ABSTRACT
We model temperature-dependent blackbody infrared radiative dissociation (BIRD) rate coefficients of Ag+(H2O) n , n = 4-6, a system with loosely bound water molecules. We employ a master equation modeling (MEM) approach with consideration of absorption and emission of blackbody radiation, comparing single and multiple-well descriptions. The unimolecular dissociation rate coefficients are obtained using the Rice-Ramsperger-Kassel-Marcus (RRKM) theory, employing two approaches to model the sum of states in the transition state, the rigid activated complex (RAC) and the phase space limit (PSL) approach. A genetic algorithm is used to find structures of low-lying isomers for the kinetic modeling. We show that the multiple-well MEM approach with PSL RRKM in the All Wells and Transition States Are Relevant (AWATAR) variant provides a reliable description of Ag+(H2O) n BIRD, in agreement with previously published experimental data. Higher-lying isomers contribute significantly to the overall dissociation rate coefficient, underlying the importance of the multiple-well ansatz in which all isomers are treated on the same footing.
ABSTRACT
Molecular cluster ions, which are stored in an electromagnetic trap under ultra-high vacuum conditions, undergo blackbody infrared radiative dissociation (BIRD). This process can be simulated with master equation modeling (MEM), predicting temperature-dependent dissociation rate constants, which are very sensitive to the dissociation energy. We have recently introduced a multiple-well approach for master equation modeling, where several low-lying isomers are taken into account. Here, we experimentally measure the BIRD of CO4â-(H2O)1,2 and model the results with a slightly modified multiple-well MEM. In the experiment, we exclusively observe loss of water from CO4â-(H2O), while the BIRD of CO4â-(H2O)2 leads predominantly to loss of carbon dioxide, with water loss occurring to a lesser extent. The MEM of two competing reactions requires empirical scaling factors for infrared intensities and the sum of states of the loose transition states employed in the calculation of unimolecular rate constants so that the simulated branching ratio matches the experiment. The experimentally derived binding energies are ΔH0(CO4â--H2O) = 45 ± 3 kJ/mol, ΔH0(CO4â-(H2O)-H2O) = 41 ± 3 kJ/mol, and ΔH0(CO2-O2â-(H2O)2) = 37 ± 3 kJ/mol. Quantum chemical calculations on the CCSD(T)/aug-cc-pVTZ//CCSD/aug-cc-pVDZ level, corrected for the basis set superposition error, yield binding energies that are 2-5 kJ/mol higher than experiment, within error limits of both experiment and theory. The relative activation energies for the two competing loss channels are as well fully consistent with theory.
ABSTRACT
In this study, we identified three novel compound classes with potent activity against Plasmodium falciparum, the most dangerous human malarial parasite. Resistance of this pathogen to known drugs is increasing, and compounds with different modes of action are urgently needed. One promising drug target is the enzyme 1-deoxy-d-xylulose-5-phosphate synthase (DXPS) of the methylerythritol 4-phosphate (MEP) pathway for which we have previously identified three active compound classes against Mycobacterium tuberculosis. The close structural similarities of the active sites of the DXPS enzymes of P. falciparum and M. tuberculosis prompted investigation of their antiparasitic action, all classes display good cell-based activity. Through structure-activity relationship studies, we increased their antimalarial potency and two classes also show good metabolic stability and low toxicity against human liver cells. The most active compound 1 inhibits the growth of blood-stage P. falciparum with an IC50 of 600 nM. The results from three different methods for target validation of compound 1 suggest no engagement of DXPS. All inhibitor classes are active against chloroquine-resistant strains, confirming a new mode of action that has to be further investigated.
