New CYP154C4 from Streptomyces cavourensis YBQ59 performs regio- and stereo- selective 3β-hydroxlation of nootkatone.

Nootkatone, a sesquiterpenoid widely used in the food and cosmetics industries, exhibits diverse biological activities and pharmaceutical prospects. Modification of nootkatone to create new derivatives with desirable activities has attracted significant attention. For this purpose, cytochrome P450 monooxygenases (P450 or CYP) are attractive candidates due to their ability to perform regio- and stereoselective hydroxylation at allylic C-H bonds.

High Target Homology Does Not Guarantee Inhibition: Aminothiazoles Emerge as Inhibitors of .

In this study, we identified three novel compound classes with potent activity against , 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 .

Spotlight on CYP4B1.

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.

Differential Multimolecule Fingerprint for Similarity Search─Making Use of Active and Inactive Compound Sets in Virtual Screening.

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.

Resurrection and characterization of ancestral CYP11A1 enzymes.

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.

Metabolism of oral turinabol by the human brain cholesterol 24-hydroxylase CYP46A1.

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-17β-dihydroxy,17α-methylandrosta-1,4-dien-3-one), which is a doping substance.

Molecular evolution of a cytochrome P450 for the synthesis of potential antidepressant (2R,6R)-hydroxynorketamine.

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.

Highly regio- and stereoselective hydroxylation of vitamin D2 by CYP109E1.

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.

Identification and circumvention of bottlenecks in CYP21A2-mediated premedrol production using recombinant Escherichia coli.

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.

Novel insights into oxidation of fatty acids and fatty alcohols by cytochrome P450 monooxygenase CYP4B1.

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.

Conservation and Relevance of Pharmacophore Point Types.

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.

Novel approach to improve progesterone hydroxylation selectivity by CYP106A2 via rational design of adrenodoxin binding.

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.

CYP109E1 from Bacillus megaterium Acts as a 24- and 25-Hydroxylase for Cholesterol.

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.

Binding modes of CYP106A2 redox partners determine differences in progesterone hydroxylation product patterns.

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 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.

The current limits in virtual screening and property prediction.

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.

Structure-Based Engineering of Steroidogenic CYP260A1 for Stereo- and Regioselective Hydroxylation of Progesterone.

The production of regio- and stereoselectively hydroxylated steroids is of high pharmaceutical interest and can be achieved by cytochrome P450-based biocatalysts. CYP260A1 from Sorangium cellulosum strain So ce56 catalyzes hydroxylation of C19 or C21 steroids at the very unique 1α-position. However, the conversion of progesterone (PROG) by CYP260A1 is very unselective.

Engineering of versatile redox partner fusions that support monooxygenase activity of functionally diverse cytochrome P450s.

Most bacterial cytochrome P450 monooxygenases (P450s or CYPs) require two redox partner proteins for activity. To reduce complexity of the redox chain, the Bacillus subtilis flavodoxin YkuN (Y) was fused to the Escherichia coli flavodoxin reductase Fpr (R), and activity was tuned by placing flexible (GGGGS) or rigid ([E/L]PPPP) linkers (n = 1-5) in between. P-linker constructs typically outperformed their G-linker counterparts, with superior performance of YR-P5, which carries linker ([E/L]PPPP).

Biotransformation of prednisone and dexamethasone by cytochrome P450 based systems - Identification of new potential drug candidates.

Prednisone and dexamethasone are synthetic glucocorticoids widely used as anti-inflammatory and immunosuppressive drugs. Since their hydroxylated derivatives could serve as novel potential drug candidates, our aim was to investigate their biotransformation by the steroid hydroxylase CYP106A2 from Bacillus megaterium ATCC13368. In vitro we were able to demonstrate highly selective 15β-hydroxylation of the steroids with a reconstituted CYP106A2 system.

Regioselective Acetylation of C21 Hydroxysteroids by the Bacterial Chloramphenicol Acetyltransferase I.

Chloramphenicol acetyltransferase I (CATI) detoxifies the antibiotic chloramphenicol and confers a corresponding resistance to bacteria. In this study we identified this enzyme as a steroid acetyltransferase and designed a new and efficient Escherichia-coli-based biocatalyst for the regioselective acetylation of C21 hydroxy groups in steroids of pharmaceutical interest. The cells carried a recombinant catI gene controlled by a constitutive promoter.

Mechanistic details for anthraniloyl transfer in PqsD: the initial step in HHQ biosynthesis.

PqsD mediates the conversion of anthraniloyl-coenzyme A (ACoA) to 2-heptyl-4-hydroxyquinoline (HHQ), a precursor of the Pseudomonas quinolone signal (PQS) molecule. Due to the role of the quinolone signaling pathway of Pseudomonas aeruginosa in the expression of several virulence factors and biofilm formation, PqsD is a potential target for controlling this nosocomial pathogen, which exhibits a low susceptibility to standard antibiotics. PqsD belongs to the β-ketoacyl-ACP synthase family and is similar in structure to homologous FabH enzymes in E.

Batch tautomer generation with MolTPC.

Besides all their conformational degrees of freedom, drug-like molecules and natural products often also undergo tautomeric interconversions. Compared to the huge efforts made in experimental investigation of tautomerism, open and free algorithmic solutions for prototropic tautomer generation are surprisingly rare. The few freely available software packages limit their output to a subset of the possible configurational space by sometimes unwanted prior assumptions and complete neglection of ring-chain tautomerism.

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.

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.