RESUMEN
It has long been known that small changes to the structure of the R(2) side chain of nitrogen-containing bisphosphonates can dramatically affect their potency for inhibiting bone resorption in vitro and in vivo, although the reason for these differences in antiresorptive potency have not been explained at the level of a pharmacological target. Recently, several nitrogen-containing bisphosphonates were found to inhibit osteoclast-mediated bone resorption in vitro by inhibiting farnesyl diphosphate synthase, thereby preventing protein prenylation in osteoclasts. In this study, we examined the potency of a wider range of nitrogen-containing bisphosphonates, including the highly potent, heterocycle-containing zoledronic acid and minodronate (YM-529). We found a clear correlation between the ability to inhibit farnesyl diphosphate synthase in vitro, to inhibit protein prenylation in cell-free extracts and in purified osteoclasts in vitro, and to inhibit bone resorption in vivo. The activity of recombinant human farnesyl diphosphate synthase was inhibited at concentrations > or = 1 nM zoledronic acid or minodronate, the order of potency (zoledronic acid approximately equal to minodronate > risedronate > ibandronate > incadronate > alendronate > pamidronate) closely matching the order of antiresorptive potency. Furthermore, minor changes to the structure of the R(2) side chain of heterocycle-containing bisphosphonates, giving rise to less potent inhibitors of bone resorption in vivo, also caused a reduction in potency up to approximately 300-fold for inhibition of farnesyl diphosphate synthase in vitro. These data indicate that farnesyl diphosphate synthase is the major pharmacological target of these drugs in vivo, and that small changes to the structure of the R(2) side chain alter antiresorptive potency by affecting the ability to inhibit farnesyl diphosphate synthase.
Asunto(s)
Transferasas Alquil y Aril/antagonistas & inhibidores , Resorción Ósea/prevención & control , Difosfonatos/farmacología , Inhibidores Enzimáticos/farmacología , Compuestos de Nitrógeno/farmacología , Transferasas Alquil y Aril/metabolismo , Animales , Difosfonatos/química , Inhibidores Enzimáticos/química , Geraniltranstransferasa , Indicadores y Reactivos , Ácido Mevalónico/metabolismo , Compuestos de Nitrógeno/química , Osteoclastos/metabolismo , Conformación Proteica , Prenilación de Proteína , Conejos , Proteínas Recombinantes/química , Relación Estructura-ActividadRESUMEN
In eubacteria, green algae, and plant chloroplasts, isopentenyl diphosphate, a key intermediate in the biosynthesis of isoprenoids, is synthesized by the methylerythritol phosphate pathway. The five carbons of the basic isoprenoid unit are assembled by joining pyruvate and D-glyceraldehyde 3-phosphate. The reaction is catalyzed by the thiamine diphosphate-dependent enzyme 1-deoxy-D-xylulose 5-phosphate synthase. In Rhodobacter capsulatus, two open reading frames (ORFs) carry the genes that encode 1-deoxy-D-xylulose 5-phosphate synthase. ORF 2816 is located in the photosynthesis-related gene cluster, along with most of the genes required for synthesis of the photosynthetic machinery of the bacterium, whereas ORF 2895 is located elsewhere in the genome. The proteins encoded by ORF 2816 and ORF 2895, 1-deoxy-D-xylulose 5-phosphate synthase A and B, containing a His(6) tag, were synthesized in Escherichia coli and purified to greater than 95% homogeneity in two steps. 1-Deoxy-D-xylulose 5-phosphate synthase A appears to be a homodimer with 68 kDa subunits. A new assay was developed, and the following steady-state kinetic constants were determined for 1-deoxy-D-xylulose 5-phosphate synthase A and B: K(m)(pyruvate) = 0.61 and 3.0 mM, K(m)(D-glyceraldehyde 3-phosphate) = 150 and 120 microM, and V(max) = 1.9 and 1.4 micromol/min/mg in 200 mM sodium citrate (pH 7.4). The ORF encoding 1-deoxy-D-xylulose 5-phosphate synthase B complemented the disrupted essential dxs gene in E. coli strain FH11.
