固定黄素:劫持黄素转移酶以配备共价黄素辅因子
Yapei Tong1, Saniye G Kaya1, Sara Russo1
1Molecular Enzymology Group, University of Groningen, Nijenborgh 4, 9747 AG Groningen, The Netherlands.
Journal of the American Chemical Society
|December 4, 2023
概括
研究人员开发了一种方法,通过将黄素辅因子与蛋白质结合而制造共价黄蛋白. 这种工程方法提高了蛋白质的稳定性和功能,防止了辅助因子的损失.
科学领域:
- 生物化学
- 蛋白质工程
- 酵素学
背景情况:
- 黄依赖酶通常使用可分离的黄辅因子.
- 配因子解离可能会限制酶的稳定性和性能.
- 开发稳定的辅因子附着方法对于生物技术应用至关重要.
研究的目的:
- 在flavin辅因子和宿主蛋白之间设计具有共价键的黄蛋白.
- 为了证明从最初可分离的蛋白质中制造稳定,共价的黄蛋白的可行性.
- 评估共价化对蛋白质功能,稳定性和性能的影响.
主要方法:
- 改造的黄化基因变成了目标黄蛋白.
- 在体内共价化中使用了转移酶.
- 表达和纯化的工程蛋白质.
- 通过结构 (晶体) 和功能测试来表征共价蛋白.
主要成果:
- 成功实现了四种不同类型的黄蛋白 (LOV,mini-SO,NRE,OYE).
- 为高效的共价结合,优化了烯酸基的基因和表达条件.
- 工程共价蛋白保持或改善功能,热稳定性和催化性能.
- 结晶结构证实了共价三酸连接.
结论:
- 建立了一种创新的多功能方法来制造共价黄蛋白.
- 这种方法克服了辅因子解离问题,增强了蛋白质的强度.
- 改造的共价黄蛋白表现出更好的稳定性和性能,扩大了它们的潜在应用.
更多相关视频
12:08Monitoring the Reductive and Oxidative Half-Reactions of a Flavin-Dependent Monooxygenase using Stopped-Flow Spectrophotometry
Published on: March 18, 2012
15.2K
10:50Using In Vitro Fluorescence Resonance Energy Transfer to Study the Dynamics Of Protein Complexes at a Millisecond Time Scale
Published on: March 14, 2019
8.2K
相关概念视频
Role of Reduced Coenzymes NADH and FADH₂
11.7K
The energy released from the breakdown of the chemical bonds within nutrients can be stored either through the reduction of electron carriers or in the bonds of adenosine triphosphate (ATP). In living systems, a small class of compounds functions as mobile electron carriers, molecules that bind to and shuttle high-energy electrons between compounds in pathways. The principal electron carriers that will be considered originate from the B vitamin group and are derivatives of nucleotides; they are...
11.7K
Electron Transport Chain: Complex I and II
14.0K
The mitochondrial electron transport chain (ETC) is the main energy generation system in the eukaryotic cells. However, mitochondria also produce cytotoxic reactive oxygen species (ROS) due to the large electron flow during oxidative phosphorylation. While Complex I is one of the primary sources of superoxide radicals, ROS production by Complex II is uncommon and may only be observed in cancer cells with mutated complexes.
ROS generation is regulated and maintained at moderate levels necessary...
ROS generation is regulated and maintained at moderate levels necessary...
14.0K
Electron Transport Chains
98.8K
The final stage of cellular respiration is oxidative phosphorylation that consists of two steps: the electron transport chain and chemiosmosis. The electron transport chain is a set of proteins found in the inner mitochondrial membrane in eukaryotic cells. Its primary function is to establish a proton gradient that can be used during chemiosmosis to produce ATP and generate electron carriers, such as NAD+ and FAD, that are used in glycolysis and the citric acid cycle.
The ETC is comprised of...
The ETC is comprised of...
98.8K
Cofactors and Coenzymes
81.6K
Enzymes require additional components for proper function. There are two such classes of molecules: cofactors and coenzymes. Cofactors are metallic ions and coenzymes are non-protein organic molecules. Both of these types of helper molecule can be tightly bound to the enzyme or bound only when the substrate binds.
81.6K
Electron Transport Chain: Complex III and IV
7.5K
During the electron transport chain, electrons from NADH and FADH2 are first transferred to complexes I and II, respectively. These two complexes then transfer the electrons to ubiquinol, which carries them further to complex III. Complex III passes the electrons across the intermembrane space to Cyt c, which carries them further to complex IV. Complex IV donates electrons to oxygen and reduces it to water. As electrons pass through complexes I, III, and IV, the energy released aids the pumping...
7.5K
ATP Synthase: Mechanism
14.7K
In animals, the mitochondrial F1F0 ATP synthase is the key protein that synthesizes ATP molecules through a complex catalytic mechanism. While the nuclear genome encodes the majority of ATP synthase subunits, the mitochondrial genome encodes some of the enzyme's most critical components. The formation of this multi-subunit enzyme is a complex multi-step process regulated at the level of transcription, translation, and assembly. Defects in one or more of these steps can result in decreased...
14.7K
