Michael C. Hutter

Dynamic water networks in cytochrome C oxidase from Paracoccus denitrificans investigated by molecular dynamics simulations.

We present a molecular dynamics study of cytochrome c oxidase from Paracoccus denitrificans in the fully oxidized state, embedded in a fully hydrated dimyristoylphosphatidylcholine lipid bilayer membrane. Parallel simulations with different levels of protein hydration, 1.125 ns each in length, were carried out under conditions of constant temperature and pressure using three-dimensional periodic boundary conditions and full electrostatics to investigate the distribution and dynamics of water molecules and their corresponding hydrogen-bonded networks inside cytochrome c oxidase. The majority of the water molecules had residence times shorter than 100 ps, but a few water molecules are fixed inside the protein for up to 1.125 ns. The hydrogen-bonded network in cytochrome c oxidase is not uniformly distributed, and the degree of water arrangement is variable. The average number of solvent sites in the proton-conducting K- and D-pathways was determined. In contrast to single water files in narrow geometries we observe significant diffusion of individual water molecules along these pathways. The highly fluctuating hydrogen-bonded networks, combined with the significant diffusion of individual water molecules, provide a basis for the transfer of protons in cytochrome c oxidase, therefore leading to a better understanding of the mechanism of proton pumping.

Regioselective hydroxylation of norisoprenoids by CYP109D1 from Sorangium cellulosum So ce56

Sesquiterpenes are particularly interesting as flavorings and fragrances or as pharmaceuticals. Regio- or stereoselective functionalizations of terpenes are one of the main goals of synthetic organic chemistry, which are possible through radical reactions but are not selective enough to introduce the desired chiral alcohol function into those compounds. Cytochrome P450 monooxygenases are versatile biocatalysts and are capable of performing selective oxidations of organic molecules. We were able to demonstrate that CYP109D1 from Sorangium cellulosum So ce56 functions as a biocatalyst for the highly regioselective hydroxylation of norisoprenoids, α- and β-ionone, which are important aroma compounds of floral scents. The substrates α- and β-ionone were regioselectively hydroxylated to 3-hydroxy-α-ionone and 4-hydroxy-β-ionone, respectively, which was confirmed by 1H NMR and 13C NMR. The results of docking α- and β-ionone into a homology model of CYP109D1 gave a rational explanation for the regio-selectivity of the hydroxylation. Kinetic studies revealed that α- and β-ionone can be hydroxylated with nearly identical Vmax and Km values. This is the first comprehensive investigation of the regioselective hydroxylation of norisoprenoids by CYP109D1.

Prediction of blood-brain barrier permeation using quantum chemically derived information.

A model for the prediction of the blood–brain distribution (logBB) is obtained by multiple regression analysis of molecular descriptors for a training set of 90 compounds. The majority of the descriptors are derived from quantum chemical information using semi-empirical AM1 calculations to compute fundamental properties of the molecules investigated. The polar surface area of the compounds can be described appropriately by six descriptors derived from the molecular electrostatic potential. This set shows a strong correlation with the observed logBB. Additional quantum chemically computed properties that contribute to the final model comprise the ionization potential and the covalent hydrogen-bond basicity. Complementary descriptors account for the presence of certain chemical groups, the number of hydrogen-bond donors, and the number of rotatable bonds of the compounds. The quality of the fit is further improved by including variables derived from principal component analysis of the molecular geometry.

A structural model of the complex formed by phospholamban and the calcium pump of sarcoplasmic reticulum obtained by molecular mechanics

Phospholamban (PLN) is an intrinsic membrane protein of 52 amino acids that modulates the activity of the reticular Ca2+ ion pump. We recently solved the three‐dimensional structure of chemically synthesized, unphosphorylated, monomeric PLN (C41F) by high‐resolution nuclear magnetic resonance spectroscopy in chloroform/methanol. The structure is composed of two α‐helical regions connected by a β turn (Type III). We used this structure and the crystallographic structure of the sarcoplasmic reticulum calcium pump (SERCA) recently determined by Toyoshima and co‐workers and modeled into its E2 form by Stokes (1KJU) or by Toyoshima (1FQU). We applied restrained and unrestrained energy optimizations and used the AMBER molecular mechanics force field to model the complex formed between PLN and the pump. The results indicate that transmembrane helix 6 (M6) of the SERCA pump is energetically favored, with respect to the other transmembrane helices, as the PLN binding partner within the membrane and is the only one of these helices that also permits contact between the N‐terminal residues of PLN and the critical cytosolic binding loop region of the pump. This result is in agreement with published biochemical data and with the predictions of previous mutagenesis work on the membrane sector of the pump. The model reveals that PLN does not span the entire width of the membrane, that is, its hydrophobic C‐terminal end is located near the center of the transmembrane region of the SERCA pump. The model also shows that interaction with M6 is stabilized by additional contacts made by PLN to M4. The contact between the N‐terminal portion of PLN and the pump is stabilized by a number of salt and hydrogen‐bond bridges, which may be abolished by phosphorylation of PLN. The contacts between the cytosolic portions of PLN and the pump are only observed in the E2 conformation of the pump. Our model of the complex also offers a plausible structural explanation for the preference of protein kinase A for phosphorylation of Ser16 of PLN.

