Limonene Hydroxylases as a Model for Cytochrome P450 Catalysis

Washington State University, Pullman WA

Investigators

Abstract

Terpenoids constitute the largest family of natural products and are extremely diverse in structure and biological function. Most terpenoids are formed by a common core pathway in which an initial cyclization reaction defines the basic structural type and subsequent cytochrome P450-mediated hydroxylation reactions determine the oxygenation pattern(s) of the derived family of metabolites. The oxygenation reactions play primary roles in the diversification to terpenoid end products and it is the oxygenated terpenoids, rather than their hydrocarbon precursors, that most often possess the relevant biological properties. The P450 heme-thiolate enzymes represent a very large class of catalysts important in both biosynthetic and catabolic processes, as well as in detoxification reactions. Eukaryotic forms of these enzymes have proven difficult to characterize, and the physical definition of active site structure has been precluded by a size too large for NMR-based approaches and by the recalcitrance of the solubilized membranous enzymes to yield suitable crystals for X-ray diffraction. A uniquely informative model system, that exploits a set of regio and stereospecific monoterpene hydroxylases from very closely related mint species, has been developed under the prior NSF grant, and these recombinant enzymes provide the foundation for the next phase of the research which involves synthetic, biochemical, molecular, modeling and spectrometric tools to address fundamental questions concerning active site structure-function relationships of this important enzyme class. The objectives of this project are: 1. to develop a functional overexpression system for the wild-type and mutant hydroxylases; 2. to optimize the preparation of the recombinant hydroxylases for spectrometric evaluation; 3. to determine substrate binding geometry at the active sites of the different hydroxylases using spatially defined fluorinated and deuterated substrate analogs and electron-nuclear double resonance (ENDOR) spectrometry; and 4. to employ model-guided mutagenesis, coupled with the use of kinetic probes, to determine the minimum change in active site structure required to alter hydroxylation regio and stereochemistry. Completion of these experimental objectives will test the hypothesis that subtle differences in active site topography underlie alterations in the regiochemistry and stereochemistry of production formation, and will provide a detailed level of structural understanding that has not been possible with other types of P450 enzymes. Additionally, the ability to exploit and redesign these important biosynthetic enzymes will permit their use in 'green' chemistry for difficult synthetic transformations and will provide an unprecedented opportunity to engineer terpene metabolic pathways in plants. The latter has broad implications for the manipulation of economic factors in plant performance, ranging from the generation of novel products, such as pharmaceuticals, agrochemicals and synthetic intermediates, to disease, insect and herbicide resistance.