Subject(s)
Antimalarials , Malaria, Falciparum , Thiazoles , Humans , Plasmodium falciparum , Malaria, Falciparum/drug therapy , Malaria, Falciparum/parasitology , Chloroquine , Antimalarials/pharmacology , Antimalarials/chemistryABSTRACT
The mammalian cytochrome P450 monooxygenase CYP4B1 can bioactivate a wide range of xenobiotics, such as its defining/hallmark substrate 4-ipomeanol leading to tissue-specific toxicities. Similar to other members of the CYP4 family, CYP4B1 has the ability to hydroxylate fatty acids and fatty alcohols. Structural insights into the enigmatic role of CYP4B1 with functions in both, xenobiotic and endobiotic metabolism, as well as its unusual heme-binding characteristics are now possible by the recently solved crystal structures of native rabbit CYP4B1 and the p.E310A variant. Importantly, CYP4B1 does not play a major role in hepatic P450-catalyzed phase I drug metabolism due to its predominant extra-hepatic expression, mainly in the lung. In addition, no catalytic activity of human CYP4B1 has been observed owing to a unique substitution of an evolutionary strongly conserved proline 427 to serine. Nevertheless, association of CYP4B1 expression patterns with various cancers and potential roles in cancer development have been reported for the human enzyme. This review will summarize the current status of CYP4B1 research with a spotlight on its roles in the metabolism of endogenous and exogenous compounds, structural properties, and cancer association, as well as its potential application in suicide gene approaches for targeted cancer therapy.
Subject(s)
Aryl Hydrocarbon Hydroxylases , Cytochrome P-450 Enzyme System , Fatty Acids , Animals , Humans , Rabbits , Aryl Hydrocarbon Hydroxylases/metabolism , Cytochrome P-450 Enzyme System/genetics , Cytochrome P-450 Enzyme System/metabolism , Fatty Acids/metabolism , Mammals/metabolism , Xenobiotics/pharmacologyABSTRACT
Understanding the structural basis of the selectivity of steroid hydroxylation requires detailed structural and functional investigations on various steroid hydroxylases with different selectivities, such as the bacterial cytochrome P450 enzymes. Here, the crystal structure of the cytochrome P450 CYP106A1 from Priestia megaterium was solved. CYP106A1 exhibits a rare additional structural motif of a cytochrome P450, a sixth ß-sheet. The protein was found in different unusual conformations corresponding to both open and closed forms even when crystallized without any known substrate. The structural comparison of CYP106A1 with the previously investigated CYP106A2, including docking studies for both isoforms with the substrate cortisol, reveals a completely different orientation of the steroid molecule in the active sites. This distinction convincingly explains the experimentally observed differences in substrate conversion and product formation by the two enzymes.
Subject(s)
Cytochrome P-450 Enzyme System , Steroids , Cytochrome P-450 Enzyme System/genetics , Cytochrome P-450 Enzyme System/metabolism , Catalytic Domain , Hydroxylation , Steroid Hydroxylases/metabolismABSTRACT
In conventional fingerprint methods, the similarity between two molecules is calculated using the Tanimoto index as a numerical criterion. Thus, the query molecules in virtual screening should be most representative of the wanted compound class at hand. In the concept introduced here, all available active molecules form a multimolecule fingerprint in which the appearing features are weighted according to their respective frequency. The features of inactive molecules are treated likewise and the resulting values are subtracted from those of the active ones. The obtained differential multimolecule fingerprint (DMMFP) is thus specific for the respective class of compounds. To account for the noninteger representation within this fingerprint, a modified Sørensen-Dice coefficient is used to compute the similarity. Potentially active molecules yield positive scores, whereas presumably inactive ones are denoted by negative values. The concept was applied to Angiotensin-converting enzyme (ACE) inhibitors, ß2-adrenoceptor ligands, leukotriene A4 hydrolase inhibitors, dopamine D3 antagonists, and cytochrome CYP2C9 substrates, for which experimental binding affinities are known and was tested against decoys from DUD-E and a further background database consisting of molecules from the dark chemical matter, which comprises compounds that appear as frequent hitters across multiple assays. Using the 166 publicly available keys of the MACCS fingerprint and the larger PubChem fingerprint, actives were recovered with very high sensitivity. Furthermore, three marketed ACE inhibitors as well as the carbonic anhydrase II inhibitor dorzolamide were detected in the dark chemical matter data set. For comparison, the DMMFP was also used with a Bayesian classifier, for which the specificity (correctly classified inactives) and likewise the accuracy was superior. Conversely, the similarity score produced by the Sørensen-Dice coefficient showed its potential for the early recognition of (potentially) active molecules.
