Cellini Barbara, Bertoldi Mariarita, Montioli Riccardo, Laurents Douglas V, Paiardini Alessandro, Voltattorni Carla Borri
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.
Biochemistry. 2006 Nov 28;45(47):14140-54. doi: 10.1021/bi061496l.
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.
溶胞素是导致牙周炎的齿垢密螺旋体中的关键毒力因子，是一种同二聚体的磷酸吡哆醛（PLP）-C-S裂解酶。将全酶形式的溶胞素的二聚化过程和尿素诱导的去折叠平衡与脱辅基形式的进行了比较。PLP的存在使单体-二聚体平衡解离常数降低了约4倍。通过使用各种光谱和分析方法，我们证明了它们的去折叠谱存在差异。脱辅基溶胞素在1至2 M尿素之间发生单体化时，在2.5至4 M尿素范围内，具有非常高β-折叠含量的自缔合平衡中间体得以稳定，在更高尿素浓度下产生完全去折叠的单体。另一方面，PLP释放和单体化需要高度不稳定的条件，并伴有大量不溶性聚集体的形成。复性研究以及对溶胞素解离/缔合过程的分析，揭示了复性条件下蛋白质浓度以及PLP的存在与否如何影响活性二聚体酶的恢复和不溶性聚集体的产生。当蛋白质完全变性时，全酶形式和脱辅基溶胞素的最佳再活化产率分别约为50%和25%。已将溶胞素的二聚化和折叠过程与另一种PLP C-S裂解酶、大肠杆菌的MalY的进行了比较，并讨论了它们的PLP结合模式在这些过程中的可能相关性。
