Evolutionary History of a Specialized P450 Propane Monooxygenase

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• 作者：Rudi Fasan ; Yergalem T. Meharenna ; Christopher D. Snow ; Thomas L. Poulos ; Frances H. Arnold
• 关键词：alkane oxidation ; protein evolution ; directed evolution ; propane monooxygenase ; cytochrome P450 BM3
• 刊名：Journal of Molecular Biology
• 出版年：2008
• 出版时间：28 November 2008
• 年：2008
• 卷：383
• 期：5
• 页码：1069-1080
• 全文大小：1288 K

文摘

The evolutionary pressures that shaped the specificity and catalytic efficiency of enzymes can only be speculated. While directed evolution experiments show that new functions can be acquired under positive selection with few mutations, the role of negative selection in eliminating undesired activities and achieving high specificity remains unclear. Here we examine intermediates along the ‘lineage’ from a naturally occurring C₁₂–C₂₀ fatty acid hydroxylase (P450_BM3) to a laboratory-evolved P450 propane monooxygenase (P450_PMO) having 20 heme domain substitutions compared to P450_BM3. Biochemical, crystallographic, and computational analyses show that a minimal perturbation of the P450_BM3 fold and substrate-binding pocket accompanies a significant broadening of enzyme substrate range and the emergence of propane activity. In contrast, refinement of the enzyme catalytic efficiency for propane oxidation ( 9000-fold increase in k_cat/K_m) involves profound reshaping and partitioning of the substrate access pathway. Remodeling of the substrate-recognition mechanisms ultimately results in remarkable narrowing of the substrate profile around propane and enables the acquisition of a basal iodomethane dehalogenase activity as yet unknown in natural alkane monooxygenases. A highly destabilizing L188P substitution in a region of the enzyme that undergoes a large conformational change during catalysis plays an important role in adaptation to the gaseous alkane. This work demonstrates that positive selection alone is sufficient to completely respecialize the cytochrome P450 for function on a nonnative substrate.