Subject(s)
Research , Bayes Theorem , Databases, Factual , LigandsABSTRACT
Seaweeds emerge as promising third-generation renewable for sustainable bioproduction. In the present work, we valorized brown seaweed to produce l-lysine, the world's leading feed amino acid, using Corynebacterium glutamicum, which was streamlined by systems metabolic engineering. The mutant C. glutamicum SEA-1 served as a starting point for development because it produced small amounts of l-lysine from mannitol, a major seaweed sugar, because of the deletion of its arabitol repressor AtlR and its engineered l-lysine pathway. Starting from SEA-1, we systematically optimized the microbe to redirect excess NADH, formed on the sugar alcohol, towards NADPH, required for l-lysine synthesis. The mannitol dehydrogenase variant MtlD D75A, inspired by 3D protein homology modelling, partly generated NADPH during the oxidation of mannitol to fructose, leading to a 70% increased l-lysine yield in strain SEA-2C. Several rounds of strain engineering further increased NADPH supply and l-lysine production. The best strain, SEA-7, overexpressed the membrane-bound transhydrogenase pntAB together with codon-optimized gapN, encoding NADPH-dependent glyceraldehyde 3-phosphate dehydrogenase, and mak, encoding fructokinase. In a fed-batch process, SEA-7 produced 76 g L-1l-lysine from mannitol at a yield of 0.26 mol mol-1 and a maximum productivity of 2.1 g L-1 h-1. Finally, SEA-7 was integrated into seaweed valorization cascades. Aqua-cultured Laminaria digitata, a major seaweed for commercial alginate, was extracted and hydrolyzed enzymatically, followed by recovery and clean-up of pure alginate gum. The residual sugar-based mixture was converted to l-lysine at a yield of 0.27 C-mol C-mol-1 using SEA-7. Second, stems of the wild-harvested seaweed Durvillaea antarctica, obtained as waste during commercial processing of the blades for human consumption, were extracted using acid treatment. Fermentation of the hydrolysate using SEA-7 provided l-lysine at a yield of 0.40 C-mol C-mol-1. Our findings enable improvement of the efficiency of seaweed biorefineries using tailor-made C. glutamicum strains.
Subject(s)
Corynebacterium glutamicum , Seaweed , Corynebacterium glutamicum/genetics , Humans , Lysine/genetics , Metabolic Engineering , NADPABSTRACT
Mitochondrial cytochromes P450 presumably originated from a common microsomal P450 ancestor. However, it is still unknown how ancient mitochondrial P450s were able to retain their oxygenase function following relocation to the mitochondrial matrix and later emerged as enzymes specialized for steroid hormone biosynthesis in vertebrates. Here, we used the approach of ancestral sequence reconstruction (ASR) to resurrect ancient CYP11A1 enzymes and characterize their unique biochemical properties. Two ancestral CYP11A1 variants, CYP11A_Mammal_N101 and CYP11A_N1, as well as an extant bovine form were recombinantly expressed and purified to homogeneity. All enzymes showed characteristic P450 spectral properties and were able to convert cholesterol as well as other sterol substrates to pregnenolone, yet with different specificities. The vertebrate CYP11A_N1 ancestor preferred the cholesterol precursor, desmosterol, as substrate suggesting a convergent evolution of early cholesterol metabolism and CYP11A1 enzymes. Both ancestors were able to withstand increased levels of hydrogen peroxide but only the ancestor CYP11A_N1 showed increased thermostability (Ë 25 °C increase in T50 ) compared with the extant CYP11A1. The extraordinary robustness of ancient mitochondrial P450s, as demonstrated for CYP11A_N1, may have allowed them to stay active when presented with poorly compatible electron transfer proteins and resulting harmful ROS in the new environment of the mitochondrial matrix. To the best of our knowledge, this work represents the first study that describes the resurrection of ancient mitochondrial P450 enzymes. The results will help to understand and gain fundamental functional insights into the evolutionary origins of steroid hormone biosynthesis in animals.
