PLP (I) and the α-amino group of alanine to
produce the external aldimine II; (ii) abstraction of the α-hydrogen from alanine to produce a
resonance-stabilized quinonoid intermediate (III); (iii) reprotonation at the α-carbon of the
quinonoid intermediate III on the side opposite to that where the α-hydrogen was abstracted;
(IV) second transaldimination between IV and Lys 238 to release the product enantiomer of
112 Barbara Cellini, Riccardo Montioli and Carla Borri Voltattorni
alanine (V). In this mechanism racemization and transamination of alanine share the step
leading to the quinonoid intermediate. When an half-transamination occurs, the C4’ position
of the cofactor moiety is reprotonated, thus generating the pyruvate-PMP ketimine
intermediate (VI). The hydrolysis of the intermediate VI leads to the formation of PMP and
Interestingly, it has been demonstrated that in the reverse transamination of pyruvate
catalyzed by cystalysin, the cleavage of the C-H bond at C4’ of PMP and the reprotonation of
the α-carbon of the anionic intermediate take place in a non-stereospecific manner. This is
similar to what has been observed with alanine racemase. However cystalysin is the first
Molecular modeling studies have been undertaken in order to rationalize the
experimental data and identify possible acid-base catalysts involved in the two-base
racemization mechanism. The putative binding modes of L- and D-alanine at the active site
of cystalysin are shown in Figure 10A and 10B, respectively. The inspection of the model
have indicated that for both substrates, according to the Dunathan hypothesis, the leaving
group is antiperiplanar to the aromatic moiety of PLP. For L-alanine, the structure has
revealed that Lys 238 is located close to the Cα-hydrogen of the substrate and to the C4’ of
the cofactor. Thus this residue seems to have the proper orientation to act as a catalytic base
on the si face of PLP. For D-alanine, two tyrosines (Tyr 123 and Tyr124) and a water
PLP ring, is placed in a proper position to act as a catalyst. However, it should rotate out from
its hydrophobic environment for the proton abstraction. Also a water molecule, held in place
by Tyr 123 and, to a lesser extent, by Tyr124, could bridge this gap for proton abstraction,
acting as a general acid-base catalyst [26].
Site-directed mutagenesis studies have been employed to gain insight into the mechanism
of racemization and transamination of both enantiomers of alanine catalyzed by cystalysin.
As a first step, Lys 238 and Tyr 123 were selected as target for mutagenesis, and the
functional properties of the active-site mutants K238A and Y123F were analyzed to probe the
hypothetical role of the mutated residues in racemase and transaminase activities.
The K238A mutant neither shows detectable racemase activity in both directions, nor
catalyzes the transamination of L-alanine thus indicating that Lys 238 is the base located on
the si face of PLP specifically abstracting the α-hydrogen from L-alanine [27]. It can be
observed that this residue plays in the racemization the same role that has been already
proposed for it in the α,β-elimination reaction [18]. In addition, it has been found that K238A
catalyzes the overall transamination of D-alanine. This strongly suggests that on the re face
of PLP is located an acid-base catalyst whose role in the forward reaction is proton
abstraction from the D-alanine-PLP external aldimine complex and reprotonation at the C4’
the pyruvate-PMP ketimine intermediate to the Cα of the quinonoid to regenerate D-alanine.
On the basis of molecular modeling studies, the possibility that Tyr 123 is the acid-base
catalyst located on the re face of PLP has been checked. However, Y123F mutant retains
Cystalysin: An Example of the Catalytic Versatility… 113
poor racemase and transaminase activities, thus suggesting that Tyr 123 is not essential for
catalysis. The possibility that the catalytic function of Tyr 123 is replaced by Tyr 124 in the
Y123F mutant has been excluded by the spectral and kinetic characterization of the
Y123F/Y124F mutant. In fact, the catalytic efficiencies of the racemization and
transamination reactions are weakly altered in the double mutant with respect to the single
mutant [27]. On this basis, it has been hypothesized that the water molecule held in place by
Tyr 123 and Tyr 124 may function as the acid-base catalyst on the re face of the cofactor.
Following this view, the reduction of the racemase activity of Y123F has been ascribed to the
mispositioning of the water molecule upon the mutation of tyrosine 123 to phenylalanine, as
confirmed by molecular modelling. A second hypothesis advanced is that Tyr 123 could have
a direct role in proton abstraction/donation being its function replaced by the water molecule
in the Y123F mutant. Available data did not allow to unequivocally identify the acid-base
catalysts on the re face of PLP in cystalysin; it has only been proposed that water molecules
and their hydrogen bond interactions with Tyr 123 are required for an efficient proton
β-DESULFINATION AND β-DECARBOXYLATION CATALYZED
When the interaction of cystalysin with L-cysteine sulfinic acid and L-aspartic acid was
studied, it was found that the enzyme does not catalyze the α,β-elimination of these ligands.
