ABSTRACT
A high-throughput screening protocol for evaluating chimeric, self-sufficient P450 biocatalysts and their mutants against a panel of substrates was developed, leading to the identification of a number of novel biooxidation activities.
ABSTRACT
Cytochromes P450 (P450s) are a family of haem-containing oxidases with considerable potential as tools for industrial biocatalysis. Organismal genomes are revealing thousands of gene sequences that encode P450s of as yet unknown function, the exploitation of which will require high-throughput tools for their isolation and characterisation. In this report, a ligationindependent cloning vector "LICRED" is described that enables the high-throughput generation of libraries of redox-self-sufficient P450s by fusing a range of P450 haem domains to the reductase of P450RhF (RhF-Red) in a robust and generically applicable way. Cloning and expression of fusions of RhF-Red with the haem domains of P450cam and P450-XplA resulted in soluble, active, redox-self-sufficient, chimeric enzymes. In vitro studies also revealed that electron transfer from NADPH to haem was primarily intramolecular. The general applicability of the LICRED platform was then demonstrated through the creation of a library of RhF-Red fusion constructs by using the diverse complement of P450 haem domains identified in the genome of Nocardia farcinica. The resultant fusion-protein library was then screened against a panel of substrates; this revealed chimeric enzymes competent for the hydroxylation of testosterone and methyltestosterone, and the dealkylation of 7-ethoxycoumarin.
Subject(s)
Cytochrome P-450 Enzyme System/metabolism , Cytochrome P-450 Enzyme System/genetics , Electron Transport , Enzymes , Genetic Vectors , Heme/chemistry , Kinetics , NADP/chemistry , Oxidation-Reduction , Protein Structure, Tertiary , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolismABSTRACT
A chimeric oxygenase, in which the P450cam domain was fused to the reductase host domains of a P450RhF from Rhodococcus sp. strain NCIMB 9784 was optimised to allow for a biotransformation at 30 mM substrate in 80% overall yield, with the linker region between P450 and FMN domain proving to be important for the effective biotransformation of (+)-camphor to 5-exo-hydroxycamphor.
Subject(s)
Camphor 5-Monooxygenase/chemistry , Camphor 5-Monooxygenase/genetics , Oxidoreductases/chemistry , Oxidoreductases/genetics , Protein Engineering/methods , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/genetics , Protein Structure, Tertiary , Rhodococcus/enzymology , Transformation, GeneticABSTRACT
The aquaporin protein family performs fundamental tasks in the physiology of several organs in the human body. Their roles in several disorders known to involve water movement make them attractive targets for the development of novel drug therapies.This chapter describes assays commonly used to study the water permeability across AQPs. It also describes the effect of some known inhibitors of aquaporins on water permeability, such as mercury, gold, silver, copper, phloretin, tetraethyl ammonium salts and acetazolamide compounds.
Subject(s)
Aquaporins/antagonists & inhibitors , Cell Membrane Permeability/drug effects , Drug Design , Water/metabolism , Animals , Aquaporins/chemistry , Aquaporins/metabolism , Biological Assay/methods , Fluorescent Dyes , Humans , Indicator Dilution Techniques , Metals/pharmacology , Molecular Structure , Phloretin/pharmacology , Protein Conformation , Quaternary Ammonium Compounds/pharmacology , Structure-Activity Relationship , Sulfonamides/pharmacologyABSTRACT
A series of 1-aminopropan-2-ols were synthesized and evaluated against two strains of malaria, Plasmodium falciparum FCR3 (chloroquine-resistant) and 3D7 (chloroquine-sensitive). Microwave-assisted ring opening of epoxides (aryl and alkyl glycidyl ethers, glycidol, epichlorohydrin) with various amines without catalysts generated the desired library of beta-amino alcohols rapidly and efficiently. Most of the compounds showed micromolar potency against malaria, with seven of them having IC50 values between 1 and 10 microM against both Plasmodium falciparum strains.
Subject(s)
Antimalarials/chemical synthesis , Epoxy Compounds/radiation effects , Microwaves , Plasmodium falciparum/drug effects , Propanolamines/chemical synthesis , Animals , Antimalarials/chemistry , Antimalarials/pharmacology , Cell Line, Tumor , Cells, Cultured , Chloroquine/pharmacology , Drug Resistance , Epoxy Compounds/chemistry , Erythrocytes/drug effects , Erythrocytes/parasitology , Humans , Inhibitory Concentration 50 , Propanolamines/chemistry , Propanolamines/pharmacology , Structure-Activity RelationshipABSTRACT
We report here an efficient synthesis for pyrimidine nucleoside analogues by [4 + 2] cycloaddition reaction. These compounds were obtained by convergent chemistry from glycosyl isothiocyanates 3a-f (pyranoses, furanoses, and dissaccharides) and diazadienium salt 5. In fact, diazapentadienium iodide 5 prepared from vinylthioamide 4 is an efficient intermediate in heterocyclic synthesis and reacts with isothiocyanates 3a-f affording beta-D-uracil analogues 7a-f in good yields and with total regiocontrol. All compounds were fully characterized by IR, HRMS, and 13C and 1H NMR (COSY and HMQC).