Subject(s)
Cholesterol Side-Chain Cleavage Enzyme , Cholesterol Side-Chain Cleavage Enzyme/genetics , Cholesterol Side-Chain Cleavage Enzyme/isolation & purification , Cholesterol Side-Chain Cleavage Enzyme/metabolism , Humans , PhylogenyABSTRACT
The human microsomal cytochrome P450 enzyme CYP46A1 plays a crucial role in cholesterol elimination from the brain. It performs a 24-hydroxylation of cholesterol and is of outstanding significance for memory and cognition. This study demonstrates the catalytic activity of human CYP46A1 towards an anabolic androgenic steroid, oral turinabol (dehydrochloromethyltestosterone, 4-chloro-17ß-dihydroxy,17α-methylandrosta-1,4-dien-3-one), which is a doping substance. CYP46A1 is the first human microsomal steroid-converting P450 showing activity towards this xenobiotic compound. Furthermore, the inhibitory effect of oral turinabol on the cholesterol conversion has been investigated in vitro demonstrating competition of the two substrates on the active site of CYP46A1 which might be of importance for potential pathogenic effects of oral turinabol. The conversion of oral turinabol was found to be selective resulting in the formation of only one product, as shown by HPLC analysis. To produce sufficient amounts of this product for NMR analysis, a system expressing human full-length CYP46A1 and CPR on a bicistronic vector was successfully developed realizing the selective cholesterol 24-hydroxylation in E. coli in mg amounts. Using this novel whole-cell system, the conversion of oral turinabol was performed and the product of this conversion by CYP46A1 was isolated and identified as 16ß-hydroxy oral turinabol by NMR.
Subject(s)
Anabolic Agents/pharmacology , Cholesterol 24-Hydroxylase/metabolism , Testosterone/analogs & derivatives , Brain/enzymology , Cholesterol 24-Hydroxylase/genetics , Escherichia coli/enzymology , Escherichia coli/genetics , Humans , Molecular Docking Simulation , Oxandrolone/pharmacology , Testosterone/pharmacologyABSTRACT
Saturation mutagenesis at seven first-sphere residues of the cytochrome P450 monooxygenase 154E1 (CYP154E1) from Thermobifida fusca YX was applied to construct a variant with only three substitutions that enabled the effective two-step synthesis of the potential antidepressant (2R,6R)-hydroxynorketamine. A recombinant E. coli whole-cell system was essential for GC/MS based medium-throughput screening and at the same time facilitated the oxidation of the substrate (R)-ketamine at a higher scale for product isolation and subsequent NMR analysis.
