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In: Vitamin B: New Research ISBN 978-1-60021-782-1
Editor: Charlyn M. Elliot, pp. 99-119 © 2008 Nova Science Publishers, Inc.
CYSTALYSIN: AN EXAMPLE OF THE CATALYTIC
VERSATILITY OF PYRIDOXAL 5’-PHOSPHATE
Barbara Cellini∗ , Riccardo Montioli and Carla Borri Voltattorni
Dipartimento di Scienze Morfologico-Biomediche, Sezione di Chimica Biologica,
Facoltà di Medicina e Chirurgia, Università degli Studi di Verona, Strada Le Grazie, 8,
Pyridoxal 5’-phosphate (PLP) is the catalitically active form of the water-soluble
vitamin B6, and hence the cofactor of a number of enzymes essential to the human body.
PLP-dependent enzymes are unique for the variety of reactions on amino acids that they
occur simultaneously, but the protein moiety drives the catalytic power of the coenzyme
toward a specific reaction. However, this specificity is not absolute; most PLP-enzymes
catalyze indeed side-reactions which can have physiological significance and provide
interesting mechanistic and stereochemical information about the structure of the enzyme
Cystalysin is a PLP-dependent Cβ-Sγ lyase present in Treponema denticola, and its
main reaction is the α,β-elimination of L-cysteine to produce pyruvate, ammonia and
H2S. The latter is probably responsible for the hemolytic and hemoxidative activity
associated with the enzyme catalysis. Cystalysin is one of the most representative
examples of the high catalytic versatility of PLP-dependent enzymes. Recently, indeed, it
has been shown that cystalysin is also able to catalyze the racemization of both
100 Barbara Cellini, Riccardo Montioli and Carla Borri Voltattorni
seconds, and the transamination of L- and D-alanine with turnover numbers measured in
Extensive biochemical investigations have uncovered several interesting features of
cystalysin, including the binding mode of the cofactor, its substrate specificity, the
formation of reaction intermediates characteristic of most PLP-enzymes, and the
involvement of some active-site residues in the primary and secondary catalytic
Vitamin B6 is a water-soluble compound discovered about 70 years ago whose major
active chemical form is pyridoxal 5’-phosphate (PLP), that plays a vital role as a cofactor of a
large number of enzymes in all organisms [1]. Overall, the Enzyme Commission (EC;
http://www.chem.qmul.ac.uk/iubmb/enzyme/) has listed more than 140 PLP-dependent
enzymatic activities, corresponding to about 4% of all classified activities. Additionally,
several putative PLP-binding proteins have been identified in genome sequencing projects
[2]. PLP is considered to be one of the nature’s most versatile cofactors, and PLP-dependent
enzymes mediate different cellular processes mainly involving amino compounds and
ranging from the biosynthesis of amino acids and amino acids-derived metabolites, to the
biosynthesis of amino sugars and other amino-containing compounds [3]. They catalyze a
which enzymes control substrate and reaction specificity [4]. In all PLP-enzymes, the
cofactor is covalently bound to the apoprotein through a Schiff base linkage between the
aldehydic group of the coenzyme and the ε-amino group of an active site lysine residue
(internal aldimine). With the exception of phosphorilases, which utilize PLP in a different
way and will not be considered here, the first step is common to all PLP-catalyzed reactions
and consists in the displacement of the active site lysine by an incoming substrate amino
group to form the external aldimine [1]. From this point on, the catalytic pathways differ
among the enzymes according to their reaction specificity. In fact, in the next step of the
reaction, each one of the three bonds at Cα of the external aldimine may be broken resulting
in the formation of a quinonoid intermediate. This process is facilitated by the electron-sink
properties of the pyridine moiety of the coenzyme, which stabilizes the developing negative
charge. On the basis of the Dunathan’s hypothesis [5], advanced in 1966 and later confirmed
by the resolution of the aspartate aminotransferase/phosphopyridoxyl aspartate complex [6],
the bond to be cleaved is the one aligned perpendicularly to the pyridine ring of the cofactor.
