Hoeven Robin, Hardman Samantha J O, Heyes Derren J, Scrutton Nigel S
Centre for Synthetic Biology of Fine and Speciality Chemicals, Manchester Institute of Biotechnology, The University of Manchester , 131 Princess Street, Manchester M1 7DN, U.K.
Biochemistry. 2016 Feb 16;55(6):903-13. doi: 10.1021/acs.biochem.5b01355. Epub 2016 Feb 3.
Experimental interrogation of the relationship between protein dynamics and enzyme catalysis is challenging. Light-activated protochlorophyllide oxidoreductase (POR) is an excellent model for investigating this relationship because photoinitiation of the reaction cycle enables coordinated turnover in a "dark-assembled" ternary enzyme-substrate complex. The catalytic cycle involves sequential hydride and proton transfers (from NADPH and an active site tyrosine residue, respectively) to the substrate protochlorophyllide. Studies with a limited cross-species subset of POR enzymes (n = 4) have suggested that protein dynamics associated with hydride and proton transfer are distinct [Heyes, D. J., Levy, C., Sakuma, M., Robertson, D. L., and Scrutton, N. S. (2011) J. Biol. Chem. 286, 11849-11854]. Here, we use steady-state assays and single-turnover laser flash spectroscopy to analyze hydride and proton transfer dynamics in an extended series of POR enzymes taken from many species, including cyanobacteria, algae, embryophytes, and angiosperms. Hydride/proton transfer in all eukaryotic PORs is faster compared to prokaryotic PORs, suggesting active site architecture has been optimized in eukaryotic PORs following endosymbiosis. Visible pump-probe spectroscopy was also used to demonstrate a common photoexcitation mechanism for representative POR enzymes from different branches of the phylogenetic tree. Dynamics associated with hydride transfer are localized to the active site of all POR enzymes and are conserved. However, dynamics associated with proton transfer are variable. Protein dynamics associated with proton transfer are also coupled to solvent dynamics in cyanobacterial PORs, and these networks are likely required to optimize (shorten) the donor-acceptor distance for proton transfer. These extended networks are absent in algal and plant PORs. Our analysis suggests that extended networks of dynamics are disfavored, possibly through natural selection. Implications for the evolution of POR and more generally for other enzyme catalysts are discussed.
对蛋白质动力学与酶催化之间关系进行实验性探究具有挑战性。光激活原叶绿素酸氧化还原酶（POR）是研究这种关系的一个极佳模型，因为反应循环的光引发能够在“暗组装”的三元酶 - 底物复合物中实现协同周转。催化循环涉及氢化物和质子依次转移（分别来自NADPH和一个活性位点酪氨酸残基）至底物原叶绿素酸。对POR酶的一个有限跨物种子集（n = 4）的研究表明，与氢化物和质子转移相关的蛋白质动力学是不同的[海耶斯，D. J.，利维，C.，佐久间，M.，罗伯逊，D. L.，以及斯克鲁顿，N. S.（2011年）《生物化学杂志》286卷，11849 - 11854页]。在此，我们使用稳态测定法和单周转激光闪光光谱法来分析一系列来自许多物种（包括蓝细菌、藻类、胚植物和被子植物）的POR酶中的氢化物和质子转移动力学。与原核生物的POR相比，所有真核生物的POR中的氢化物/质子转移更快，这表明在共生之后真核生物的POR中活性位点结构已得到优化。可见泵浦 - 探测光谱法也被用于证明来自系统发育树不同分支的代表性POR酶具有共同的光激发机制。与氢化物转移相关的动力学局限于所有POR酶的活性位点且是保守的。然而，与质子转移相关的动力学是可变的。与质子转移相关的蛋白质动力学在蓝细菌的POR中也与溶剂动力学相耦合，并且这些网络可能是优化（缩短）质子转移供体 - 受体距离所必需的。这些扩展网络在藻类和植物的POR中不存在。我们的分析表明扩展的动力学网络可能通过自然选择而不受青睐。文中讨论了对POR进化以及更广泛地对其他酶催化剂进化的影响。
