RESUMEN
Some trans-prenyltransferases, such as long-chain C40 octaprenyl diphosphate synthase (OPPS), short-chain C15 farnesyl diphosphate synthase (FPPS), and C20 geranylgeranyl diphosphate synthase (GGPPS), are important drug targets. These enzymes catalyze chain elongation of FPP or geranyl diphosphate (GPP) through condensation reactions with isopentenyl diphosphate (IPP), forming designated numbers of trans-double bonds in the final products. To facilitate drug discovery, we report here a sensitive and reliable fluorescence-based assay for monitoring their activities in real time. MANT-O-GPP, a fluorescent analogue of FPP, was used as an alternative substrate and converted by the wild-type OPPS and the engineered FPPS and GGPPS into sufficiently long products with enhanced fluorescence intensities. This fluorescence probe was used to reveal the inhibitory mechanism of zoledronate, a bisphosphonate drug that targets human FPPS and possibly GGPPS.
Asunto(s)
Dimetilaliltranstransferasa/antagonistas & inhibidores , Dimetilaliltranstransferasa/química , Colorantes Fluorescentes/química , Sondas Moleculares/química , Fosfatos de Poliisoprenilo/química , Sesquiterpenos/química , Transferasas Alquil y Aril/antagonistas & inhibidores , Transferasas Alquil y Aril/química , Transferasas Alquil y Aril/genética , Sustitución de Aminoácidos , Dimetilaliltranstransferasa/genética , Difosfonatos/farmacología , Descubrimiento de Drogas , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Farnesiltransferasa/antagonistas & inhibidores , Farnesiltransferasa/química , Farnesiltransferasa/genética , Geraniltranstransferasa/antagonistas & inhibidores , Geraniltranstransferasa/química , Geraniltranstransferasa/genética , Humanos , Imidazoles/farmacología , Cinética , Modelos Moleculares , Técnicas de Sonda Molecular , Mutagénesis Sitio-Dirigida , Conformación Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas de Saccharomyces cerevisiae/antagonistas & inhibidores , Especificidad por Sustrato , Ácido ZoledrónicoRESUMEN
We present here how two amino acid residues in the first helix distal from the main dimer interface modulate the dimerization and activity of a geranylgeranyl diphosphate synthase (GGPPs). The enzyme catalyzes condensation of farnesyl diphosphate and isopentenyl diphosphate to generate a C(20) product as a precursor for chlorophylls, carotenoids, and geranylgeranylated proteins. The 3D structure of GGPPs from Saccharomyces cerevisiae reveals an unique positioning of the N-terminal helix A, which protrudes into the other subunit and stabilizes dimerization, although it is far from the main dimer interface. Through a series of mutants that were characterized by analytic ultracentrifugation (AUC), the replacement of L8 and I9 at this helix with Gly was found sufficient to disrupt the dimer into a monomer, leading to at least 10(3)-fold reduction in activity. Molecular dynamics simulations and free energy decomposition analyses revealed the possible effects of the mutations on the protein structures and several critical interactions for maintaining dimerization. Further site-directed mutagenesis and AUC studies elucidated the molecular mechanism for modulating dimerization and activity by long-range interactions.