Subject(s)
Antidepressive Agents/chemical synthesis , Cytochrome P-450 Enzyme System/chemistry , Ketamine/analogs & derivatives , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Catalytic Domain/genetics , Cytochrome P-450 Enzyme System/genetics , Cytochrome P-450 Enzyme System/metabolism , Evolution, Molecular , Hydroxylation , Ketamine/chemical synthesis , Ketamine/chemistry , Ketamine/metabolism , Molecular Docking Simulation , Mutation , Oxidation-Reduction , Protein Binding , Streptomyces coelicolor/enzymology , Thermobifida/enzymologyABSTRACT
Vitamin D2 is a form of vitamin D derived from mushrooms and plants which is structurally modified in the body due to the action of several enzymes. The resulting metabolites represent important compounds with potential bioactive properties. However, they are poorly studied and their availability is mostly limited. In order to identify new enzymes capable of producing vitamin D2 metabolites, we investigated a bacterial P450 monooxygenase, CYP109E1, which was previously shown to be a vitamin D3 hydroxylase. It was found that CYP109E1 catalyzes a vitamin D2 two-step hydroxylation at positions C24 and C25 resulting in the generation of 24(R),25-diOH VD2. Interestingly, the enzyme showed high selectivity towards vitamin D2, whereas it showed an unselective product pattern for the structurally similar vitamin D3. Our docking results for vitamin D2 and D3 revealed favorable hydroxylation positions for both substrates and suggested an explanation for the high selectivity of CYP109E1 towards vitamin D2. In addition, we established a whole-cell biocatalyst expressing CYP109E1 in Bacillus megaterium to produce 24(R),25-diOH VD2 and a production yield of 12.3 ± 1.2 mg/L was obtained after 48 h. To the best of our knowledge, this is the first report on the generation of 24(R),25-diOH VD2 by a microbial biocatalyst allowing a low-cost and eco-friendly production of this pharmaceutically interesting and expensive metabolite from the relatively cheap substrate, VD2.
Subject(s)
Bacillus megaterium/metabolism , Bacterial Proteins/metabolism , Cytochrome P-450 Enzyme System/metabolism , Ergocalciferols/metabolism , Bacillus megaterium/enzymology , Hydroxylation , Molecular Docking Simulation , Stereoisomerism , Substrate SpecificityABSTRACT
Synthetic glucocorticoids such as methylprednisolone are compounds of fundamental interest to the pharmaceutical industry as their modifications within the sterane scaffold lead to higher inflammatory potency and reduced side effects compared with their parent compound cortisol. In methylprednisolone production, the complex chemical hydroxylation of its precursor medrane in position C21 exhibits poor stereo- and regioselectivity making the process unprofitable and unsustainable. By contrast, the use of a recombinant E. coli system has recently shown high suitability and efficiency. In this study, we aim to overcome limitations in this biotechnological medrane conversion yielding the essential methylprednisolone-precursor premedrol by optimizing the CYP21A2-based whole-cell system on a laboratory scale. We successfully improved the whole-cell process in terms of premedrol production by (a) improving the electron supply to CYP21A2; here we use the N-terminally truncated version of the bovine NADPH-dependent cytochrome P450 reductase (bCPR-27 ) and coexpression of microsomal cytochrome b5 ; (b) enhancing substrate access to the heme by modification of the CYP21A2 substrate access channel; and (c) circumventing substrate inhibition which is presumed to be the main limiting factor of the presented system by developing an improved fed-batch protocol. By overcoming the presented limitations in whole-cell biotransformation, we were able to achieve a more than 100% improvement over the next best system under equal conditions resulting in 691 mg·L-1 ·d-1 premedrol.
Subject(s)
Escherichia coli/genetics , Metabolic Engineering/methods , Methylprednisolone , Recombinant Proteins/metabolism , Steroid 21-Hydroxylase/metabolism , Animals , Biotransformation , Cattle , Escherichia coli/metabolism , Hydroxylation , Methylprednisolone/analogs & derivatives , Methylprednisolone/analysis , Methylprednisolone/chemistry , Methylprednisolone/metabolism , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Steroid 21-Hydroxylase/chemistry , Steroid 21-Hydroxylase/geneticsABSTRACT
CYP4B1 is an enigmatic mammalian cytochrome P450 monooxygenase acting at the interface between xenobiotic and endobiotic metabolism. A prominent CYP4B1 substrate is the furan pro-toxin 4-ipomeanol (IPO). Our recent investigation on metabolism of IPO related compounds that maintain the furan functionality of IPO while replacing its alcohol group with alkyl chains of varying structure and length revealed that, in addition to cytotoxic reactive metabolite formation (resulting from furan activation) non-cytotoxic ω-hydroxylation at the alkyl chain can also occur. We hypothesized that substrate reorientations may happen in the active site of CYP4B1. These findings prompted us to re-investigate oxidation of unsaturated fatty acids and fatty alcohols with C9-C16 carbon chain length by CYP4B1. Strikingly, we found that besides the previously reported ω- and ω-1-hydroxylations, CYP4B1 is also capable of α-, ß-, γ-, and δ-fatty acid hydroxylation. In contrast, fatty alcohols of the same chain length are exclusively hydroxylated at ω, ω-1, and ω-2 positions. Docking results for the corresponding CYP4B1-substrate complexes revealed that fatty acids can adopt U-shaped bonding conformations, such that carbon atoms in both arms may approach the heme-iron. Quantum chemical estimates of activation energies of the hydrogen radical abstraction by the reactive compound 1 as well as electron densities of the substrate orbitals led to the conclusion that fatty acid and fatty alcohol oxidations by CYP4B1 are kinetically controlled reactions.
