The aim of this study was to rationalize the regioselectivity of hydroxylation of octane and lauric acid by WT CYP102A1 (BM3) and three active site mutants—F87A, L188Q/A74G, and F87V/L188Q/A74G. In Figure 5 an energy diagram of intermediate states considered is presented, going from unbound substrate to product. The ratio of hydroxylated products formed depends on the relative rate of each of the corresponding reactions. The rate of the reaction depends on the activation energy (ΔEproduct formation) and the measured product ratios, and it is, therefore, corresponding to the differences in free energy of activation (ΔΔGproduct formation). In the present study, this process has been described in two discrete steps: first, the free energy of orientation (ΔGorientation) of the bound ligand in the active site cavity and, second, the subsequent initiation of the reaction by overcoming the activation energy for H-abstraction (ΔEH-abstraction) of the substrate by activated iron–oxygen species. The sum of these last two energies, therefore, should correspond to the overall rate of product formation, and differences between them correspond to observed product ratios.
Figure Figure 5.. Schematic representation of different states and corresponding (free) energy differences considered in this study. Docking was used to go from unbound to bound state, but since the free energy of binding (ΔGbinding) would be identical for all products formed from a substrate, no free energy barriers (ΔGbinding) were calculated for this step. MD was used to calculate the free energy associated with substrate orientation (ΔGorientation). QM was used to calculated the activation barrier for H-abstraction (ΔEH-abstraction). Experimental product formation corresponds to the (free) energy barrier of product formation (ΔEproduct formation), and can be calculated as the sum of the orientation free energy barrier and the energy barrier of H-abstraction (ΔGorientation + ΔEH-abstraction). Finally, product ratios correspond to the relative free energy of product formation (ΔΔGproduct formation).
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Docking has been used to obtain starting conformations of bound substrates. For a given substrate, the change in free energy associated with the first step, i.e., substrate binding (ΔGbinding), is identical for all possible products of that substrate and is, therefore, not considered in the current study. Statistics of substrate binding orientations were obtained from MD simulations and H-abstraction activation energy barriers (ΔEH-abstraction) at different positions in the substrates from QM calculations. The combination of substrate binding orientation and H-abstraction activation is subsequently used as a basis for a better understanding of the BM3 monooxygenase activity toward aliphatic substrates, and provides an important step toward the quantitative prediction of product formation.
Binding and dynamics
In Cytochromes P450, the substrate is believed to bind before molecular oxygen binds to the iron (Guengerich 2002b). Recent advances in experimental techniques that shed light on details of the P450 catalytic cycle are impressive (e.g., Davydov et al. 2005; Sono et al. 2005); however, it is still impossible to determine whether the substrate binding orientation is influenced by the presence of oxygen at the heme–iron. Although several accurate force field parameter sets for this type of atomic species are available, none is validated for use with the GROMOS “43a1” force field (Daura et al. 1996; Van Gunsteren et al. 1996). Recent work on catalytic site prediction in CYP2D6 has also been successful using the same force field without explicit inclusion of the iron–oxygen (De Graaf et al. 2005a). During the MD simulations described in the current study, therefore, no atomic or molecular oxygen was located at the sixth coordination position of the heme iron.
By using MD simulations, entropic factors in the determination of product formation are explicitly taken into account. In the simulations presented, octane and lauric acid were found to bind in a broad distribution of conformations (see Fig. 1B, C; Table 2), corresponding with the high internal flexibility of typical fatty acid substrates for BM3 and their small size with respect to the binding cavity (Ravichandran et al. 1993; Sevrioukova et al. 1999). This substrate flexibility corresponds with large substrate movements that have been implicated in the catalytic mechanism of BM3 (Modi et al. 1996; Appel et al. 2001).
The binding orientations found were strongly biased toward the ω position. For the other positions (ω−1, ω−2, and ω−3), the trends observed in measured product formation of octane and lauric acid between WT and the mutants were qualitatively reproduced by the statistics of substrate binding orientations derived from the MD simulations. In particular, Phe87 (see also Fig. 1A) is implicated in determining regioselectivity (Graham-Lorence et al. 1997; Cowart et al. 2001) and plays a key role in hydrophobic interactions shifting the substrates away from a ω-orientation. For lauric acid, the anchoring of its carboxyl group to Arg47 and Tyr51, postulated for crucial ligand interactions in BM3 (Haines et al. 2001; Glieder et al. 2002; see Fig. 1A), was indeed a dominant factor in determining the binding statistics of this substrate.
In the current MD simulations, the dynamics of the active site as analyzed with ED were influenced by the active site mutations investigated (Fig. 3). The dynamics of the rest of the protein appeared unaffected by the mutations, indicating that the influence of the mutations on the protein dynamics does not extend much beyond the active site. The dynamics of neither the active site nor the whole protein were affected by binding of octane and lauric acid. Conversely, ligand binding itself did not seem to be affected by the changes in protein dynamics resulting from the mutations.
