Shinichi, T.; Padmakumar, R.; Lai M., Liu H.; Banerjee, R. (1994). Inhibition of the human

methylmalonyl-CoA mutase by various CoA-esters. J. Biol. Chem., 269, 31630-31634.

98 Thore Rohwerder and Roland H. Müller

Solana-Serena, F.; Marchal, R.; Heiss, S.; Vandecasteele, J.-P. (2004). Degradation of

isooctane by Mycobacterium austroafricanum IFP 2173: growth and catabolic pathway.

J. Appl. Microbiol., 97, 629-639.

Steffan, R. J.; McClay, K.; Vainberg, S.; Condee, C. W.; Zhang, D. (1997). Biodegradation

of the gasoline oxygenates methyl tert-butyl ether, ethyl tert-butyl ether, and tert-amyl

methyl ether by propane-oxidizing bacteria. Appl. Environ. Microbiol., 63, 4216-4222.

Takada, A. & Sudoh, M. (2003). Solution of N-[O-(P-pivaloyloxybenzenesulfonylamino)

benzoyl]glycine monosodium salt tetrahydrate and drug product thereof. United States

Patent, No. US 6,552,082 B2.

Textor, S.; Wendisch, V. F.; De Graaf, A. A.; Müller, U.; Linder, M. I.; Linder, D.; Buckel,

W. (1997). Propionate oxidation in Escherichia coli: evidence for operation of a

methylcitrate cycle in bacteria. Arch. Microbiol., 168, 428–436.

Tholozan, J.-L.; Samain, E.; Grivet, J.-P. (1988). Isomerization between n-butyrate and

isobutyrate in enrichment cultures. FEMS Micobiol. Lett., 53, 187-191.

Trevor, C. C. & Punita, A. (1999). Methylmalonyl-CoA mutase encoding gene of

Sinorhizobium meliloti. Gene, 226, 121–127.

Wilkes, H.; Kühner, S.; Bolm, C.; Fischer, T.; Classen, A.; Widdel, F.; Rabus, R. (2003).

Formation of n-alkane- and cycloalkane-derived organic acids during anaerobic growth

of a denitrifying bacterium with crude oil. Org. Geochem., 34, 1313-1323.

Wilkes, H.; Rabus, R.; Fischer, T.; Armstroff, A.; Behrends, A.; Widdel, F. (2002).

Anaerobic degradation of n-hexane in a denitrifying bacterium: further degradation of the

initial intermediate (1-methylpentyl)succinate via C-skeleton rearrangement. Arch.

Microbiol., 177, 235-243.

Willard, H. F. & Rosenberg, L. E. (1980). Inherited methylmalonyl-CoA mutase apoenzyme

deficiency in human fibroblasts. J. Clin. Invest., 65, 690-698.

Wu, W.-M.; Jain, M. K.; Zeikus, J. G. (1994). Anaerobic degradation of normal and

branched-chain fatty acids with four or more carbons to methane by a syntrophic

methanogenic triculture. Appl. Environ. Microbiol., 60, 2220-2226.

Zerbe-Burkhardt, K.; Ratnatilleke, A.; Philippon, N.; Birch, A.; Leiser, A.; Vrijbloed, J. W.;

Hess, D.; Hunziker, P.; Robinson, J. A. (1998). Cloning, sequencing, expression, and

insertional inactivation of the gene for the large subunit of the coenzyme B12-dependent

isobutyryl-CoA mutase from Streptomyces cinnamonensis. J. Biol. Chem., 273, 6508-

6517.

In: Vitamin B: New Research ISBN 978-1-60021-782-1

Editor: Charlyn M. Elliot, pp. 99-119 © 2008 Nova Science Publishers, Inc.

Chapter VI

CYSTALYSIN: AN EXAMPLE OF THE CATALYTIC

VERSATILITY OF PYRIDOXAL 5’-PHOSPHATE

DEPENDENT ENZYMES

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,

37134 Verona, Italy.

ABSTRACT

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

are able to catalyze (transamination, decarboxylation, racemization, β- or γreplacement/elimination). In the absence of the apoenzyme, different reactions would

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

active site.

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

∗

 Correspondence concerning this article should be addressed to: Barbara Cellini, Dipartimento di Scienze

Morfologico-Biomediche, Sezione di Chimica Biologica, Facoltà di Medicina e Chirurgia, Università degli Studi

di Verona, Strada Le Grazie, 8, 37134 Verona, Italy. Tel.: +39-045-8027-293; Fax: +39-045-8027-170; E-mail:

barbara.cellini@univr.it.

100 Barbara Cellini, Riccardo Montioli and Carla Borri Voltattorni

enantiomers of alanine, the β-desulfination of L-cysteine sulfinic acid, and the βdecarboxylation of L-aspartate and oxalacetate with turnover numbers measured in

seconds, and the transamination of L- and D-alanine with turnover numbers measured in

minutes.

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

reactions.

INTRODUCTION

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

wide variety of reactions, including transamination, racemization, decarboxylation, β- or γreplacement/elimination, and provide a unique model to understand the mechanisms by

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.

This allows the resulting carbanion to be stabilized by conjugation with the extended πsystem of PLP. The topology of the external aldimine is one of the major determinants of

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.

Internal

aldimine

External

aldimine

Quinonoid

Quinonoid Quinonoid

Quinonoid

Quinonoid β,γ−unsatured

aldimine

α,β−unsatured

aldimine

H+

 from Cα

R from Cα CO2 from Cα

H+ H to C4’ +

 to Cα

X from Cγ

H+

 from Cβ

H+

 to Cγ

X from Cβ

R to Cβ

H+

 to Cα

H+ H to C4’ +

 to Cα

R to Cα

H+

 to Cα

H+

 to Cα

R to Cγ

β−Eliminases

Racemases

α−Synthases

Serine

hydroxymethyl

transferase

Transaminating

decarboxylases

Decarboxylases

β−Synthases

Aminotransferases

γ−Synthases

Figure 1. Schematic representation of the catalytic versatility of pyridoxal 5’-phosphate (PLP)

dependent enzymes. Each reaction begins with conversion of the internal to external aldimine. Covalent

modifications occurring at successive steps are indicated on the arrows connecting the intermediates.

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

[12].

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

molecules [14].