However, both L-cysteine sulfinic acid and L-aspartic acid induce time-dependent changes in
the protein-bound coenzyme which strongly suggest an active-site directed event and thus the
occurrence of a reaction between cystalysin and each of these ligands [28]. Unexpectedly, it
amount of L-alanine far exceeding that of the D-alanine. The kinetic parameters for these
catalytic activities, reported in Table 3, reveal that both reactions take place with high
turnover numbers. In particular, the kcat value for the β-desulfination reaction is about 2-3
114 Barbara Cellini, Riccardo Montioli and Carla Borri Voltattorni
potassium phosphate buffer pH 7.4 at 25°C
L-cysteine sulfinic acid L-aspartate Oxalacetate
kcat (s-1) 89 ± 7 0.8 ± 0.1 0.15 ± 0.01
Km (mM) 49 ± 9 280 ± 70 13 ± 2
-1) 1.8 ± 0.3 0.0028 ± 0.0008 0.011 ± 0.002
gradual conversion of PLP bound to PMP. This event is due to a half-transamination reaction,
which occurs at a lower rate with respect to the rate of cleavage of the β-substituent for both
Figure 11. Reaction mechanism for β-desulfination and β-decarboxylation reactions catalyzed by
Cystalysin: An Example of the Catalytic Versatility… 115
On the basis of all the results, the reaction of cystalysin with L-cysteine sulfinic acid and
L-aspartic acid has been interpreted according to the mechanism depicted in Figure 11. After
a first transaldimination step (IÆII), a Cβ-R-
cleavage occurs where the negatively charged
side chains of L-cysteine sulfinic acid and L-aspartic acid are eliminated without
deprotonation at Cα. Thus, the electrophilic displacement of the negatively charged
substituent at Cβ is not in the main pathway of the α,β-elimination catalyzed by cystalysin.
cleavage leads to the formation of the L-alanine aldimine complex (III), which can
undergo either a racemization or a transamination reaction. On the basis of the proposed
mechanism, PMP formation could be due to a half-transamination which can occur either
from the substrate-aldimine complex (II), or from the alanine-aldimine complex (III). The
comparison between the rate of transamination of L-cysteine sulfinic acid with that of
alanine, has provided evidence that the formation of PMP is due to the direct transamination
formed by β-decarboxylation [28].
1500-fold lower than that of cystalysin [29]. Notably, E.coli aspartate aminotransferase
belongs to the aminotransferases subgroup Ia which includes enzymes that undergo a large
conformational change from an open to a closed form upon substrate binding [30]. It has
been reported that enzymic forms with enhanced β-desulfinase and β-decarboxylase activities
result from mutations that prevent the transition to the closed conformation. Cystalysin
belongs to the aminotransferases subgroup Ib, which are unable to undergo that
conformational change. Indeed, the complex of cystalysin with the inhibitor
aminoethoxyvinylglycine [14] does not reveal any large conformational change with respect
to the enzyme in the internal aldimine form. Thus, it has been suggested that the absence of a
ligand-induced closure of the active site in cystalysin could be at least partially responsible
for the high catalytic versatility of the enzyme by favoring the possibility of side-reactions to
It is noteworthy that among PLP-dependent enzymes able to perform β-displacement
reactions, also some Cβ-Sγ lyases, such as NifS and NifS-like proteins, catalyze the
lyase able to perform both a desulfhydrase and a desulfinase reaction.
An additional and even more unexpected result is the finding that cystalysin in the PMP
form catalyzes the β-decarboxylation of oxalacetate. In fact, when the apoenzyme in the
presence of PMP was allowed to react with oxalacetate, a gradual conversion of PMP into
PLP was observed. These data were indicative of a reverse half-transamination reaction,
which would convert oxalacetate into aspartate and PMP into PLP. However, no formation of
aspartate was found in the reaction mixture. Unexpectedly, the reaction of the PMP form of
cystalysin with oxalacetate leads to the production of pyruvate, thus indicating that
oxalacetate undergoes a PMP-dependent β-decarboxylation. The kinetic parameters of this
reaction are reported in Table 3. Thus, the PMP to PLP conversion observed during the
116 Barbara Cellini, Riccardo Montioli and Carla Borri Voltattorni
reaction of cystalysin with oxalacetate is due to the reverse transamination of pyruvate
generated by β-decarboxylation, rather than to the direct half-transamination of oxalacetate.
From a mechanicistic point of view, it can be postulated that the binding of oxalacetate to
the active site of cystalysin in the PMP form generates a ketimine intermediate which is
potentially susceptible to β-decarboxylation because the imine bond is in β-position with
respect to the carboxylate group. The decarboxylation step leads to the formation of the
pyruvate ketimine intermediate which can either be hydrolyzed to pyruvate and PMP, or
undergo a half-transamination reaction with the production of alanine and PLP (Figure 12).
ketimine intermediate pyruvate ketimine
Figure 12. Proposed mechanism for the β-decaboxylation of oxalacetate catalyzed by cystalysin.
The quite large turnover number which characterizes the β-decarboxylase activity of the
PMP-form of cystalysin, has lead to propose a possible physiological role of this reaction for
Treponema denticola. A recent study [31] indicates that glutathione metabolism plays a role
in nutrition and potential virulence expression of Treponema denticola. Indeed, it has been
found that pyruvate, one of the end products of glutathione metabolism, promotes bacterial
growth. In addition, it should be taken into account that some anaerobic bacteria are able to
grow using the decarboxylation of saturated dicarboxylic acids as the only source of energy
Cystalysin: An Example of the Catalytic Versatility… 117
[32]. Several biochemical studies on fermenting bacteria suggest that two different
mechanisms exist for the synthesis of adenosine triphosphate (ATP). In one case, the
decarboxylation energy is directly converted into a Na+ ions electrochemical gradient across
the plasma membrane; in a second case, an electrochemical gradient is generated by the
association between an electrogenic dicarboxylate/monocarboxylate antiporter and a soluble
decarboxylase. Notably, all the soluble decarboxylases identified so far require thiamine
The study of the reaction specificity of cystalysin have highlighted the high catalytic
versatility of this enzyme. This makes cystalysin a useful model of the wide catalytic
potential of PLP-dependent proteins and a suitable model to understand the relationship
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