Structural evidence: a single charged residue affects substrate binding in cytochrome P450 BM-3

Abstract

Cytochrome P450 BM-3 is a bacterial enzyme with sequence similarity to mammalian P450s that catalyzes the hydroxylation of fatty acids with high efficiency. Enzyme-substrate binding and dynamics has been an important topic of study for cytochromes P450 because most of the crystal structures of substrate-bound structures show the complex in an inactive state. We have determined a new crystal structure for cytochrome P450 BM-3 in complex with N-palmitoylglycine (NPG), which unexpectedly showed a direct bidentate ion pair between NPG and arginine 47 (R47). We further explored the role of R47, the only charged residue in the binding pocket in cytochrome P450 BM-3, through mutagenesis and crystallographic studies. The mutations of R47 to glutamine (R47Q), glutamic acid (R47E), and lysine (R47K) were designed to investigate the role of its charge in binding and catalysis. The oppositely charged R47E mutation had the greatest effect on activity and binding. The crystal structure of R47E BMP shows that the glutamic acid side chain is blocking the entrance to the binding pocket, accounting for NPG's low binding affinity and charge repulsion. For R47Q and R47K BM-3, the mutations caused only a slight change in kcat and a large change in Km and Kd, which suggests that R47 mostly is involved in binding and that our crystal structure, 4KPA , represents an initial binding step in the P450 cycle.

Figure 1

Structure of cytochrome P450 BMP with NPG bound. The F and G loops form one side of the substrate entrance channel, whereas the other side of the channel is formed by the 3₁₀ helix (residues 16–20) and the R47 β-sheet. The binding pocket and access channel are colored in blue, NPG, green, heme, black, and R47, red. This figure shows that arginine is in a key position to interact with substrates at the entrance of the binding pocket. This structure was prepared in PyMOL with PDB file 1JPZ.

Figure 2

Structure of N-palmitoylglycine (NPG). NPG is used as a substrate because of its increased water solubility and tight binding to BM-3. P450 BM-3 in the presence of NADPH and O₂ catalyzes the hydroxylation of fatty acids, such as NPG, at positions ω-1, ω-2, and ω-3.

Figure 3

Comparison of 4KPA and 1JPZ. (A) Cα alignment of 1JPZ (gray and green) and 4KPA (red and pink) with an rmsd of 0.34 Å showing that the overall fold is the same. (B) Interaction of NPG with R47 in 4KPA, which is in a different conformation than it is in 1JPZ. The structures were prepared in PyMOL.

Figure 4

Hydrogen-bonding networks of NPG bound to BM-3. The comparison of the hydrogen-bonding networks between NPG and BMP from the crystal structures is as follows: 1JPZ (green), 3CBD (blue), 4KPA (red), and the omit Fo-Fc maps (gray). In 1JPZ, the carboxylate (C1) of NPG is close enough to hydrogen bond to the backbone of Q73 and A74, and the amide carbonyl (C3) is hydrogen bonded to Y51. However, in our structure, 4KPA, C1 of NPG is in a direct bidentate ion-pair interaction with R47, and it does not appear that C3 is hydrogen bonding to any residue. In 3CBD, only one of the oxygens of carboxylate (C1) is close enough to hydrogen bond to R47. The structures were prepared in PyMOL.

Figure 5

Spin state temperature dependence for BM-3 wild type (A), R47E (B), R47K (C), and R47Q (D). The optical absorbance spectra with a 30-fold excess of NPG was collected at temperatures of 37 (red), 27 (orange), 17 (green), 7 (blue), and −3 °C (purple). The resting state of the enzyme without NPG is shown in black at room temperature. A temperature-dependent change in the spin state of the heme iron was observed with excess NPG because the absorbance at 418 nm (low spin) decreased and at 393 nm (high spin) increased as the temperature increased for wild-type, R47K, and R47Q. There was no temperature-dependent spin state change observed for R47E BM-3 with NPG bound. The percentage of the population in high spin at 37 °C is 97% for the wild-type, 80% for R47K, 50% for R47Q, and 8% for R47E.

Figure 6

Crystal structure of R47E BMP. The structure of R47E BMP (4KPB, cyan) is compared to our crystal structure of NPG bound to BMP (4KPA, red) and to wild-type BMP (1BU7, orange). (A) Comparison of NPG–BMP with R47E BMP. (B) The same structure from A rotated 180°. The largest differences in structure are the F, G, and 3₁₀ helices as well as residue 47, which forms the entrance to the substrate-binding channel and is known to close upon substrate binding. (C) Comparison of R47E BMP with wild-type BMP free enzyme. (D) The same structure from C rotated 180°. R47E BMP more closely resembles substrate-free structure 1BU7. These two structures are very similar with the exception of E47’s position, which appears to be blocking the entrance of the channel, and the electron density of E47 indicates that there may be multiple confirmations. The structures were prepared in PyMOL.

Figure 7

Comparison of the position of arginine in NPG bound to BMP in 4KPA (4KPA, red and green) and flurbiprofen bound to CYP2C9 (1R90, blue and orange). Both proteins have the same overall fold (A); however, the arginine residues in the binding pocket are in different locations to accommodate the difference in the size of the substrates (B). The structures were prepared in PyMOL.