In Silico Prediction of Drug Properties

Drug design has become inconceivable without the assistance of computer-aided methods. In this context in silico was chosen as designation to emphasize the relationship to in vitro and in vivo testing. Nowadays, virtual screening covers much more than estimation of solubility and oral bioavailability of compounds. Along with the challenge of parsing virtual compound libraries, the necessity to model more specific metabolic and toxicological aspects has emerged. Here, recent developments in prediction models are summarized, covering optimization problems in the fields of cytochrome P450 metabolism, blood-brain-barrier permeability, central nervous system activity, and blockade of the hERG-potassium channel. Aspects arising from the use of homology models and quantum chemical calculations are considered with respect to the biological functions. Furthermore, approaches to distinguish drug-like substances from nondrugs by the means of machine learning algorithms are compared in order to derive guidelines for the design of new agents with appropriate properties.

Gradual in Silico Filtering for Druglike Substances

The suitability of decision trees in comparison to support vector machines for the classification of chemical compounds into drugs and nondrugs was investigated. To account for the requirements upon screening virtual compound libraries, schemes for successive filtering steps with gradual increasing computational cost are outlined. The obtained prediction accuracy was similar between decision trees and support vector machine approaches for the applied compound data sets. By using rapidly computable variables such as druglikeness indices, XlogP, and the molar refractivity, at least 39% of the nondrugs can be filtered out, while retaining more than 83% of the actual drugs. Computationally more demanding descriptors such as specific substructure queries and quantum chemically derived variables can be postponed to subsequent classification schemes for the reduced set of compounds, whereby up to 92% of the nondrugs can be sorted out without loosing considerably more drugs. Using all available computed descriptors simultaneously in the first step did not yield significantly better results. Furthermore, the generated decision trees are used to derive guidelines for the design of druglike substances. The numerical margins found at the branching points suggest several criteria that separate drugs from nondrugs: a molecular weight higher than 230, a molar refractivity higher than 40, and the presence of one or more rings as well as one or more functional groups. Also reported are additionally required parameters to compute values for XlogP, SlogP, and the molar refractivity of boron and silicon containing compounds.

CYP264B1 from Sorangium cellulosum So ce56: a fascinating norisoprenoid and sesquiterpene hydroxylase.

Many terpenes and terpenoid compounds are known as bioactive substances with desirable fragrance and medicinal activities. Modification of such compounds to yield new derivatives with desired properties is particularly attractive. Cytochrome P450 monooxygenases are potential enzymes for these reactions due to their capability of performing different reactions on a variety of substrates. We report here the characterization of CYP264B1 from Sorangium cellulosum So ce56 as a novel sesquiterpene hydroxylase. CYP264B1 was able to convert various sesquiterpenes including nootkatone and norisoprenoids (α-ionone and β-ionone). Nootkatone, an important grapefruit aromatic sesquiterpenoid, was hydroxylated mainly at position C-13. The product has been shown to have the highest antiproliferative activity compared with other nootkatone derivatives. In addition, CYP264B1 was found to hydroxylate α- and β-ionone, important aroma compounds of floral scents, regioselectively at position C-3. The products, 3-hydroxy-β-ionone and 13-hydroxy-nootkatone, were confirmed by 1H and 13C NMR. The kinetics of the product formation was analyzed by high-performance liquid chromatography, and the Km and kcat values were calculated. The results of docking α-/β-ionone and nootkatone into a homology model of CYP264B1 revealed insights into the structural basis of these selective hydroxylations.

Human aldosterone synthase: recombinant expression in E. coli and purification enables a detailed biochemical analysis of the protein on the molecular level.