Asunto(s)
Hemiterpenos , Sistemas de Lectura Abierta , Rhodobacter capsulatus/enzimología , Transferasas/genética , Secuencia de Aminoácidos , Dimerización , Escherichia coli/enzimología , Escherichia coli/genética , Genes Bacterianos , Prueba de Complementación Genética , Ácido Mevalónico/metabolismo , Datos de Secuencia Molecular , Compuestos Organofosforados/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Rhodobacter capsulatus/genética , Alineación de Secuencia , Transferasas/química , Transferasas/aislamiento & purificación , Transferasas/metabolismoRESUMEN
Isopentenyl diphosphate isomerase catalyzes the interconversion of isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). In eukaryotes, archaebacteria, and some bacteria, IPP is synthesized from acetyl coenzyme A by the mevalonate pathway. The subsequent isomerization of IPP to DMAPP activates the five-carbon isoprene unit for subsequent prenyl transfer reactions. In Escherichia coli, the isoprene unit is synthesized from pyruvate and glyceraldehyde-3-phosphate by the recently discovered nonmevalonate pathway. An open reading frame (ORF696) encoding a putative IPP isomerase was identified in the E. coli chromosome at 65.3 min. ORF696 was cloned into an expression vector; the 20.5 kDa recombinant protein was purified in three steps, and its identity as an IPP isomerase was established biochemically. The gene for IPP isomerase, idi, is not clustered with other known genes for enzymes in the isoprenoid pathway. E. coli FH12 was constructed by disruption of the chromosomal idi gene with the aminoglycoside 3'-phosphotransferase gene and complemented by the wild-type idi gene on plasmid pFMH33 with a temperature-sensitive origin of replication. FH12/pFMH33 was able to grow at the restrictive temperature of 44 degrees C and FH12 lacking the plasmid grew on minimal medium, thereby establishing that idi is a nonessential gene. Although the V(max) of the bacterial protein was 20-fold lower than that of its yeast counterpart, the catalytic efficiencies of the two enzymes were similar through a counterbalance in K(m)s. The E. coli protein requires Mg(2+) or Mn(2+) for activity. The enzyme contains conserved cysteine and glutamate active-site residues found in other IPP isomerases.
Asunto(s)
Isomerasas de Doble Vínculo Carbono-Carbono/genética , Escherichia coli/enzimología , Escherichia coli/genética , Genes Bacterianos , Sistemas de Lectura Abierta , Isomerasas de Doble Vínculo Carbono-Carbono/aislamiento & purificación , Isomerasas de Doble Vínculo Carbono-Carbono/metabolismo , Mapeo Cromosómico , Cromosomas Bacterianos , Escherichia coli/crecimiento & desarrollo , Hemiterpenos , Cinética , Reacción en Cadena de la Polimerasa , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Mapeo RestrictivoRESUMEN
Two Arabidopsis thaliana cDNAs (IPP1 and IPP2) encoding isopentenyl diphosphate isomerase (IPP isomerase) were isolated by complementation of an IPP isomerase mutant strain of Saccharomyces cerevisiae. Both cDNAs encode enzymes with an amino terminus that may function as a transit peptide for localization in plastids. At least 31 amino acids from the amino terminus of the IPP1 protein and 56 amino acids from the amino terminus of the IPP2 protein are not essential for enzymatic activity. Genomic DNA blot analysis confirmed that IPP1 and IPP2 are derived from a small gene family in A. thaliana. Based on northern analysis expression of both cDNAs occurs predominantly in roots of mature A. thaliana plants grown to the pre-flowering stage.
Asunto(s)
Arabidopsis/enzimología , Arabidopsis/genética , Isomerasas de Doble Vínculo Carbono-Carbono/genética , Genes de Plantas , Familia de Multigenes , Secuencia de Aminoácidos , Secuencia de Bases , Isomerasas de Doble Vínculo Carbono-Carbono/biosíntesis , Isomerasas de Doble Vínculo Carbono-Carbono/química , Cartilla de ADN , Hemiterpenos , Datos de Secuencia Molecular , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Alineación de Secuencia , Homología de Secuencia de AminoácidoRESUMEN
Isopentenyl diphosphate (IPP):dimethylallyl diphosphate isomerase catalyzes an essential activation step in the isoprenoid biosynthetic pathway. A human cDNA sequence [J. Xuan, J. Kowalski, A.F. Chambers, and D.T. Denhardt (1994) Genomics 20, 129-131] containing a 684-base-pair open reading frame was recently reported that encoded a protein with a significant degree of similarity to two fungal IPP isomerases [F.M. Hahn and C.D. Poulter (1995) J. Biol. Chem. 270, 11298-11303]. The human cDNA sequence was cloned into expression plasmid pFMH12. The encoded protein was overproduced in Escherichia coli and purified to > 90% homogeneity in two steps by ion-exchange and hydrophobic interaction chromatography. The recombinant protein catalyzed the isomerization of IPP to dimethylallyl diphosphate and was maximally active at pH 7.0 in the presence of Mg2+. The Michaelis constant for IPP was 33 microM, similar to the value of 43 microM reported for yeast IPP isomerase; Vmax = 4.1 mumol min-1 mg-1 for recombinant human IPP isomerase, approximately fivefold less than reported for the yeast enzyme [I.P. Street and C.D. Poulter (1990) Biochemistry 29, 7531-7538].