reaction specificity in PLP-dependent enzymes; however, several other factors such as
hydrogen bonding interactions, torsion and orientation of the cofactor, appear to be important
[7]. The unique environment provided by the apoprotein of a PLP-dependent enzyme drives
the catalytic power of the coenzyme so that the required reaction is optimized, while all the
Cystalysin: An Example of the Catalytic Versatility… 101
other possibilities are almost completely prevented. However, due to the large number of
alternatives, “mistakes” may occur. As a consequence, most PLP-enzymes are able to
catalyze side reactions which have a limited efficiency, but sometimes assume a
physiological meaning [1]. A schematic representation of the different reactions catalyzed by
PLP-dependent enzymes is shown in Figure 1.
Figure 1. Schematic representation of the catalytic versatility of pyridoxal 5’-phosphate (PLP)
Cystalysin is a PLP-dependent lyase which catalyzes the α,β-elimination of L-cysteine to
pyruvate, ammonia and sulfidric acid. The protein is produced by T.denticola, an oral
pathogen found at elevated concentrations in the gingival crevice of patients affected by
adulte periodontitis. T. denticola produces a large number of virulence factors including
several proteolytic and cytotoxic enzymes, required for bacterial growth in the periodontal
pocket and disease progression [8]. Cystalysin was identified in 1994, when Holt and
coworkers, while studying the hemolytic and hemoxidative properties of T. denticola, found
that both activities were dependent on a 45 KDa cell-associated protein encoded by the hly
gene [9]. After cloning of the gene, it was possible to demonstrate that the hemolysin is a
cysteine Cβ-Sγ lyase homologous to PLP-dependent aminotransferases [10]. Cystalysin is
able to interact with human red blood cells causing spikes and protrusion in the erythrocyte
membrane, and leading to the formation of irregular holes. Furthermore, the protein causes
the oxidation and sulfuration of hemoglobin to methemoglobin and sulfhemoglobin,
respectively [11]. Various studies have suggested that cystalysin induces haemolysis by a
novel mechanism, possibly dependent on its catalytic activity which determines production of
H2S. This compound is toxic for most cells and, by lysing erythrocytes, it allows the delivery
of many nutrition factors, including various amino acids and the iron of the haem [12].
Moreover, T. denticola belongs to a limited number of oral pathogens able to produce and
102 Barbara Cellini, Riccardo Montioli and Carla Borri Voltattorni
tolerate high concentrations (mM) of H2S found in periodontal disease pockets [13]. This
ability gives selective advantages to the bacterium allowing the formation of an ecological
niche in the periodontal pocket. Thus, the major function of cystalysin seems to be the
production of H2S and the protein can be regarded as a true PLP-dependent virulence factor
The crystal structure of cystalysin and cystalysin-L-aminoethoxyvinylglycine complex,
solved in 2000 by Krupka and coworkers, reveals that the protein belongs to Fold Type I or
L-aspartate aminotransferase family of PLP-dependent enzymes [14] (Figure 2). The protein
is a homodimer with 399 amino acids per subunit. Each monomer folds into two domains: i) a
large domain, consisting of residues 48-288 and carrying the PLP cofactor covalently bound
to Lys 238; ii) a small domain, consisting of the two terminal regions of the polypeptide
chain. In the centre of each cystalysin monomer, PLP is bound in a wide catalytic cleft
formed by both domains of one subunit and parts of the large domain of the other subunit.
The cofactor is bound by different types of interactions including the Schiff base linkage with
Lys 238 and ring-stacking interactions of the pyridine ring with the phenol ring of Tyr 123.
In addition, PLP is strongly anchored to the apoprotein through its phosphate group, which
forms six hydrogen bonds with protein residues and two hydrogen bonds with two water
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