The current QM calculations were used to quantify relative regioselective H-abstraction barriers as a measure for reactivity of the octane and lauric acid (see Table 3). Although in the present QM calculations no distinction could be made between the WT and the mutants studied, the method would be able to distinguish mutations involving addition or removal of charged residues.
For octane and neutral lauric acid, inclusion of the electrostatic protein environment lowered H-abstraction barriers overall, and addition of explicit counterions lowered them even further. In contrast, for deprotonated lauric acid, the H-abstraction barriers for ω increased with inclusion of the protein charges or counterions. This suggests that neutralizing the anionic charge of the carboxylic acid group in lauric acid may be a crucial factor for the reaction to proceed at the ω position. The modulations of the H-abstraction barrier height by the surrounding electrostatics reflect the importance of the electrostatic surroundings for the H-abstraction step and may be a contributing factor in the actual catalytic function of the enzyme.
The observed differences in H-abstraction barriers show that modifications of the electrostatic properties of the enzyme (e.g., by introducing or removing charged side chains, or changing pH) (Ost et al. 2000; Li et al. 2001) could be used to tune the reactivity of different positions in alkane substrates. Indeed, several BM3 mutants which change turnover and product ratios of polycyclic aromatic hydrocarbons without changing the iron spin state have been reported (Carmichael and Wong 2001), which supports the suggestion that substrate binding orientations and electrostatic interactions between substrate and enzyme also affect the enzymatic action.
Predicting product formation
The relatively high H-abstraction barrier energies (ΔEH-abstraction) for the ω position (see Table 3), may explain the absence of ω-hydroxylated product but not the observed differences between WT and the mutants (Table 1). On the other hand, the distribution of binding orientations (see Table 2) explains most of the differences between the WT and the three mutants, but not the absence of ω-hydroxylated product (Table 1).
From the MD simulations, free energies associated with different orientations of the substrates (ΔGorientation) were calculated based on the statistics of all simulations of each combination of substrate and enzyme, as explained further in Materials and Methods. In Figure 6A the calculated free energies of orientation (ΔGorientation) are shown. A lower energy corresponds to a higher overall fraction oriented (cf. Table 2). The QM calculations yield the activation energies of the H-abstraction reaction (ΔEH-abstraction). In Figure 6B the calculated H-abstraction barriers (cf. Table 3) are also shown. Subsequent to H-abstraction the product is formed and finally released from the enzyme; these steps are not taken into account in this study.
Figure Figure 6.. Orientation, dynamics, H-abstraction, and product formation of octane and lauric acid for BM3 WT and mutants expressed as free energies and activation energies. For clarity, traces of WT and the mutants have been given slight offsets in the horizontal direction; WT, single, double, and triple mutant from left to right. (A) Free energy of orientation (ΔGorientation) of octane and lauric acid for the WT and the three mutants cf. the population of orientations in Table 2, and standard deviation shown as error bars. (B) Activation energy of H-abstraction (ΔEH-abstraction) for octane and lauric acid cf. Table 3. (C) The calculated activation energy for product formation (ΔGproduct formation = ΔGorientation + ΔEH-abstraction) for octane and lauric acid. (D) The relative free energy corresponding to the experimentally determined product formation (ΔΔGproduct formation) for octane and lauric acid, cf. Table 1. For the ω positions an approximate detection limit of 0.1% was used for calculating the apparent activation energy.
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Taking together the free energy of orientation (ΔGorientation) and activation energy of H-abstraction (ΔEH-abstraction) into the calculated activation energy of product formation (ΔEproduct formation) is proposed to rationalize product formation for octane and lauric acid hydroxylation by BM3, as shown in Figure 6C. Taking a cutoff value of 7.5 kcal/mol as the maximum activation barrier that could be overcome by the enzyme, leading to product formation, the overall absence of ω hydroxylated product for octane and lauric acid can be explained. In addition, the nearly absent hydroxylation of octane by BM3 WT found experimentally can thus also be explained.
For octane, using the orientational energy (ΔGorientation) for BM3 WT and mutants and the H-abstraction barriers (ΔEH-abstraction) in the protein without counterions, the calculated activation energy (ΔEproduct formation) for ω hydroxylation was only slightly higher than for the other positions (Fig. 6C). Although the trend is correct, this relatively low activation energy (ΔEproduct formation) for the ω position would lead to a predicted ω-hydroxylation that does not accurately match the experimental results, where no ω-hydroxylation was measured (Table 1). Furthermore, for the ω−1 position, a slightly lower activation energy (ΔEproduct formation) was calculated than for ω−2, which neither completely matches the experimental observation. For lauric acid, on the other hand, using the H-abstraction barriers (ΔEH-abstraction) obtained with the protonated carboxylic acid (—COOH) in the protein with counterions, the relative ranking of ω, ω−1, ω−2, and ω−3 hydroxylation was reproduced rather well (Table 1). Also, the relatively high amount of ω−1 hydroxylated lauric acid formed by the WT was reproduced well, in that the calculated activation energy (ΔEproduct formation) for ω−1 hydroxylation in the WT was lower than in the L188Q/A74G and F87V/L188Q/A74G mutants.
