RESUMEN

RNA interference (RNAi) has considerable promise for developing novel pest control techniques, especially because of the threat of the development of resistance against current strategies. For this purpose, the key is to select pest control genes with the greatest potential for developing effective pest control treatments. The present study demonstrated that the 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoA reductase; HMGR) gene is a potential target for insect control using RNAi. HMGR is a key enzyme in the mevalonate pathway in insects. A complete cDNA encoding full length HMGR (encoding an 837-aa protein) was cloned from Helicoverpa armigera (Lepidoptera: Noctuidae). The HaHMGR (H. armigera HMGR) knockdown using systemic RNAi in vivo inhibited the fecundity of the females, effectively inhibited ovipostion, and significantly reduced vitellogenin (Vg) mRNA levels. Moreover, the oviposition rate of the female moths was reduced by 98% by silencing HaHMGR compared to the control groups. One-pair experiments showed that both the proportions of valid mating and fecundity were zero. Furthermore, the HaHMGR-silenced females failed to lay eggs (approximate 99% decrease in oviposition) in the semi-field cage performance. The present study demonstrated the potential implications for developing novel pest management strategies using HaHMGR RNAi in the control of H. armigera and other insect pests.

Asunto(s)

Hidroximetilglutaril-CoA Reductasas/metabolismo , Control de Insectos/métodos , Proteínas de Insectos/antagonistas & inhibidores , Larva/genética , Mariposas Nocturnas/genética , Oviposición/genética , ARN Interferente Pequeño/genética , Animales , Femenino , Gossypium/parasitología , Hidroximetilglutaril-CoA Reductasas/clasificación , Hidroximetilglutaril-CoA Reductasas/genética , Proteínas de Insectos/clasificación , Proteínas de Insectos/genética , Proteínas de Insectos/metabolismo , Larva/enzimología , Mariposas Nocturnas/enzimología , Filogenia , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Proteínas Recombinantes/clasificación , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Vitelogeninas/antagonistas & inhibidores , Vitelogeninas/genética , Vitelogeninas/metabolismo

RESUMEN

HMG-CoA reductase (HMGR) is an enzyme critical for cellular cholesterol synthesis in mammals and isoprenoid synthesis in certain eubacteria, catalyzing the NAD(P)H-dependent reduction of HMG-CoA to mevalonate. We have isolated the gene encoding HMG-CoA reductase from Listeria monocytogenes and expressed the recombinant 6x-His-tagged form in Escherichia coli. Using NAD(P)(H), the enzyme catalyzes HMG-CoA reduction approximately 200-fold more efficiently than mevalonate oxidation in vitro. The purified enzyme exhibits dual coenzyme specificity, utilizing both NAD(H) and NADP(H) in catalysis; however, catalytic efficiency using NADP(H) is approximately 200 times greater than when using NAD(H). The statins mevinolin and mevastatin are weak inhibitors of L. monocytogenes HMG-CoA reductase, requiring micromolar concentrations for inhibition. Three-dimensional modeling reveals that the overall structure of L. monocytogenes HMG-CoA reductase is likely similar to the known structure of the class II enzyme from Pseudomonas mevalonii. It appears that the enzyme has catalytic amino acids in analogous positions that likely play similar roles and also has a flap domain that brings a catalytic histidine into the active site. However, in L. monocytogenes HMG-CoA reductase histidine 143 and methionine 186 are present in the putative NAD(P)(H)-selective site, possibly interacting with the 2' phosphate of NADP(H) or 2' hydroxyl of NAD(H) and providing the active site architecture necessary for dual coenzyme specificity.

Asunto(s)

Hidroximetilglutaril-CoA Reductasas/química , Hidroximetilglutaril-CoA Reductasas/metabolismo , Listeria monocytogenes/enzimología , Secuencia de Aminoácidos , Aminoácidos/química , Aminoácidos/genética , Catálisis , Hemiterpenos/biosíntesis , Humanos , Hidroximetilglutaril-CoA Reductasas/clasificación , Hidroximetilglutaril-CoA Reductasas/genética , Cinética , Listeria monocytogenes/genética , Modelos Moleculares , Datos de Secuencia Molecular , Compuestos Organofosforados , Péptidos/química , Péptidos/genética , Unión Proteica , Estructura Cuaternaria de Proteína , Estructura Terciaria de Proteína , Sensibilidad y Especificidad , Alineación de Secuencia , Homología de Secuencia de Aminoácido

Asunto(s)

