Terpenoid compounds constitute one of the largest groups of biological molecules. The parent terpene hydrocarbons are often readily available and their oxygenation gives derivatives which are fragrance and flavoring compounds, insect pheromones, and precursors to pharmaceuticals. The monoterpene (+)-a-pinene is structurally related to camphor. The Y96F mutant of P450cam showed strengthened monoterpene binding and increased oxidation rates and coupling . Pinene was oxidized mainly to verbenol and pinene oxide while limonene gave mainly isopiperitenol and some limonene oxide. The proportion of epoxides was higher for mutations that increased the active site volume, indicating that substrate mobility would result in the reactive olefinic bond being referentially attacked.
The F87W and V247L mutations were introduced as a means of reducing substrate mobility. Despite weakening substrate binding, both mutations substantially increased pinene oxidation rates and improved coupling efficiency while enhancing the selectivity for verbenol formation (70% for the F87W/Y96F/V247L mutant). The limonene oxidation rate of the F87W/Y96F/V247L mutant was lower than those for the F87W/Y96F and Y96F/V247L mutants but it showed substantially higher selectivity (~90%) for isopiperitenol formation. The crystal structure of the triple mutant complexed with (+)-a-pinene revealed two substrate binding orientations. In one orientation C3 was positioned over the heme iron (Fig. 4.4a), consistent with verbenol formation. In the other the heme iron was closest to the allylic C10 methyl group, a configuration that should give myrtenol
Fig. 4.4 The active site structure of the F87W/Y96F/V247L mutant of P450cam with showing the two binding orientations observed for (+)-a-pinene. The orientation in (a) is related to the camphor binding orientation in the wild type, with the bridgehead methyl group interacting with V295. The C3 allylic carbon is closest to the heme iron and (+)-verbenol is the predicted
Fig. 4.4 The active site structure of the F87W/Y96F/V247L mutant of P450cam with showing the two binding orientations observed for (+)-a-pinene. The orientation in (a) is related to the camphor binding orientation in the wild type, with the bridgehead methyl group interacting with V295. The C3 allylic carbon is closest to the heme iron and (+)-verbenol is the predicted product. The orientation in (b) should give rise to (+)-myrtenol but this is not observed in the products. Rapid interconversion between the two orientations results in the more reactive C—H bond at C3 to be preferentially attacked. There are subtle differences in the L244 and V247 side-chain conformations between the two orientations.
(Fig. 4.4b). Since no myrtenol was detected in practice, it was proposed that the two orientations interconvert, leading predominantly to oxidation of the allylic CH2 group at C3, which is significantly more reactive than the methyl group at C10. Analysis of enzyme/substrate interactions indicated that the F87W and V247L mutations had opposing effects on pinene orientation within the active site, and that the L244 side-chain contacted multiple substrate carbons. The F87W/Y96F/L244A mutant gave 86% (+)-cis-verbenol and the Y96F/L244A/ V247L mutant gave similar selectivity for C3 oxidation but 32% of (+)-verbenone was formed with 55% (+)-cis-verbenol (Scheme 4.8) . Both verbenol and ver-benone are active pheromones against various beetle species.
(+)-Valencene, a sesquiterpene found in orange oil, is the likely biological precursor of (+) -nootkatone, a fragrance found in grapefruit juice. Valencene is larger and more hydrophobic than camphor. No valencene oxidation activity was found for wild type P450cam or the Y96A and Y96F mutants even though the Y96A mutant oxidized diphenylmethane which is of comparable size . The total active site volume in the Y96A might be sufficient for valencene, but the topology was incorrect. The Y96F mutation was used to render the active site more hydrophobic, and smaller amino acids were introduced at F87 to alter the topology. Ideally the space created at the 87 side-chain should accommodate the isopropenyl group of valencene, which would place C2 closest to the heme iron for oxidation to nootkatol and thence to nootkatone. The 87/96 double mutants oxidized valen-cene at C2 and the effect of side-chain volume at 87 was subtle. The F87V mutation favored the oxidation of nootkatol but not of nootkatone while the F87A and F87L mutations both retarded the oxidation of nootkatol to nootkatone.
Additional substitutions were introduced with a view to improving selectivity. The V247L mutation retarded the further oxidation of nootkatol while the L244A mutation had the opposite effect, promoting the oxidation of nootkatol and nootkatone. In combination studies the F87A/Y96F/L244A/V247L mutant gave 86% (+)-trans-nootkatol, 4% nootkatone and 10% of products from nootkatone oxidation (mainly 9-hydroxynootkatone) (Scheme 4.9) while the F87V/Y96F/L244A mutant gave 38% nootkatol and 47% nootkatone.
I JL - P450cam i 5 T mutants
(+)-Valencene (+)-Nootkatol (+)-Nootkatone (+)-irans-Nootkaton-9-ol
(+)-Valencene (+)-Nootkatol (+)-Nootkatone (+)-irans-Nootkaton-9-ol
(+)-Valencene oxidation by a series of R47L/Y51F mutants of P450BM-3 has been investigated . Compared with the P450cam mutants, the turnover activities of the P450BM-3 mutants were higher but they were much less selective. Nootkatol and nootkatone were minor products in some mutants but most gave many products. Moreover, nootkatone was oxidized more rapidly than valencene and hence the steady state concentration of nootkatone was low. The R47L/Y51F series of P450BM-3 mutants also oxidized pinene and limonene with higher activity than P450cam mutants but again the selectivity was low (Sowden and Wong, unpublished results). However, P450BM-3 mutants showed excellent regioselec-tivity for the oxidation of P-ionone (a sesquiterpenoids analog) to 4-hydroxy-P-ionone, with stereoselectivity of up to 39% ee for the R-isomer (Scheme 4.10) . Such ionone oxidation products are flavor components and synthetic intermediates. The F87V mutant was 100 times more active than the wild type, and addition of the R47L/Y51F combination increased the activity 3-fold further. Screening after two rounds of random mutagenesis of the F87V mutant identified the triple mutant A74E/F87V/P386S which was also 300 times more active than the wild type. P386 is located on the surface of the enzyme molecule. Nevertheless, its substitution by serine dramatically increased the NADPH turnover rate during P-ionone hydroxylation. The crystal structure of the complex between the heme and FMN-containing domains of P450BM-3 suggested that the region between P382 and E387 might affect electron transfer from the FMN to the heme iron . Therefore, mutations of P386 could influence the interaction between the monooxygenase domain and an FMN-binding reductase domain. Also remarkable was the effect of the substitution A74E which apparently narrowed the substrate channel and provided a better orientation of the substrate in the binding pocket.
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