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Published November 28, 2008 | Accepted Version + Supplemental Material
Journal Article Open

Evolutionary History of a Specialized P450 Propane Monooxygenase

Abstract

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 (P450BM3) to a laboratory-evolved P450 propane monooxygenase (P450PMO) having 20 heme domain substitutions compared to P450BM3. Biochemical, crystallographic, and computational analyses show that a minimal perturbation of the P450BM3 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 (not, vert, similar 9000-fold increase in kcat/Km) 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.

Additional Information

© 2008 Elsevier. Received 22 April 2008; revised 13 June 2008; accepted 19 June 2008. Available online 28 June 2008. We are grateful to Huiying Li for assistance in collecting diffraction data. This work was supported by a Swiss National Science Foundation postdoctoral fellowship to R.F., a Jane Coffin Childs postdoctoral fellowship to C.S.F., NIH Grant GM32688 to T.L.P., and DOE grant (DE-FG02-06ER15762) and U.S. Army Research Office grant (AROICB DAAD19-03-D-0004) to F.H.A. Graphics were prepared using PyMOL Molecular Graphics System. Coordinates and structure factors have been deposited in the Protein Data Bank with accession number 3CBD. Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.jmb.2008.06.060

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Accepted Version - nihms77103.pdf

Supplemental Material - FASjmb08supp.doc

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