Archaeoglobus fulgidus/enzimología , Hidroximetilglutaril-CoA Reductasas , Ácido Mevalónico/metabolismo , Pseudomonas/enzimología , Staphylococcus aureus/enzimología , Secuencia de Aminoácidos , Hidroximetilglutaril-CoA Reductasas/química , Hidroximetilglutaril-CoA Reductasas/clasificación , Hidroximetilglutaril-CoA Reductasas/genética , Hidroximetilglutaril-CoA Reductasas/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Alineación de Secuencia

RESUMEN

In higher plants, fungi, and animals isoprenoids are derived from the mevalonate pathway. The carboxylic acid mevalonate is formed from acetyl-CoA and acetoacetyl-CoA via the intermediate 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA). The four-electron reduction of HMG-CoA to mevalonate, which utilizes two molecules of NADPH, is the committed step in the biosynthesis of isoprenoids. This reaction is catalyzed by HMG-CoA reductase (HMGR). The activity of HMGR is controlled through synthesis, degradation and phosphorylation. The human enzyme has also been targeted successfully by drugs, known as statins, in the clinical treatment of high serum cholesterol levels. The crystal structure of the catalytic portion of HMGR has been determined recently with bound reaction substrates and products. The structure illustrates how HMG-CoA and NADPH are recognized and suggests a catalytic mechanism. Catalytic portions of human HMGR form tight tetramers, explaining the influence of the enzyme's oligomeric state on the activity and suggesting a mechanism for cholesterol sensing.

Asunto(s)

Hidroximetilglutaril-CoA Reductasas/química , Animales , Sitios de Unión , Activación Enzimática , Humanos , Hidroximetilglutaril-CoA Reductasas/clasificación , Inhibidores de Hidroximetilglutaril-CoA Reductasas/química , Modelos Moleculares , Estructura Molecular , NADP/química , Fosforilación , Conformación Proteica , Especificidad por Sustrato

RESUMEN

The enzyme 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, which plays a key role in isoprenoid biosynthesis, catalyses the synthesis of mevalonate from HMG-CoA. Insects do not synthesize cholesterol de novo, rather mevalonate derivatives lead to non-sterol isoprenoids which are essential for development and reproduction. In this paper, we describe an HMG-CoA reductase of the moth Agrotis ipsilon and we report its expression in fat body, ovary, muscle, brain and corpora allata tissues of adult specimens. The analysis of the cDNA reveals that it encodes a polypeptide of 833 amino acids (Mr = 89785). Alignments of this HMG-CoA reductase from A. ipsilon with the homologous sequences of other eukaryotes shows a high degree of conservation in all species studied. Parsimony analysis based on these alignments produced dendrograms congruent with the current systematic schemes. This suggests that, during eukaryote evolution, HMG-CoA reductase diversified in parallel with taxonomic splitting.

Asunto(s)

Hidroximetilglutaril-CoA Reductasas/genética , Mariposas Nocturnas/enzimología , Secuencia de Aminoácidos , Aminoácidos/análisis , Animales , Secuencia de Bases , Clonación Molecular , Expresión Génica , Biblioteca de Genes , Genes de Insecto , Hidroximetilglutaril-CoA Reductasas/clasificación , Hidroximetilglutaril-CoA-Reductasas NADP-Dependientes , Datos de Secuencia Molecular , Mariposas Nocturnas/genética , ARN Mensajero , Análisis de Secuencia de Proteína

RESUMEN

Both in eukaryotes and in archaebacteria the enzyme 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase (E.C. 1.1. 1.34) is known to catalyze an early reaction unique to isoprenoid biosynthesis. In humans, the HMG-CoA reductase reaction is rate-limiting for the biosynthesis of cholesterol and therefore constitutes a prime target of drugs that reduce serum cholesterol levels. Recent advances in genome sequencing that permitted comparison of 50 HMG-CoA reductase sequences has revealed two previously unsuspected classes of this enzyme. Based on sequence and phylogenetic considerations, we propose the catalytic domain of the human enzyme and the enzyme from Pseudomonas mevalonii as the canonical sequences for Class I and Class II HMG-CoA reductases, respectively. These sequence comparisons have revealed, in addition, that certain true bacteria, including several human pathogens, probably synthesize isoprenoids by reactions analogous to those of eukaryotes and that there therefore exist two distinct pathways for isoprenoid biogenesis in true bacteria.