The combination of almost exclusive “ω” binding orientation (ΔGorientation) of octane in the WT and a high activation barrier (ΔEH-abstraction) at the “ω” position explains the near absence of activity of BM3 WT toward octane. In correspondence with this, the closely homologous CYP102A3 WT (from Bacillus subtilis) does not convert octane, while a CYP102A3 triple mutant containing the F-V mutation corresponding to the BM3 (CYP102A1) F87V produces only 11% 1-octanol, and a CYP102A3 double mutant containing the “wild-type” F produces 45% 1-octanol, although at a very low turnover and coupling efficiency (Lentz et al. 2004, 2006). Another crucial mutation in CYP102A3 is A330V (corresponding to A328 in BM3), which narrows the binding pocket and hence restricts variations in the substrate binding modes considerably. The corresponding A328V mutation is reported as well for BM3 in an 11-mutant (most nonactive-site residues), which produces modest amounts (10%) of 1-octanol, but at even lower turnover and coupling (Peters et al. 2003). It is interesting to note the existence of isoforms and mutants that are able to form ω-hydroxylated alkane products and, thus, would be useful to be investigated whether indeed in those cases the electrostatic properties of the isoform or mutant also modulates the activation barrier at the ω-position to enable ω-hydroxylation.
The high activation barriers (ΔEH-abstraction) for the charged (unprotonated) lauric acid at the ω position indicate a possible important role of binding of the carboxyl group to the Arg47/Tyr51 anchoring point in neutralizing the negative carboxylic charge. For deprotonated octanoic acid (—COO–), similarly high activation barriers were found (data not presented), but octanoic acid is shorter than lauric acid and therefore unable to reach from the Arg47/Tyr51 binding site to the vicinity of the heme–iron (data not presented). This is in correspondence with the very low (or no) turnover of octanoic acid by BM3 WT. A recently reported L75T/L181K mutant that introduces a cationic/H-bonding site closer to the heme results in a fivefold increased catalytic efficiency for octanoic acid (Ost et al. 2000).
Our relatively simple approach is capable of capturing several important features of the BM3 catalyzed hydroxylation of alkanes and alkanoic acids. Clearly, there is a certain room for improvement of our methodology, and several recent and well-established QM/MM approaches may be applied for this purpose.
Understanding the oxygenation activity of CYP biotransformation enzymes is crucial to the prediction of ADME-Tox properties of drugs (Clark and Grootenhuis 2002; Vermeulen 2003; Hou and Xu 2004) and the successful application of these enzymes in (industrial) biocatalysis (Ost et al. 2000; Li et al. 2001). Trends observed in experimentally determined product formation for octane and lauric acid by the WT enzyme and three active site mutants were qualitatively explained by our calculations including MD simulations to obtain relative free energies for alternate binding orientations (ΔGorientation) and QM calculations to obtain the activation energies for the rate-limiting H-abstraction (ΔEH-abstraction).
Our computational approach helps explain the recently reported regioselective product formation for octane and lauric acid of wild-type and engineered CYP102A1 (F87A, L188Q/A74G, and F87V/L188Q/A74G mutants), some of which already show some preference for hydroxylation at the ω-position in octane (Peters et al. 2003). In addition to the known carboxylic anchoring site Arg47/Tyr51, hydrophobic interactions and hydrophobic interactions and steric exclusion, mainly by Phe87, play a determining role in the binding modes of the substrates. Electrostatic interactions between the protein and the substrate strongly modulate the substrate's regiodependent H-abstraction barriers. The fine details of the electrostatic and steric interactions between the BM3 enzyme and the substrate are important to capture the effects of mutations, changes in protein conformation, substrate orientations, and protonation state, as we have shown in our current calculations.
More quantitative estimates of the H-abstraction barrier energies (ΔEH-abstraction) could be obtained by extending the method applied here to include a more accurate description of the protein, heme, and possibly the solvent surrounding the substrate during calculation of the H-abstraction. Nevertheless, the current approach has provided a better understanding of the mechanisms underlying the substrate binding, H-abstraction, and product formation in CYP102A1, and constitutes an important step toward more quantitative predictions of product ratios and substrate turnover.