Hydroxylation of limonene enantiomers and analogs by recombinant (-)-limonene 3- and 6-hydroxylases from mint (Mentha) species: Evidence for catalysis within sterically constrained active sites

Limonene enantiomers and substrate analogs, including specifically fluorina ted derivatives, were utilized to probe active site interactions with recom binant (-)-(4S)-limonene-3-hydroxylase (CYP71D13) and (-)-(4S)-limonene-6-h ydroxylase (CYP71D18) from mint (Mentha) species. (-)-(4S)-Limonene is hydr oxylated by both enzymes at the designated C3- and C6-allylic positions, wi th strict reso- and stereospecificity and without detectable allylic rearra ngement, to give the corresponding products (-) -trans-isopiperitenol and ( -)-trans-carveol. CYP71D13-catalyzed hydroxylation of (+)-(4R)-limonene als o yields the corresponding trans-3-hydroxylated product ((+)-trans-isopiper itenol); however, the C6-hydroxylase converts (+)-(4R)-limonene to a comple tely different product profile dominated by the enantiopure cis-6-hydroxyla ted product (+)-cis-carveol along with several minor products, including bo th enantiomers of the trans-6-hydroxylated product ((+/-)-trans-carveol), i ndicating allylic rearrangement during catalysis, These results demonstrate that the regiospecificity and facial stereochemistry of oxygen insertion i s dictated by the absolute configuration of the substrate. Fluorinated limo nene analogs are also tightly bound by both enzymes and hydroxylated at the topologically congruent positions in spite of the polarizing effect of the fluorine atom on substrate reactivity. This strict retention of oxygenatio n geometry suggests a rigid substrate orientation imposed by multiple hydro phobic active site contacts. Structurally simplified substrate analogs are hydroxylated at slower rates and with substantial loss of regiospecificity, consistent with a loss of active site complementarity. Evaluation of the p roduct profiles generated allowed assessment of the role of hydrophobic con tacts in orienting the substrate relative to the activated oxygen species. (C) 2001 Academic Press.