Asunto(s)

Hidroximetilglutaril-CoA Reductasas/química , Hidroximetilglutaril-CoA Reductasas/genética , Secuencia de Aminoácidos , Animales , Arabidopsis/enzimología , Archaea/enzimología , Bacterias/enzimología , Drosophila melanogaster/enzimología , Evolución Molecular , Hongos/enzimología , Humanos , Hidroximetilglutaril-CoA Reductasas/clasificación , Datos de Secuencia Molecular , Filogenia , Plantas/enzimología , Pseudomonas/enzimología , Saccharomyces cerevisiae/enzimología , Alineación de Secuencia , Homología de Secuencia de Aminoácido

RESUMEN

A previous report identified and classified a small family of gram-negative bacterial drug and heavy metal efflux permeases, now commonly referred to as the RND family (TC no. 2.6). We here show that this family is actually a ubiquitous superfamily with representation in all major kingdoms. We report phylogenetic analyses that define seven families within the RND superfamily as follows: (1) the heavy metal efflux (HME) family (gram negative bacteria), (2) the hydrophobe/amphiphile efflux-1 (HAE1) family (gram negative bacteria), (3) the nodulation factor exporter (NFE) family (gram negative bacteria), (4) the SecDF protein-secretion accessory protein (SecDF) family (gram negative and gram positive bacteria as well as archaea), (5) the hydrophobe/amphiphile efflux-2 (HAE2) family (gram positive bacteria), (6) the eukaryotic sterol homeostasis (ESH) family, and (7) the hydrophobe/amphiphile efflux-3 (HAE3) family (archaea and spirochetes). Functionally uncharacterized proteins were identified that are members of the RND superfamily but fall outside of these seven families. Some of the eukaryotic homologues function as enzymes and receptors instead of (or in addition to) transporters. The sizes and topological patterns exhibited by members of all seven families are shown to be strikingly similar, and statistical analyses establish common descent. Multiple alignments of proteins within each family allow derivation of family-specific signature sequences. Structural, functional, mechanistic and evolutionary implication of the reported results are discussed.

Asunto(s)

Proteínas Potenciadoras de Unión a CCAAT , Proteínas Portadoras , Glicoproteínas de Membrana , Proteínas de Transporte de Membrana/metabolismo , Enfermedades de Niemann-Pick/enzimología , Factores de Transcripción , Secuencia de Aminoácidos , Animales , Archaea/enzimología , Proteínas Bacterianas/clasificación , Proteínas Bacterianas/metabolismo , Proteínas de Unión al ADN/clasificación , Células Eucariotas , Bacterias Gramnegativas/enzimología , Bacterias Grampositivas/enzimología , Humanos , Hidroximetilglutaril-CoA Reductasas/clasificación , Hidroximetilglutaril-CoA Reductasas/metabolismo , Péptidos y Proteínas de Señalización Intracelular , Proteínas de la Membrana/clasificación , Proteínas de la Membrana/metabolismo , Proteínas de Transporte de Membrana/clasificación , Datos de Secuencia Molecular , Proteína Niemann-Pick C1 , Proteínas Nucleares/clasificación , Proteínas/clasificación , Proteínas/metabolismo , Proteína 1 de Unión a los Elementos Reguladores de Esteroles

RESUMEN

3-Hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) catalyzes a key step in isoprenoid metabolism leading to a range of compounds that are important for the growth, development and health of the plant. We have isolated 7 classes of genomic clones encoding HMGR from a potato genomic library. Comparison of nucleic acid sequences reveals a high degree of identity between all seven classes of clones and the potato hmg 1 gene described by Choi et al. (Plant Cell 4: 1333, 1992), indicating that all are members of the same subfamily in potato. A representative member (hmg 1.2) of the most abundant class of genomic clones was selected for further characterization. Transgenic tobacco and potato containing the beta-glucuronidase (GUS) reporter gene under the control of the hmg 1.2 promoter expressed GUS activity constitutively at a low level in many plant tissues. High levels of GUS activity were observed only in the pollen. GUS assays of isolated pollen, correlations of GUS activity with the HMGR activity of anthers, hmg 1.2 promoter deletion studies, and segregation analysis of the expression of hmg 1.2::GUS among the R2 pollen of R1 progeny plants demonstrated that the hmg 1.2 promoter controls pollen expression.

Asunto(s)

Genes de Plantas/genética , Hidroximetilglutaril-CoA Reductasas/genética , Familia de Multigenes/genética , Solanum tuberosum/genética , Secuencia de Aminoácidos , Secuencia de Bases , Northern Blotting , Southern Blotting , ADN Complementario/genética , Genes Reporteros , Biblioteca Genómica , Glucuronidasa/genética , Hidroximetilglutaril-CoA Reductasas/clasificación , Datos de Secuencia Molecular , Plantas Modificadas Genéticamente , Plantas Tóxicas , Polen/genética , Regiones Promotoras Genéticas/genética , ARN Mensajero/genética , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido , Homología de Secuencia de Ácido Nucleico , Distribución Tisular , Nicotiana/genética , Transformación Genética