stereoselectivity Dotaz Zobrazit nápovědu
The sesquiterpenoid juvenile hormone (JH) is vital to insect development and reproduction. Intracellular JH receptors have recently been established as basic helix-loop-helix transcription factor (bHLH)/PAS proteins in Drosophila melanogaster known as germ cell-expressed (Gce) and its duplicate paralog, methoprene-tolerant (Met). Upon binding JH, Gce/Met activates its target genes. Insects possess multiple native JH homologs whose molecular activities remain unexplored, and diverse synthetic compounds including insecticides exert JH-like effects. How the JH receptor recognizes its ligands is unknown. To determine which structural features define an active JH receptor agonist, we tested several native JHs and their nonnative geometric and optical isomers for the ability to bind the Drosophila JH receptor Gce, to induce Gce-dependent transcription, and to affect the development of the fly. Our results revealed high ligand stereoselectivity of the receptor. The geometry of the JH skeleton, dictated by two stereogenic double bonds, was the most critical feature followed by the presence of an epoxide moiety at a terminal position. The optical isomerism at carbon C11 proved less important even though Gce preferentially bound a natural JH enantiomer. The results of receptor-ligand-binding and cell-based gene activation assays tightly correlated with the ability of different geometric JH isomers to induce gene expression and morphogenetic effects in the developing insects. Molecular modeling supported the requirement for the proper double-bond geometry of JH, which appears to be its major selective mechanism. The strict stereoselectivity of Gce toward the natural hormone contrasts with the high potency of synthetic Gce agonists of disparate chemistries.
- MeSH
- Drosophila melanogaster chemie genetika metabolismus MeSH
- juvenilní hormony chemie metabolismus MeSH
- molekulární modely MeSH
- proteiny Drosophily metabolismus MeSH
- receptory buněčného povrchu metabolismus MeSH
- stereoizomerie MeSH
- transkripční faktory bHLH metabolismus MeSH
- transkripční faktory metabolismus MeSH
- vazba proteinů MeSH
- vývojová regulace genové exprese MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
FTIR, circular dichroism (CD) and fluorescence spectroscopies were used to characterize conformational changes in horse liver alcohol dehydrogenase (HLADH) and ketoreductase (KRED 117) upon physical and covalent immobilizations on silica particles (functionalized with amino, epoxy and thiol groups) of different sizes. Conformational changes for immobilized enzymes were associated with high and low frequency shifts of the amide I and II bands. CD spectra of native HLADH and KRED 117 characterized with a negative peak at 222nm indicating a α-helical structure. The disappearance of the negative peak in the CD spectra of immobilized enzymes and appearance of a positive peak at 222nm supported these observations. These findings demonstrated unfolding of folded enzymes and exposure of the amino acid residues during denaturation with a red shift in tryptophan fluorescence. The decrease in specific activities (by 60-70% in all cases) for both immobilized enzymes was correlated to those of conformational changes. Silica-attached enzyme-NADH systems were evaluated for enantioselective reduction of 1-(p-methoxyphenyl)-propan-2-one. Conformational changes enhanced the enantioselectivity of immobilized HLADH with a switch in its stereoselectivity. In the case of immobilized KRED 117, kinetic values (V(max) and K(m)) were lower than that of the free enzyme, without enhancing enzyme enantio- and stereoselectivity.
- MeSH
- alkoholdehydrogenasa chemie MeSH
- alkoholoxidoreduktasy chemie MeSH
- cirkulární dichroismus MeSH
- enzymy imobilizované chemie MeSH
- fluorescenční spektrometrie MeSH
- játra enzymologie MeSH
- kinetika MeSH
- koenzymy chemie MeSH
- koně MeSH
- NAD chemie MeSH
- nanočástice chemie ultrastruktura MeSH
- oxid křemičitý chemie MeSH
- rozbalení proteinů MeSH
- sekundární struktura proteinů MeSH
- spektroskopie infračervená s Fourierovou transformací MeSH
- stereoizomerie MeSH
- substrátová specifita MeSH
- terciární struktura proteinů MeSH
- tryptofan chemie MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
A series of keto steroids were reduced with sodium borohydride in the presence of cerium(III) chloride or samarium(III) iodide (Luche reduction). The ratios of axial and equatorial alcohols were determined by HPLC and the results were compared with those obtained by a standard sodium borohydride reduction. The best results were obtained with the 2-keto derivative 1, 7-keto derivatives 5 and 6, and 12-keto derivative 8 where the cerium(III) ion addition resulted in the inversion of the axial/equatorial ratios. The Luche reduction of the 20-keto derivative 11 improved the proportion of the (20S)-alcohol in a mixture of (20S)/(20R) alcohols up to 35% from 5% in a standard sodium borohydride reduction.
