Dimerization and Folding Processes of Treponema denticola Cystalysin: The Role of Pyridoxal 5'-Phosphate
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- 作者：Barbara Cellini ; Mariarita Bertoldi ; Riccardo Montioli ; Douglas V. Laurents ; Alessandro Paiardini ; Carla Borri Voltattorni
- 刊名：Biochemistry
- 出版年：2006
- 出版时间：November 28, 2006
- 年：2006
- 卷：45
- 期：47
- 页码：14140 - 14154
- 全文大小：546K
- 年卷期：v.45,no.47(November 28, 2006)
- ISSN：1520-4995
文摘
Cystalysin, the key virulence factor in the bacterium Treponema denticola responsible forperiodontitis, is a homodimeric pyridoxal 5'-phosphate (PLP)-C-S lyase. The dimerization process andthe urea-induced unfolding equilibrium of holocystalysin were compared with those of the apo form. Thepresence of PLP decreases ~4 times the monomer-dimer equilibrium dissociation constant. By using avariety 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 equilibriumintermediate with a very high 
-sheet content is stabilized over the 2.5-4 M urea range, giving rise to afully 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 releaseand monomerization. Refolding studies, together with analysis of the dissociation/association process ofcystalysin, shed light on how the protein concentration and the presence or absence of PLP under refoldingconditions 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 ~50% and 25% for holoand apocystalysin, respectively. The dimerization and folding processes of cystalysin have been comparedwith those of another PLP C-S lyase, MalY from E. coli, and the possible relevance of their PLP bindingmode in these processes has been discussed.
-sheet content is stabilized over the 2.5-4 M urea range, giving rise to afully 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 releaseand monomerization. Refolding studies, together with analysis of the dissociation/association process ofcystalysin, shed light on how the protein concentration and the presence or absence of PLP under refoldingconditions 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 ~50% and 25% for holoand apocystalysin, respectively. The dimerization and folding processes of cystalysin have been comparedwith those of another PLP C-S lyase, MalY from E. coli, and the possible relevance of their PLP bindingmode in these processes has been discussed.