Institut de Biotecnologia i de Biomedicina and Departamento de Bioquimica i Biologia Molecular, Universitat Autonoma de Barcelona, 08193 Bellaterra, Spain; Instituto de Bioquímica Médica Leopoldo de Meis, Programa de Biologia Estrutural, Universidade Federal do Rio de Janeiro, 21941-902 Rio de Janeiro, RJ, Brazil.
Instituto de Química, Programa de Pós-Graduação em Bioquímica, Universidade Federal do Rio de Janeiro, 21941-909 Rio de Janeiro, Brazil.
Structure. 2018 Feb 6;26(2):199-208.e3. doi: 10.1016/j.str.2017.12.004. Epub 2018 Jan 4.
Lipases and esterases constitute a group of enzymes that catalyze the hydrolysis or synthesis of ester bonds. A major biotechnological interest corresponds to thermophilic esterases, due to their intrinsic stability at high temperatures. The Pf2001 esterase from Pyrococcus furiosus reaches its optimal activity between 70°C and 80°C. The crystal structure of the Pf2001 esterase shows two different conformations: monomer and dimer. The structures reveal important rearrangements in the "cap" subdomain between monomer and dimer, by the formation of an extensive intertwined helical interface. Moreover, the dimer interface is essential for the formation of the hydrophobic channel for substrate selectivity, as confirmed by mutagenesis and kinetic analysis. We also provide evidence for dimer formation at high temperatures, a process that correlates with its enzymatic activation. Thus, we propose a temperature-dependent activation mechanism of the Pf2001 esterase via dimerization that is necessary for the substrate channel formation in the active-site cleft.
脂肪酶和酯酶构成了一组能够催化酯键水解或合成的酶。嗜热酯酶由于其在高温下的固有稳定性，具有重要的生物技术应用价值。来自 Pyrococcus furiosus 的 Pf2001 酯酶在 70°C 到 80°C 之间达到最佳活性。Pf2001 酯酶的晶体结构显示出两种不同的构象：单体和二聚体。结构揭示了单体和二聚体之间“帽”亚结构域的重要重排，形成了广泛的交错螺旋界面。此外，二聚体界面对于底物选择性的疏水通道的形成是必不可少的，这一点通过突变和动力学分析得到了证实。我们还提供了在高温下形成二聚体的证据，这与酶的激活过程相关。因此，我们提出了 Pf2001 酯酶通过二聚化的温度依赖性激活机制，这对于活性位点裂缝中底物通道的形成是必要的。
