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OleD is shown to play a key reductive role in the generation of alkenes (olefins) from acyl thioesters in Stenotrophomonas maltophilia. The gene coding for OleD clusters with three other genes, oleABC, and all appear to be transcribed in the same direction as an operon in various olefin producing bacteria. In this study, a series of substrates varying in chain length and stereochemistry were synthesized and used to elucidate the functional role and substrate specificity of OleD. We demonstrated that OleD, which is an NADP(H) dependent reductase, is a homodimer which catalyzes the reversible stereospecific reduction of 2-alkyl-3-ketoalkanoic acids. Maximal catalytic efficiency was observed with syn-2-decyl-3-hydroxytetradecanoic acid, with a k(cat)/K(m) 5- and 8-fold higher than for syn-2-octyl-3-hydroxydodecanoic acid and syn-2-hexyl-3-hydroxydecanoic acid, respectively. OleD activity was not observed with syn-2-butyl-3-hydroxyoctanoic acid and compounds lacking a 2-alkyl group such as 3-ketodecanoic and 3-hydroxydecanoic acids, suggesting the necessity of the 2-alkyl chain for enzyme recognition and catalysis. Using diastereomeric pairs of substrates and 4 enantiopure isomers of 2-hexyl-3-hydroxydecanoic acid of known stereochemistry, OleD was shown to have a marked stereochemical preference for the (2R,3S)-isomer. Finally, experiments involving OleA and OleD demonstrate the first 3 steps and stereochemical course in olefin formation from acyl thioesters; condensation to form a 2-alkyl-3-ketoacyl thioester, subsequent thioester hydrolysis, and ketone reduction. 相似文献
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Soon RL Velkov T Chiu F Thompson PE Kancharla R Roberts K Larson I Nation RL Li J 《Analytical biochemistry》2011,(2):273-283
Fluorescence assays employing semisynthetic or commercial dansyl-polymyxin B have been widely employed to assess the affinity of polycations, including polymyxins, for bacterial cells and lipopolysaccharide (LPS). The five primary γ-amines on diaminobutyric acid residues of polymyxin B are potentially derivatized with dansyl-chloride. Mass spectrometric analysis of the commercial product revealed a complex mixture of di- or tetra-dansyl-substituted polymyxin B. We synthesized a mono-substituted fluorescent derivative, dansyl[Lys]1polymyxin B3. The affinity of polymyxin for purified gram-negative LPS and whole bacterial cells was investigated. The affinity of dansyl[Lys]1polymyxin B3 for LPS was comparable to polymyxin B and colistin, and considerably greater (Kd < 1 μM) than for whole cells (Kd ∼ 6–12 μM). Isothermal titration calorimetric studies demonstrated exothermic enthalpically driven binding between both polymyxin B and dansyl[Lys]1polymyxin B3 to LPS, attributed to electrostatic interactions. The hydrophobic dansyl moiety imparted a greater entropic contribution to the dansyl[Lys]1polymyxin B3–LPS reaction. Molecular modeling revealed a loss of electrostatic contact within the dansyl[Lys]1polymyxin B3–LPS complex due to steric hindrance from the dansyl[Lys]1 fluorophore; this corresponded with diminished antibacterial activity (MIC ?16 μg/mL). Dansyl[Lys]1polymyxin B3 may prove useful as a screening tool for drug development. 相似文献
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Yepuri Vijay Jalali Saakshi Kancharla Nagesh Reddy V. B. Arockiasamy S. 《Molecular biology reports》2020,47(7):5091-5099
Molecular Biology Reports - Jatropha curcas is a potential biodiesel crop and a highly adaptable species to various agro-climatic conditions. In this study, we have utilized transposable... 相似文献
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