Aldosterone, the most important human mineralocorticoid, is involved in the regulation of the blood pressure and has been reported to play a key role in the formation of arterial hypertension, heart failure and myocardial fibrosis. Aldosterone synthase (CYP11B2) catalyzes the biosynthesis of aldosterone by successive 11β- and 18-hydroxylation followed by an 18-oxidation of 11-deoxycorticosterone and thus comprises an important drug target. For more than 20 years, all attempts to purify recombinant human CYP11B2 in significant amounts for detailed analysis failed due to its hydrophobic nature as a membrane protein. Here, we present the successful expression of the protein in E. coli yielding approx. 90 nmol/l culture, its purification and detailed enzymatic characterization. Biochemical analyses have been performed using in vitro conversion assays which revelead a V(max) of 238±8 nmol products/nmol hCYP11B2/min and a K(m) of 103±8 μM 11-deoxycorticosterone. Furthermore, binding analyses indicated a very loose binding of the first intermediate of the reaction, corticosterone with a K(d) value of 115±6 μM whereas for 11-deoxycorticosterone a K(d) of 1.34±0.13 μM was estimated. Upon substrate conversion of 11-deoxycorticosterone, new intermediates have been identified as 19- and 18-hydroxylated products not described before for the human enzyme. To understand the differences in substrate conversion, we constructed a new homology model based on the 3D structure of CYP11A1, performed docking studies and calculated the activation energy for hydrogen abstraction of the different ligands. The data demonstrated that the 11β-hydroxylation requires much less abstraction energy than hydroxylation at C18 and C19. However, the C18 and C19 hydroxylated products might be of clinical importance. Finally, purified CYP11B2 represents a suitable tool for the investigation of potential inhibitors of this protein for the development of novel drugs against hypertension and heart failure as was shown using ketoconazole.

Resin Acid Conversion with CYP105A1: An Enzyme with Potential for the Production of Pharmaceutically Relevant Diterpenoids

Cytochrome P450s are very versatile enzymes with great potential for biotechnological applications because of their ability to oxidize unactivated CH bonds. CYP105A1 from Streptomyces griseolus was first described as a herbicide‐inducible sulfonylurea hydroxylase, but it is also able to convert other substrates such as vitamin D3. To extend the substrate pool of this interesting enzyme further, we screened a small diterpenoid compound library and were able to show the conversion of several resin acids. Binding of abietic acid, dehydroabietic acid, and isopimaric acid to the active site was assayed, and Vmax and Km values were calculated. The products were analyzed by NMR spectroscopy and identified as 15‐hydroxyabietic acid, 15‐hydroxydehydroabietic acid, and 15,16‐epoxyisopimaric acid. As the observed products are difficult to obtain by chemical synthesis, CYP105A1 has proved to be a promising candidate for biotechnological applications that combine bioconversion and chemical synthesis to obtain functionalized resin acids.

Re-Face Stereospecificity of Methylenetetrahydromethanopterin and Methylenetetrahydrofolate Dehydrogenases is Predetermined by Intrinsic Properties of the Substrate

Four different dehydrogenases are known that catalyse the reversible dehydrogenation of N5,N10‐methylenetetrahydromethanopterin (methylene‐H4MPT) or N5,N10‐methylenetetrahydrofolate (methylene‐H4F) to the respective N5,N10‐methenyl compounds. Sequence comparison indicates that the four enzymes are phylogenetically unrelated. They all catalyse the Re‐face‐stereospecific removal of the pro‐R hydrogen atom of the coenzymes methylene group. The Re‐face stereospecificity is in contrast to the finding that in solution the pro‐S hydrogen atom of methylene‐H4MPT and of methylene‐H4F is more reactive to heterolytic cleavage. For a better understanding we determined the conformations of methylene‐H4MPT in solution and when enzyme‐bound by using NMR spectroscopy and semiempirical quantum mechanical calculations. For the conformation free in solution we find an envelope conformation for the imidazolidine ring, with the flap at N10. The methylene pro‐S C−H bond is anticlinal and the methylene pro‐R C−H bond is synclinal to the lone electron pair of N10. Semiempirical quantum mechanical calculations of heats of formation of methylene‐H4MPT and methylene‐H4F indicate that changing this conformation into an activated one in which the pro‐S C−H bond is antiperiplanar, resulting in the preformation of the leaving hydride, would require a ΔΔH