Hydroxylation Regiochemistry Is Robust to Active Site Mutations in Cytochrome P450cam (CYP101A1)

Abstract

Cytochrome P450_cam (CYP101A1) catalyzes the hydroxylation of d-camphor by molecular oxygen. The enzyme-catalyzed hydroxylation exhibits a high degree of regioselectivity and stereoselectivity, with a single major product, d-5-exo-hydroxycamphor, suggesting that the substrate is oriented to facilitate this specificity. In previous work, we used an elastic network model and perturbation response scanning to show that normal deformation modes of the enzyme structure are highly responsive not only to the presence of a substrate but also to the substrate orientation. This work examines the effects of mutations near the active site on substrate localization and orientation. The investigated mutations were designed to promote a change in substrate orientation and/or location that might give rise to different hydroxylation products, while maintaining the same carbon and oxygen atom balances as in the wild type (WT) enzyme. Computational experiments and parallel in vitro site-directed mutations of CYP101A1 were used to examine reaction products and enzyme activity. ¹H-¹⁵N TROSY-HSQC correlation maps were used to compare the computational results with detectable perturbations in the enzyme structure and dynamics. We found that all of the mutant enzymes retained the same regio- and stereospecificity of hydroxylation as the WT enzyme, with varying degrees of efficiency, which suggests that large portions of the enzyme have been subjected to evolutionary pressure to arrive at the appropriate sequence-structure combination for efficient 5-exo hydroxylation of camphor.

Figure 1:

Regio and stereospecific hydroxylation of camphor catalyzed by CYP101A1.

Figure 2.

Solution structure of CYP101A1 (PDB entry 2L8M (1)), with secondary structural features referred to in the text labeled according to the scheme of Raag and Poulos (2). Heme is shown as magenta sticks, and camphor as blue. Residue numbers are as follows: A helix (V38–E47), β1 (L53–A65), B helix (R67–D77), B′ helix (P89–E94), C helix (Q108–V119), D helix (D125–R143), E helix (F150–G168), F helix (P170–T185), G helix (T192–K214), H helix (A219–A224), β2 (Q227–P232), I helix (T234–A265), J helix (S267–R277), K helix (R280–F292), β3 (G298–L301 and Q317–L319), β4 (Y305–L312), K′ helix (P321–L327), L helix (Q360–I378), β5 (S382–V405).

Figure 3.

Mutation sites in the CYP101A1 structure (PDB entry 2L8M).

Figure 4.

Distribution of distance between camphor CAM(C5) and C atom of carbon monoxide (CMO(C)). The turquoise vertical line shows the maximum in the WT distribution, around 3.98 Å. From top to bottom: WT, 2S, 3M, 4M and 4M2S. A representative structure is shown above the peaks of each distribution.

Figure 5.

Angle θ is defined between an internal vector connecting the camphor center of mass (CM) and C5, and a vector that connects camphor CM and CMO(C).

Figure 6.

2D density plots, regions in yellow represent the most populated states after equilibration. Angle θ on the vertical axis against distance between camphor center of mass (CM) and CMO(C) on the horizontal axis. From left to right: top: WT, 2S and 3M, bottom: 4M and 4M2S.

Figure 7.

Hydrogen bond pattern in WT and 2S. Left: WT enzymatic complex. The hydrogen bond between the carbonyl oxygen of the camphor and the hydroxyl group of Tyr 96 is thought to be critical for camphor orientation. Right: 2S enzymatic complex. A hydrogen bond between the carbonyl oxygen of the camphor and Thr 101 side chain hydroxyl group.

Figure 8.

Distance between camphor center of mass CM and CMO(C) (horizontal axis) and angle θ (vertical). From top to bottom: WT, 2S, 3M, 4M and 4M2S. The results for the restricted protein trajectories are shown in black, while the colored points represent the results after removal of positional restraints on the protein. The red point within the black points shows the coordinates of the initial structure for each MD run.

Figure 9.

Distribution of NMR-detected perturbations in the four mutants described (upper left, 2S; upper right, 3M; lower left, 4M; lower right, 4M2S). Color coding: yellow, slight resonance shifts (< 0.1 ppm in either the ¹H or ¹⁵N dimension); orange, significant shifts (> 0.1 ppm in either dimension); red, strongly broadened or not observable. The positions of the mutations are indicated in blue for each structure. All structures are identical, and in approximately the same orientation as in Figure 2.

Figure 10.

Distance distributions of carbon atoms of substrate camphor relative to carbonmonoxy carbon (CMO(C)) in WT and 2S mutants. C8, C9 and C10 are methyls (CH₃), C5 and C6 are methylenes (CH₂).

Figure 11.

Turnover efficiency (%) relative to WT plotted as a function of the percentage of time substrate spends with C5-CMO(C) distance less than 4 Å.