Dimerization and folding processes of Treponema denticola cystalysin: the role of pyridoxal 5'-phosphate.
Cystalysin, the key virulence factor in the bacterium Treponema denticola responsible for periodontitis, is a homodimeric pyridoxal 5'-phosphate (PLP)-C-S lyase. The dimerization process and the urea-induced unfolding equilibrium of holocystalysin were compared with those of the apo form. The presence of PLP decreases approximately 4 times the monomer-dimer equilibrium dissociation constant. By using a variety of spectroscopic and analytical procedures, we demonstrated a difference in their unfolding profiles. Upon the monomerization of apocystalysin, occurring between 1 and 2 M urea, a self-associated equilibrium intermediate with a very high beta-sheet content is stabilized over the 2.5-4 M urea range, giving rise to a fully unfolded monomer at higher urea concentrations. On the other hand, highly destabilizing conditions, accompanied by the formation of a significant amount of insoluble aggregates, are required for PLP release and monomerization. Refolding studies, together with analysis of the dissociation/association process of cystalysin, shed light on how the protein concentration and the presence or absence of PLP under refolding conditions could affect the recovery of the active dimeric enzyme and the production of insoluble aggregates. When the protein is completely denatured, the best reactivation yield found was approximately 50% and 25% for holo and apocystalysin, respectively. The dimerization and folding processes of cystalysin have been compared with those of another PLP C-S lyase, MalY from E. coli, and the possible relevance of their PLP binding mode in these processes has been discussed.
Cellini
Barbara
B
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.
Bertoldi
Mariarita
M
Montioli
Riccardo
R
Laurents
Douglas V
DV
Paiardini
Alessandro
A
Voltattorni
Carla Borri
CB
D003542:Cystathionine gamma-Lyase / Q000737:chemistry / Q000378:metabolism*; D019281:Dimerization; D004591:Electrophoresis, Polyacrylamide Gel; D008958:Models, Molecular; D019906:Nuclear Magnetic Resonance, Biomolecular; D011487:Protein Conformation; D011489:Protein Denaturation; D011732:Pyridoxal Phosphate / Q000378:metabolism*; D048373:Treponema denticola / Q000201:enzymology*
D011732:Pyridoxal Phosphate; C400145:cystalysin; D003542:Cystathionine gamma-Lyase