Selective allylic hydroxylation of acyclic terpenoids by CYP154E1 from Thermobifida fusca YX

Summary Allylic alcohols are valuable precursors in the synthesis of pharmaceutical intermediates, agrochemicals and natural products. Regioselective oxidation of parental alkenes is a challenging task for chemical catalysts and requires several steps including protection and deprotection. Many cytochrome P450 enzymes are known to catalyse selective allylic hydroxylation under mild conditions. Here, we describe CYP154E1 from Thermobifida fusca YX that enables this type of oxidation. Several acyclic terpenoids were tested as possible substrates for CYP154E1, and the regio- and chemoselectivity of their oxidation was investigated. Using a previously established bioinformatics approach we identified position 286 in the active site of CYP154E1 which is putatively involved in substrate binding and thereby might have an effect on enzyme selectivity. To tune regio- and chemoselectivity of the enzyme three mutants at position 286 were constructed and used for substrate oxidation. All formed products were analysed with GC–MS and identified using chemically synthesised authentic samples and known compounds as references. Best regioselectivity towards geraniol and nerol was observed with the wild type enzyme mainly leading to 8-hydroxy derivatives (8-hydroxygeraniol or 8-hydroxynerol) with high selectivity (100% and 96% respectively). Highest selectivities during the oxidation of geranylacetone and nerylacetone were observed with the following variants: V286F led mainly to 7-hydroxygeranylacetone (60% of the total product) and V286A produced predominantly 12-hydroxynerylacetone (75% of total product). Thus, CYP154E1 and its mutants expand the tool-box for allylic hydroxylation in synthetic chemistry.

Experimental and analytical data 1

. Chemicals
Monoterpenes and other chemicals were purchased from Fluka (Buchs, Switzerland) and Sigma (Deisenhofen, Germany). All chemicals were of analytical grade. from Thermobifida fusca YX, camA and camB (encoding for putidaredoxin reductase (PdR) and putidaredoxin (Pdx) genes from Pseudomonas putida ATCC17453, respectively) were amplified from pET-11a(+) from Mercian Corporation (Fujisawa, Japan), respectively, by PCR using primers designed to facilitate cloning into pET-28a(+) vector under control of the T7 phage promoter. The CYP154E1 gene was cloned as described earlier [1]. The three CYP154E1 mutants V286L, V286A, V286F were generated by using the "Quick change" site-directed mutagenesis method using the wild type gene as template. The following forward and reverse primers were The genes were amplified in 30 cycles of 2 min 95°C, followed by 2 min annealing at 57°C and extension for 4 min at 72°C in an Eppendorf thermal cycler. The amplified genes were subsequently cloned into the expression vector by NdeI/EcoRI restriction sites. Initial cloning has been performed in strain DH5α (Clontech, Heidelberg, Germany), which gives high transformation efficiency and good plasmid yield followed by heterologous expression in BL21(DE3) E. coli cells (Novagen , Madison, Wis., USA) which N-terminal His 6 -tag to facilitate purification of the protein via nickel affinity chromatography. Attachment of the His 6 polypeptide had no effect on the activity of the gene product.

Protein expression and purification
Expression and purification by immobilised metal chelate affinity chromatography (IMAC) of CYP154E1 and its variants was performed as described earlier [1]. The redox partner proteins Pdx and PdR could be expressed in E. coli and purified by IMAC according to established protocols [2].The purity of the enzyme was estimated by SDS-PAGE, using 12.5% polyacrylamide gels. Enzyme samples were stored at -20°C until use.

Biotransformation and analysis
The 0.5 mL reactions were carried out in 2 mL plastic reaction tubes at 30°C. where it was held for 1 min. The temperatures of the injector and the interface were set to 285°C, respectively.

Materials
All chemicals used in this work were purchased from Fluka (Buchs, Switzerland) or Sigma (Deisenhofen, Germany) and were of analytical grade or higher.

Synthesis of reference compounds
General procedure for the allylic oxidation with selenium dioxide Starting material (1.00 mmol) was added to a solution of selenium dioxide (44 mg, 0.40 mmol) and t-BuOOH (453 mg, 3.10 mmol) in dichloromethane (5 mL) at 0°C.
After stirring under nitrogen at 0°C for a time t (vide infra), the mixture was diluted with ethyl acetate (15 mL), and washed successively with water (2 x 10 mL), saturated NaHCO 3 (10 mL), water (10 mL) and brine (10 mL). The organic layer was then dried over MgSO 4 and evaporated under reduced pressure. The crude product was purified by chromatography on silica gel (hexanes / ethyl acetate).
After warming to room temperature, the mixture was stirred for 24 h. Water (20 mL) and dichloromethane (20 mL) were then added and the aqueous layer was extracted with dichloromethane (20 mL). The combined organic layers were washed successively with saturated NaHCO 3 (10 mL) and brine (10 mL Spectroscopic data were in accordance with ref. [7].