Asunto(s)
Isomerasas de Doble Vínculo Carbono-Carbono , Isomerasas/biosíntesis , Isomerasas/aislamiento & purificación , Secuencia de Aminoácidos , Secuencia de Bases , Clonación Molecular , Cartilla de ADN/genética , ADN Complementario/genética , Escherichia coli/genética , Hemiterpenos , Humanos , Concentración de Iones de Hidrógeno , Isomerasas/genética , Cinética , Datos de Secuencia Molecular , Sistemas de Lectura Abierta , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Homología de Secuencia de AminoácidoRESUMEN
Isopentenyl diphosphate (IPP) isomerase catalyzes an essential activation step in the isoprenoid biosynthetic pathway. A database search based on probes from the highly conserved regions in three eukaryotic IPP isomerases revealed substantial similarity with ORF176 in the photosynthesis gene cluster in Rhodobacter capsulatus. The open reading frame was cloned into an Escherichia coli expression vector. The encoded 20-kDa protein, which was purified in two steps by ion exchange and hydrophobic interaction chromatography, catalyzed the interconversion of IPP and dimethylallyl diphosphate. Thus, the photosynthesis gene cluster encodes all of the enzymes required to incorporate IPP into the ultimate carotenoid and bacteriochlorophyll metabolites in R. capsulatus. More recent searches uncovered additional putative open reading frames for IPP isomerase in seed-bearing plants (Oryza sativa, Arabidopsis thaliana, and Clarkia breweri), a worm (Caenorhabditis elegans), and another eubacterium (Escherichia coli). The R. capsulatus enzyme is the smallest of the IPP isomerases to be identified thus far and may consist mostly of a fundamental catalytic core for the enzyme.
Asunto(s)
Isomerasas de Doble Vínculo Carbono-Carbono , Genes Bacterianos , Isomerasas/genética , Familia de Multigenes , Sistemas de Lectura Abierta , Rhodobacter capsulatus/genética , Secuencia de Aminoácidos , Secuencia de Bases , Southern Blotting , Hemiterpenos , Isomerasas/aislamiento & purificación , Datos de Secuencia MolecularRESUMEN
Isopentenyl diphosphate (IPP) isomerase catalyzes an essential activation step in the isoprene biosynthetic pathway. The Saccharomyces cerevisiae gene for IPP isomerase, IDI1, was recently isolated and characterized (Anderson, M. S., Muehlbacher, M., Street, I. P., Proffitt, J., and Poulter, C. D. (1989) J. Biol. Chem. 264, 19169-19175), and the wild-type gene, IDI1, was disrupted with a LEU2 marker to create a diploid yeast strain heterozygous for the idi1::leu2 disruption, which revealed that IDI1 was an essential single-copy gene (Mayer, M.P., Hahn, F. M., Stillman, D. J., and Poulter, C. D. (1992) Yeast 8, 743-748). We now report the isolation of a cDNA clone from Schizosaccharomyces pombe by a plasmid shuffle-mediated complementation of the LEU2 disrupted yeast gene. The S. pombe clone encoded a 26,864-dalton polypeptide of 227 amino acids with a high degree of similarity to the S. cerevisiae IDI1 enzyme. S. pombe IPP isomerase contained the essential Cys and Glu catalytic residues identified in yeast isomerase (Street, I. P., Coffman, H. R., Baker, J., and Poulter, C. (1994) Biochemistry 33, 4212-4217) but was significantly smaller than the S. cerevisiae enzyme. The plasmid shuffle technique is an excellent procedure for screening expression libraries for IPP isomerase activity by complementation of the idi1 mutation.
Asunto(s)
Isomerasas de Doble Vínculo Carbono-Carbono , Isomerasas/biosíntesis , Isomerasas/genética , Schizosaccharomyces/enzimología , Secuencia de Aminoácidos , Secuencia de Bases , Clonación Molecular , ADN Complementario/aislamiento & purificación , Escherichia coli , Genes Fúngicos , Prueba de Complementación Genética , Hemiterpenos , Humanos , Datos de Secuencia Molecular , Plásmidos , Proteínas Recombinantes/biosíntesis , Mapeo Restrictivo , Saccharomyces cerevisiae/enzimología , Schizosaccharomyces/genética , Homología de Secuencia de AminoácidoRESUMEN
Isopentenyl diphosphate isomerase catalyses an essential activation step in the isoprene biosynthetic pathway. The Saccharomyces cerevisiae gene for isomerase, IDI1, was recently isolated and characterized (Anderson et al. J. Biol. Chem. 1989a, 264, 19169-19175). Wild-type IDI1 was disrupted with a LEU2 marker, and the resulting DNA was used to transform a yeast leucine auxotroph. Southern blots of EcoRI fragments of chromosomal DNA from the diploid strain showed the expected fragments for intact and disrupted IDI1. Dissection and analysis of tetrads demonstrated that IDI1 is an essential single-copy gene. A CHEF gel and clone grid filter analysis, followed by chromosomal mapping indicated that the gene is located on chromosome XVI approximately 55 kb centromere proximal to PEP4.