These results indicate that a Tyr137 amino acid mutation in the cytochrome P450 FgCYP51B could lead to resistance to TEC and that Y137 forms part of the tebuconazole-binding pocket. © 2017 Society of Chemical Industry.

BACKGROUND: Fusarium graminearum is the main pathogen of Fusarium head blight (FHB), a worldwide plant disease and a major disease of wheat in China. Control of FHB is mainly dependent on the application of demethylase inhibitor (DMI) fungicides. Fungal sterol 14 -demethylase enzymes (CYP51) are the main target for DMI fungicides. A molecular modeling study and biological evaluation were performed to investigate the binding mechanism between azoles and CYP51B in F. graminearum. RESULTS

Cytochrome P450 OleT has attracted much attention for its ability to catalyze the decarboxylation of long chain fatty acids to generate alkenes, which are not only biofuel molecule, but also can be used broadly for making lubricants, polymers and detergents. In this study, the molecular basis of the binding mechanism of P450 OleT for arachidic acid, myristic acid, and caprylic acid was investigated by utilizing conventional molecular dynamics simulation and binding free energy calculations. Moreover, random acceleration molecular dynamics (RAMD) simulations were performed to uncover the most probable access/egress channels for different fatty acids. The predicted binding free energy shows an order of arachidic acid < myristic acid < caprylic acid. Key residues interacting with three substrates and residues specifically binding to one of them were identified. The RAMD results suggest the most likely channel for arachidic acid, myristic acid, and caprylic acid are 2e/2b, 2a and 2f/2a, respectively. It is suggested that the reaction is easier to carry out in myristic acid bound system than those in arachidic acid and caprylic acid bound system based on the distance of Hβ atom of substrate relative to P450 OleT Compound I states. This study provided novel insight to understand the substrate preference mechanism of P450 OleT and valuable information for rational enzyme design for short chain fatty acid decarboxylation.

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This is an open access article under the terms of the Creat ive Commo ns Attri butio n-NonCo mmerc ial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made.Abbreviations: ACD, acid citrate dextrose; aCSF, artificial cerebrospinal fluids; ATP, adenosine triphosphate; BBB, blood-brain barrier; BCRP, breast cancer resistance protein; C csf , concentration in cerebrospinal fluids; CL, clearance; C m,blood/brain , unbound concentration in blood/brain measured by microdialysis; CNS, central nervous system; CSF, cerebrospinal fluids; C ss , steady-state concentration; C u,brain , unbound concentration in brain measured by equilibrium dialysis; DMEM, Dulbecco's Modified Eagle's Medium; DMSO, dimethyl sulfoxide; EDTA-K 2 , ethylenediaminetetraacetic acid dipotassium; ER, efflux ratio; FBS, Fetal bovine serum; f u,brain , unbound fraction in brain; HBSS, Hank's balanced salt solution;HPLC/MS/MS, high-performance liquid chromatography combined with tandem mass spectrometry; HP-β-CD, hydroxypropyl-β-cyclodextrin; IACUC, institutional animal care and use committee; ISF, interstitial fluids; K p,uu,brain , ratio of unbound brain concentration to unbound blood AbstractIn clinical pharmacology, the free drug hypothesis has been widely applied in the interpretation of the relationship between pharmacokinetics and pharmacodynamics (PK/ PD). The free drug hypothesis assumes that the unbound drug concentration in blood is the same as that in the site of action at steady state. The objective of this study is to demonstrate whether the free drug hypothesis is universally applicable for all drugs.The unbound concentrations of the 18 compounds in blood and in brain interstitial fluids (ISF) at steady state following constant intravenous infusion were simultaneously monitored up to 6 hours via in vivo microdialysis technique. Based on the permeability and efflux ratio (ER), the test compounds can be divided into two classes.Class I includes the compounds with good membrane permeability that are not substrates of efflux transporters (eg, P-gp, BCRP, and MRPs), whereas Class II includes the compounds that are substrates of efflux transporters. The steady-state unbound drug concentrations in blood, brain, and CSF are quantitatively very similar for Class I compounds, whereas the steady-state unbound concentrations in the brain and CSF are significantly lower than those in blood for Class II compounds. These results strongly suggest that the free drug hypothesis is not universal for all drugs but is only applicable for drugs with good permeability that are not substrates of efflux transporters. K E Y W O R D SBBB, efflux transporter, microdialysis, permeability, unbound concentration