Hydroxylation of specifically deuterated limonene enantiomers by cytochrome p450 limonene-6-hydroxylase reveals the mechanism of multiple product formation.
The regiochemistry and facial stereochemistry of the limonene-6-hydroxylase- (CYP71D18-) mediated hydroxylation of the monoterpene olefin limonene are determined by the absolute configuration of the substrate. (-)-(4S)-Limonene is hydroxylated at the C6 allylic position to give (-)-trans-carveol as the only product, whereas (+)-(4R)-limonene yields multiple hydroxylation products with (+)-cis-carveol predominating. Specifically deuterated limonene enantiomers were prepared to investigate the net stereospecificity of hydroxylation at C6 and the mechanism of multiple product formation. The results of isotopically sensitive branching experiments of competitive and noncompetitive design were consistent with a nondissociative kinetic mechanism, indicating that (4R)-limonene has sufficient freedom of motion within the active site of CYP71D18 to allow formation of either the trans-3- or cis-6-hydroxylated product. However, the kinetic isotope effects resulting from deuterium abstraction were significantly smaller than expected for an allylic hydroxylation, and they did not approach the intrinsic isotope effect. (4S)-Limonene is oxygenated with almost complete stereospecificity for hydrogen abstraction from the trans-6-position, demonstrating rigid orientation during hydrogen abstraction and hydroxyl delivery. The oxygenation of (4R)-limonene leading to the formation of (+/-)-trans-carveol is accompanied by considerable allylic rearrangement and stereochemical scrambling, whereas the formation of (+)-cis-carveol proceeds without allylic rearrangement and with nearly complete stereospecificity for hydrogen abstraction from the cis-6-position. These results demonstrate that a single cytochrome P450 enzyme catalyzes the hydroxylation of small antipodal substrates with distinct stereochemistries and reveal that substrate-dependent positional motion of the intermediate carbon radical (and, therefore, hydroxylation stereospecificity) is determined by active-site binding complementarity. Thus, epimerization and allylic rearrangement are not inherent features of these reactions but occur when loss of active-site complementarity allows increased substrate mobility.
Wüst
Matthias
M
Institute of Biological Chemistry, Washington State University, Pullman, Washington 99164-6340, USA.
Croteau
Rodney B
RB
D020134:Catalytic Domain; D053138:Cyclohexenes; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry / Q000378:metabolism*; D003903:Deuterium; D006900:Hydroxylation; D007700:Kinetics; D000077222:Limonene; D027541:Mentha / Q000201:enzymology; D006899:Mixed Function Oxygenases / Q000737:chemistry / Q000378:metabolism*; D008958:Models, Molecular; D010940:Plant Proteins; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013237:Stereoisomerism; D013379:Substrate Specificity; D013729:Terpenes / Q000138:chemical synthesis / Q000737:chemistry* / Q000378:metabolism*
D053138:Cyclohexenes; D010940:Plant Proteins; D011994:Recombinant Proteins; D013729:Terpenes; D003577:Cytochrome P-450 Enzyme System; D000077222:Limonene; D003903:Deuterium; D006899:Mixed Function Oxygenases; C120916:limonene-6-hydroxylase