Department of Biological Sciences, Columbia University, New York, NY 10027, USA.
Biochemistry. 2013 Jan 22;52(3):488-96. doi: 10.1021/bi301294d. Epub 2013 Jan 9.
Biotin carboxylase (BC) is a conserved component among biotin-dependent carboxylases and catalyzes the MgATP-dependent carboxylation of biotin, using bicarbonate as the CO₂ donor. Studies with Escherichia coli BC have suggested long-range communication between the two active sites of a dimer, although its mechanism is not well understood. In addition, mutations in the dimer interface can produce stable monomers that are still catalytically active. A homologous dimer for the BC domain is observed in the structure of the tetrameric pyruvate carboxylase (PC) holoenzyme. We have introduced site-specific mutations into the BC domain dimer interface of Staphylococcus aureus PC (SaPC), equivalent to those used for E. coli BC, and also made chimeras replacing the SaPC BC domain with the E. coli BC subunit (EcBC chimera) or the yeast ACC BC domain (ScBC chimera). We assessed the catalytic activities of these mutants and characterized their oligomerization states by gel filtration and analytical ultracentrifugation experiments. The K442E mutant and the ScBC chimera disrupted the BC dimer and were catalytically inactive, while the F403A mutant and the EcBC chimera were still tetrameric and retained catalytic activity. The R54E mutant was also tetrameric but was catalytically inactive. Crystal structures of the R54E, F403A, and K442E mutants showed that they were tetrameric in the crystal, with conformational changes near the mutation site as well as in the tetramer organization. We have also produced the isolated BC domain of SaPC. In contrast to E. coli BC, the SaPC BC domain is monomeric in solution and catalytically inactive.
生物素羧化酶（BC）是生物素依赖性羧化酶中的保守成分，催化生物素的 MgATP 依赖性羧化，使用碳酸氢盐作为 CO₂供体。对大肠杆菌 BC 的研究表明，二聚体的两个活性位点之间存在长程通讯，尽管其机制尚不清楚。此外，二聚体界面的突变可以产生仍然具有催化活性的稳定单体。在四聚体丙酮酸羧化酶（PC）全酶的结构中观察到与 BC 结构域同源的二聚体。我们已经在金黄色葡萄球菌 PC（SaPC）的 BC 结构域二聚体界面中引入了定点突变，相当于大肠杆菌 BC 中使用的突变，并且还构建了用大肠杆菌 BC 亚基（EcBC 嵌合体）或酵母 ACC BC 结构域（ScBC 嵌合体）替换 SaPC BC 结构域的嵌合体。我们评估了这些突变体的催化活性，并通过凝胶过滤和分析超速离心实验表征了它们的寡聚状态。K442E 突变体和 ScBC 嵌合体破坏了 BC 二聚体，并且没有催化活性，而 F403A 突变体和 EcBC 嵌合体仍然是四聚体并且保留催化活性。R54E 突变体也是四聚体，但没有催化活性。R54E、F403A 和 K442E 突变体的晶体结构表明，它们在晶体中是四聚体，突变部位附近以及四聚体组织发生构象变化。我们还生产了 SaPC 的分离 BC 结构域。与大肠杆菌 BC 不同，SaPC BC 结构域在溶液中是单体，没有催化活性。
