Although water molecules are small and only consist of two different atom types, they play various roles in cellular systems. This review discusses their influence on the binding process between biomacromolecular targets and small molecule ligands and how this influence can be modeled in computational drug design approaches. Both the structure and the thermodynamics of active site waters will be discussed as these influence the binding process significantly. Structurally conserved waters cannot always be determined experimentally and if observed, it is not clear if they will be replaced upon ligand binding, even if sufficient space is available. Methods to predict the presence of water in protein-ligand complexes will be reviewed. Subsequently, we will discuss methods to include water in computational drug research. Either as an additional factor in automated docking experiments, or explicitly in detailed molecular dynamics simulations, the effect of water on the quality of the simulations is significant, but not easily predicted. The most detailed calculations involve estimates of the free energy contribution of water molecules to protein-ligand complexes. These calculations are computationally demanding, but give insight in the versatility and importance of water in ligand binding.

Inversion of stereoselectivity: screening of a minimal mutant library revealed a cytochrome P450 BM3 variant M01 A82W S72I capable of producing 16 α-OH-testosterone. Remarkably, a single active site mutation S72I in M01 A82W inverted the stereoselectivity of hydroxylation from 16 β to 16 α. Introduction of S72I mutation in another 16 β-OH-selective variant M11 V87I, also resulted in similar inversion of stereoselectivity.

Abstract:The periplasmic oligopeptide binding protein A (OppA) represents a well-known example of water-mediated protein-ligand interactions. Here, we perform free-energy calculations for three different ligands binding to OppA, using a thermodynamic integration approach. The tripeptide ligands share a high structural similarity (all have the sequence KXK), but their experimentally-determined binding free energies differ remarkably. Thermodynamic cycles were constructed for the ligands, and simulations conducted in the bound and (freely solvated) unbound states. In the unbound state, it was observed that the difference in conformational freedom between alanine and glycine leads to a surprisingly slow convergence, despite their chemical similarity. This could be overcome by increasing the softness parameter during alchemical transformations. Discrepancies remained in the bound state however, when comparing independent simulations of the three ligands. These difficulties could be traced to a slow relaxation of the water network within the active site. Fluctuations in the number of water molecules residing in the binding cavity occur mostly on a timescale larger than the simulation time along the alchemical path. After extensive simulations, relative binding free energies that were converged to within thermal noise could be obtained, which agree well with available experimental data.

Abstract:The selective hydroxylation of an unactivated C À H bond is a crucial step in the synthesis of fine chemicals such as hydroxylated terpenoids. In the present study, the ability of 40 cytochrome P450 BM3 mutants to perform the regio-and stereoselective hydroxylation of a-ionone has been investigated. Based on their activity and selectivity to produce 3-hydroxy-a-ionone from racemic a-ionone, 6 BM3 mutants were selected. Out of these, 3 mutants (M01 A82W, M11 A82W and M11 V87I) showed high selectivity for trans-3-hydroxy-a-ionone formation while 3 other mutants (M11 L437N, M11 L437S and M11 L437T) formed almost equal amounts of both cis-3-hydroxy-and trans-3-hydroxy-a-ionone. Incubation with individual enantiomers showed that M11 L437N, M11 L437S and M11 L437T exhibited opposite stereoselectivity producing (3S,6S)-hydroxya-ionone with the (6S)-enantiomer and (3S,6R)-hydroxy-a-ionone with the (6R)-enantiomer. Thus for the first time, BM3 mutants that can selectively produce diastereomers of 3-hydroxy-a-ionone (> 90% de), with high turnover numbers and minimal secondary metabolism, have been identified. Docking studies have been performed to rationalize the basis of the experimentally observed selectivity. In conclusion, engineered P450 BM3s are promising biocatalysts for regio-and stereoselective production of hydroxylated a-ionones for industrial applications.

Three newly discovered drug metabolizing mutants of cytochrome P450 BM3 (van Vugt-Lussenburg et al., Identification of critical residues in novel drug metabolizing mutants of Cytochrome P450 BM3 using random mutagenesis, J Med Chem 2007;50:455-461) have been studied at an atomistic level to provide structural explanations for a number of their characteristics. In this study, computational methods are combined with experimental techniques. Molecular dynamics simulations, resonance Raman and UV-VIS spectroscopy, as well as coupling efficiency and substrate-binding experiments, have been performed. The computational findings, supported by the experimental results, enable structural rationalizations of the mutants. The substrates used in this study are known to be metabolized by human cytochrome P450 2D6. Interestingly, the major metabolites formed by the P450 BM3 mutants differ from those formed by human cytochrome P450 2D6. The computational findings, supported by resonance Raman data, suggest a conformational change of one of the heme propionate groups. The modeling results furthermore suggest that this conformational change allows for an interaction between the negatively charged carboxylate of the heme substituent and the positively charged nitrogen of the substrates. This allows for an orientation of the substrates favorable for formation of the major metabolite by P450 BM3.

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Previously, stereoselective hydroxylation of α-ionone by Cytochrome P450 BM3 mutants M01 A82W and M11 L437N was observed. While both mutants hydroxylate α-ionone in a regioselective manner at the C3 position, M01 A82W catalyzes formation of trans-3-OH-α-ionone products whereas M11 L437N exhibits opposite stereoselectivity, producing trans-(3S,6S)-OH-α-ionone and cis-(3S,6R)-OH-αionone. Here, we explore the stereoselective C3 hydroxylation of α-ionone by Cytochrome P450 BM3 mutants M01 A82W and M11 L437N using molecular dynamics-based free energy calculations to study the interaction between the enzyme and both the substrates and the products. The one-step perturbation approach is applied using an optimized reference state for substrates and products. While the free energy differences between the substrates free in solution amount to ∼0 kJ mol −1 , the differences in mutant M01 A82W agree with the experimentally obtained dissociation constants K d . Moreover, a correlation with experimentally observed trends in product formation is found in both mutants. The trans isomers show the most favorable relative binding free energy in the range of all four possible hydroxylated diastereomers for mutant M01 A82W, while the trans product from (6S)-α-ionone and the cis product from (6R)-α-ionone show highest affinity for mutant M11 L437N. Marcus theory is subsequently used to relate the thermodynamic stability to transition state energies and rates of formation.

4-Hydroxyphenylpyruvate dioxygenase is a relevant target in both pharmaceutical and agricultural research. We report on molecular dynamics simulations and free energy calculations on this enzyme, in complex with 12 inhibitors for which experimental affinities were determined. We applied the thermodynamic integration approach and the more efficient one-step perturbation. Even though simulations seem well converged and both methods show excellent agreement between them, the correlation with the experimental values remains poor. We investigate the effect of slight modifications on the charge distribution of these highly conjugated systems and find that accurate models can be obtained when using improved force field parameters. This study gives insight into the applicability of free energy methods and current limitations in force field parameterization.