Regio- and Stereoselective Steroid Hydroxylation at C7 by Cytochrome P450 Monooxygenase Mutants

Abstract

Steroidal C7β alcohols and their respective esters have shown significant promise as neuroprotective and anti-inflammatory agents to treat chronic neuronal damage like stroke, brain trauma, and cerebral ischemia. Since C7 is spatially far away from any functional groups that could direct C-H activation, these transformations are not readily accessible using modern synthetic organic techniques. Reported here are P450-BM3 mutants that catalyze the oxidative hydroxylation of six different steroids with pronounced C7 regioselectivities and β stereoselectivities, as well as high activities. These challenging transformations were achieved by a focused mutagenesis strategy and application of a novel technology for protein library construction based on DNA assembly and USER (Uracil-Specific Excision Reagent) cloning. Upscaling reactions enabled the purification of the respective steroidal alcohols in moderate to excellent yields. The high-resolution X-ray structure and molecular dynamics simulations of the best mutant unveil the origin of regio- and stereoselectivity.

Keywords: cytochromes; directed evolution; enzymes; oxidation; steroids.

Figure 1

Binding pocket of P450‐BM3 triple mutant (F87G/A328G/A330W) featuring 15‐residue randomization sites: R47, S72, K76, F77, V78, R79, D80, F81, A82, T88, M177, M185, L188, F205, and I209.

Figure 2

Time course of mutant LG‐23 catalyzed regio‐ and stereoselective C7β hydroxylation of 1. Reaction conditions: 1 mm 1 in 50 mL KPi buffer pH 8.0 for 5 h, 220 rpm, 25 °C.

Figure 3

Active site of the LG‐23 mutant⋅Heme⋅1 complex. A) Amino acids involved in heme binding are shown as a stick model in wheat, and the red spheres are water. The iron atom is shown in orange sphere. The heme (HEM), testosterone (TES) and imidazole (IMD) molecules are shown as stick model colored in magenta and cyan, respectively. 2 F _o−F _c (gray mesh, contoured at 1.0 σ) electron density map for heme. Dashed lines illustrate hydrogen‐bonding interactions involving the carboxy groups of heme with residues or water. B) The residues in the testosterone‐binding site are shown as stick models in wheat, and hydrogen bonds formed between the bound testosterone and LG‐23 mutant are shown with dashed lines.

Figure 4

MD simulations of LG‐23 mutant. A) Representative snapshot from the MD simulations showing the active site of mutant LG‐23 and testosterone substrate bound (shown in light orange). The most important polar interactions for 7β hydroxylation are marked with a black dashed line. C7 of testosterone is highlighted in violet color. Mutated residues are shown in teal color sticks. B) Zoom of the active site of LG‐23 showing the hydrogen bond between testosterone and G87 and T438 residues (black dashed line) together with the mean value and standard deviation for the 7C⋅⋅⋅O=Fe(heme) distance and the O(Fe=O)‐(1)‐H(7C)‐(1)‐C(7) angle along the whole simulation time. C) Plot of the C6,C7,C15(1)⋅⋅⋅O=Fe distances (y primary axis) and the O(Fe=O)‐(1)‐H(6C,7C,15C)‐(1)‐C(6,7,15) angles (y secondary axis) along the simulation time (x axis) for one of the replicas (see the Supporting Information for replica 2 and 3). Ideal Quantum Mechanics (QM) distances and angles are shown with dashed lines (distQM = 2.5 Å, angQM = 167°).

Scheme 1

Regio‐ and stereoselective 7β hydroxylation with the mutant LG‐23 for six different steroids, testosterone (1), nandrolone (3), 4‐androstenedione (5), adrenosterone (7), epitestosterone (9), and d‐ethylgonendione (11), towards their respective C7β alcohols.