Subject(s)
Aryl Hydrocarbon Hydroxylases/metabolism , Fatty Acids/metabolism , Fatty Alcohols/metabolism , Aryl Hydrocarbon Hydroxylases/chemistry , Cytochromes b5/metabolism , Humans , Kinetics , Molecular Docking Simulation , Oxidation-Reduction , Protein ConformationABSTRACT
Pharmacophore models in general use a variety of features for distinct chemical characteristics, such as hydrogen-bond properties, lipohilicity, and ionizability. Usually, features have to match onto their identical type. To clarify if this stringent one-to-one assignment is justified, we investigated a set of 581 unique ligands from the BindingDB with known orientation inside the respective binding pockets and conducted a statistical analysis of the likelihood of observed exchanges in between the pharmacophore features, respectively their degree of conservation. To find out if certain features are obsolete, we derived a ranking to determine the most relevant ones. We found that the most conserved one-to-one feature is the negative ionizable (acids), followed by hydrogen-bond donor, positive ionizable (basic nitrogens), hydrogen-bond acceptor, aromatic, nonaromatic π-systems, and other lipophilic characteristics. The most likely exchanges were found between carboxylate groups and hydrogen-bond acceptors and likewise between basic nitrogens and hydrogen-bond donors, which reflects the characteristics of Lewis acids and bases. Exchanges between hydrogen-bond donors and hydrogen-bond acceptors are hardly more likely than by chance. The kind of target (e.g., kinase, phosphatase, protease, phosphodiesterase, nuclear receptor, metal-containing, or transmembrane protein) did not show substantial influence on the degree of conservation. The relevance of the actual pharmacophore features was found to be strongly dependent on the applied ranking scheme. Mutual information ranks all hydrophobic features as least important, whereas the aromatic feature is put into second place by using a geometric series. Both ranking schemes see the negative ionizable feature of higher significance than the positively ionizable feature.
Subject(s)
Drug Design , Informatics/methods , Hydrogen Bonding , Hydrogen-Ion Concentration , Hydrophobic and Hydrophilic Interactions , Ligands , Likelihood FunctionsABSTRACT
Bacterial P450s have considerable potential for biotechnological applications. The P450 CYP106A2 from Bacillus megaterium ATCC 13368 converts progesterone to several hydroxylated products that are important precursors for pharmaceutical substances. As high yields of monohydroxylated products are required for biotechnological processes, improving this conversion is of considerable interest. It has previously been shown that the binding mode of the redox partner can affect the selectivity of the progesterone hydroxylation, being more stringent in case of the Etp1 compared with Adx(4-108). Therefore, in this study we aimed to improve hydroxylation selectivity by optimizing the binding of Adx(4-108) with CYP106A2 allowing for a shorter distance between both redox centers. To change the putative binding interface of Adx(4-108) with CYP106A2, molecular docking was used to choose mutation sites for alteration. Mutants at positions Y82 and P108 of Adx were produced and investigated, and confirmed our hypothesis. Protein-protein docking, as well as conversion studies, using the mutants demonstrated that the iron-sulfur(FeS) cluster/heme distance diminished significantly, which subsequently led to an approximately 2.5-fold increase in 15ß-hydroxyprogesterone, the main product of progesterone conversion by CYP106A2.
