Department of Chemistry and Biotechnology, School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan.
Biotechnol Bioeng. 2013 Jul;110(7):1858-64. doi: 10.1002/bit.24861. Epub 2013 Feb 22.
In nature, many enzymes participating in multienzyme reactions are often assembled to enhance efficiencies of multiple reactions. Therefore, much attention has been focused on self-assembly of multiple enzymes fused with a protein/peptide that interacts with a specific protein to enhance artificial multienzyme reactions. Sulfolobus solfataricus proliferating cell nuclear antigen (PCNA) is a ring-shaped symmetric heterotrimer consisting of PCNA1, PCNA2 and PCNA3. Multiple enzymes can be co-localized on the PCNA ring by fusing them to the C-termini of the three PCNA subunits. However, an advantage of the specific non-covalent complex formation is inextricably associated with the disadvantage of its concentration-dependent dissociation. In this study, disulfide bonds were introduced between the PCNA subunits by Cys substitution at the sites neighboring the interface for heterotrimerization. Selective intersubunit disulfide bond formation between PCNA1 and PCNA3 and between PCNA2 and PCNA3 by a natural oxidizing reagent successfully stabilized an artificial multienzyme complex, which is composed of a bacterial cytochrome P450 and its two redox partner proteins. The covalent stabilization of the multienzyme complex enhanced its cytochrome P450 activity because of the absence of inactive dissociated components.
在自然界中，许多参与多酶反应的酶通常被组装在一起，以提高多个反应的效率。因此，人们非常关注将与特定蛋白质相互作用的蛋白质/肽融合的多个酶的自组装，以增强人工多酶反应。嗜热硫叶菌增殖细胞核抗原（PCNA）是一种由 PCNA1、PCNA2 和 PCNA3 组成的环形对称三聚体。通过将多个酶融合到三个 PCNA 亚基的 C 末端，可以将它们共定位在 PCNA 环上。然而，特定的非共价复合物形成的优势不可避免地与浓度依赖性解离的劣势相关联。在这项研究中，通过在异三聚体化界面附近的位置用 Cys 取代，在 PCNA 亚基之间引入二硫键。通过天然氧化试剂，PCNA1 和 PCNA3 之间以及 PCNA2 和 PCNA3 之间的选择性亚基间二硫键形成成功稳定了由细菌细胞色素 P450 及其两个氧化还原伴侣蛋白组成的人工多酶复合物。多酶复合物的共价稳定化由于不存在无活性的解离成分，增强了细胞色素 P450 的活性。
