RESUMEN
Farnesyl diphosphate synthase (FPPS) catalyzes the consecutive condensation of two molecules of isopentenyl diphosphate with dimethylallyl diphosphate to form farnesyl diphosphate (FPP). In insects, FPP is used for the synthesis of ubiquinones, dolicols, protein prenyl groups, and juvenile hormone. A full-length cDNA of FPPS was cloned from the cotton boll weevil, Anthonomus grandis (AgFPPS). AgFPPS cDNA consists of 1,835 nucleotides and encodes a protein of 438 amino acids. The deduced amino acid sequence has high similarity to previously isolated insect FPPSs and other known FPPSs. Recombinant AgFPPS expressed in E. coli converted labeled isopentenyl diphosphate in the presence of dimethylallyl diphosphate to FPP. Southern blot analysis indicated the presence of a single copy gene. Using molecular modeling, the three-dimensional structure of coleopteran FPPS was determined and compared to the X-ray crystal structure of avian FPPS. The alpha-helical fold is conserved in AgFPPS and the size of the active site cavity is consistent with the enzyme being a FPPS.
Asunto(s)
Geraniltranstransferasa/genética , Proteínas de Insectos/genética , Estructura Cuaternaria de Proteína/genética , Gorgojos/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , ADN Complementario/genética , Femenino , Masculino , Modelos Moleculares , Datos de Secuencia Molecular , Alineación de Secuencia , Gorgojos/enzimologíaRESUMEN
A number of biochemical processes rely on isoprenoids, including the post-translational modification of signaling proteins and the biosynthesis of a wide array of compounds. Photoactivatable analogues have been developed to study isoprenoid utilizing enzymes such as the isoprenoid synthases and prenyltransferases. While these initial analogues proved to be excellent structural analogues with good cross-linking capability, they lack the stability needed when the goals include isolation of cross-linked species, tryptic digestion, and subsequent peptide sequencing. Here, the synthesis of a benzophenone-based farnesyl diphosphate analogue containing a stable phosphonophosphate group is described. Inhibition kinetics, photolabeling experiments, as well as X-ray crystallographic analysis with a protein prenyltransferase are described, verifying this compound as a good isoprenoid mimetic. In addition, the utility of this new analogue was explored by using it to photoaffinity label crude protein extracts obtained from Hevea brasiliensis latex. Those experiments suggest that a small protein, rubber elongation factor, interacts directly with farnesyl diphosphate during rubber biosynthesis. These results indicate that this benzophenone-based isoprenoid analogue will be useful for identifying enzymes that utilize farnesyl diphosphate as a substrate.
Asunto(s)
Dimetilaliltranstransferasa/antagonistas & inhibidores , Dimetilaliltranstransferasa/metabolismo , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/farmacología , Organofosfonatos/química , Fosfatos de Poliisoprenilo/química , Fosfatos de Poliisoprenilo/farmacología , Benzofenonas/química , Catálisis , Reactivos de Enlaces Cruzados/química , Cristalografía por Rayos X , Dimetilaliltranstransferasa/química , Inhibidores Enzimáticos/química , Hevea/metabolismo , Humanos , Concentración 50 Inhibidora , Cinética , Espectrometría de Masas , Modelos Moleculares , Estructura Molecular , Fotoquímica , Fosfatos de Poliisoprenilo/síntesis química , Relación Estructura-ActividadRESUMEN
Isolated gut tissue from male cotton boll weevil, Anthonomus grandis (Coleoptera: Curculionidae), incorporated radiolabeled acetate into components that co-eluted with monoterpenoid pheromone components on HPLC. This demonstrates that pheromone components of male A. grandis are produced de novo and strongly suggests that pheromone biosynthesis occurs in gut tissue. A central enzyme in isoprenoid biosynthesis is 3-hydroxy-3-methylglutaryl-CoA reductase (HMG-R), and a full-length HMG-R cDNA was isolated from A. grandis. The predicted translation product was 54 and 45% identical to HMG-R from Ips paraconfusus and Drosophila melanogaster, respectively. HMG-R gene expression gradually increased with age in male A. grandis, which correlates with pheromone production. However, topical application of JH III did not significantly increase HMG-R mRNA levels.