Subject(s)
Adrenodoxin/metabolism , Bacillus megaterium/metabolism , Bacterial Proteins/metabolism , Cytochrome P-450 Enzyme System/metabolism , Progesterone/metabolism , Adrenodoxin/chemistry , Adrenodoxin/genetics , Bacillus megaterium/genetics , Bacillus megaterium/growth & development , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Cytochrome P-450 Enzyme System/chemistry , Cytochrome P-450 Enzyme System/genetics , Hydroxylation , Models, Molecular , Molecular Docking Simulation , Mutation , Oxidation-Reduction , Protein ConformationABSTRACT
In this study, the ability of CYP109E1 from Bacillus megaterium DSM319 to metabolize cholesterol was investigated. This steroid was identified as a new substrate to be converted by CYP109E1 with adrenodoxin and adrenodoxin reductase as redox partners in vitro. The biotransformation was successfully reproduced in vivo by using Bacillus megaterium cells that overexpressed CYP109E1. To enhance the production of cholesterol derivatives, an Escherichia coli based whole-cell system that harbored CYP109E1 was established. This novel system showed a 3.3-fold higher activity than that of the B.â megaterium system, yielding about 45â mg L-1 of these products. Finally, the reaction products were isolated and identified to be the highly important cholesterol derivatives 24(S)- and 25-hydroxycholesterol.
Subject(s)
Bacillus megaterium/metabolism , Cytochrome P-450 Enzyme System/physiology , Escherichia coli/metabolism , Mixed Function Oxygenases/physiology , Adrenodoxin/metabolism , Biotransformation , Cholesterol/analogs & derivatives , Cholesterol/metabolism , Ferredoxin-NADP Reductase/metabolism , Hydroxylation , Oxidation-Reduction , Substrate SpecificityABSTRACT
Natural redox partners of bacterial cytochrome P450s (P450s) are mostly unknown. Therefore, substrate conversions are performed with heterologous redox partners; in the case of CYP106A2 from Bacillus megaterium ATCC 13368, bovine adrenodoxin (Adx) and adrenodoxin reductase (AdR). Our aim was to optimize the redox system for CYP106A2 for improved product formation by testing 11 different combinations of redox partners. We found that electron transfer protein 1(516-618) showed the highest yield of the main product, 15ß-hydroxyprogesterone, and, furthermore, produced a reduced amount of unwanted polyhydroxylated side products. Molecular protein-protein docking indicated that this is caused by subtle structural changes leading to alternative binding modes of both redox enzymes. Stopped-flow measurements analyzing the CYP106A2 reduction and showing substantial differences in the apparent rate constants supported this conclusion. The study provides for the first time to our knowledge rational explanations for differences in product patterns of a cytochrome P450 caused by difference in the binding mode of the redox partners.
Subject(s)
Computer-Aided Design , Data Mining/methods , Drug Design , Drug Discovery/methods , Animals , Big Data , Computer Simulation , Drug-Related Side Effects and Adverse Reactions/etiology , Humans , Machine Learning , Models, Biological , Pharmaceutical Preparations/chemistry , Pharmaceutical Preparations/metabolismABSTRACT
Beyond finding inhibitors that show high binding affinity to the respective target, there is the challenge of optimizing their properties with respect to metabolic and toxicological issues, as well as further off-target effects. To reduce the experimental effort of synthesizing and testing actual substances in corresponding assays, virtual screening has become an indispensable toolbox in preclinical development. The scope of application covers the prediction of molecular properties including solubility, metabolic liability and binding to antitargets, such as the hERG channel. Furthermore, prediction of binding sites and drugable targets are emerging aspects of virtual screening. Issues involved with the currently applied computational models including machine learning algorithms are outlined, such as limitations to the accuracy of prediction and overfitting.