RESUMEN
3-Hydroxy-3-methylglutaryl-coenzymeA reductase (HMGR), the first rate-limiting enzyme of Mevalonate (MVA) pathway was isolated from Andrographis paniculata (ApHMGR) and expressed in bacterial cells. Full length ApHMGR (1937 bp) was submitted to NCBI with accession number MG271748.1. The open reading frame (ORF) was flanked by a 31-bp 5'-UTR, 118-bp 3'-UTR and ApHMGR contained a 1787 bp ORF encoding protein of 595 amino acids. ApHMGR protein was approximately 64 kDa, with isoelectric point of 5.75. Isolated ApHMGR was cloned into pET102 vector and expressed in E. coli BL21 (DE 3) cells, and characterized by SDS-PAGE. HPLC analysis for andrographolide content in leaf, stem and root of A. paniculata revealed highest in leaf tissue. The expression patterns of ApHMGR in different plant tissues using qRT-PCR revealed high in root tissue correlating with HPLC data. Three dimensional (3D) structural model of ApHMGR displayed 90% of the amino acids in most favored regions of the Ramachandran plot with 93% overall quality factor. ApHMGR was highly conserved with plant specific N-terminal membrane domains and C-terminal catalytic regions. Phylogenetic analysis showed A. paniculata sharing common ancestor with Handroanthus impetiginosus. 3D model of ApHMGR was screened for the interaction with substrates NADPH, HMG CoA and inhibitor using Auto Dock Vina. In silico analysis revealed that full length ApHMGR had extensive similarities to other plant HMGRs. The present communication reports the isolation of full length HMGR from A. paniculata, its heterologous expression in bacterial cells and in silico structural and functional characterization providing valuable genomic information for future molecular interventions.
Asunto(s)
Andrographis , Hidroximetilglutaril-CoA Reductasas , Proteínas de Plantas , Andrographis/clasificación , Andrographis/enzimología , Andrographis/genética , Andrographis/metabolismo , ADN de Plantas/análisis , ADN de Plantas/genética , Hidroximetilglutaril-CoA Reductasas/química , Hidroximetilglutaril-CoA Reductasas/genética , Hidroximetilglutaril-CoA Reductasas/aislamiento & purificación , Hidroximetilglutaril-CoA Reductasas/metabolismo , Ácido Mevalónico/metabolismo , Simulación del Acoplamiento Molecular , Filogenia , Proteínas de Plantas/química , Proteínas de Plantas/genética , Proteínas de Plantas/aislamiento & purificación , Proteínas de Plantas/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
3-hydroxy-3-methyl glutaryl-CoA reductase, also known as HMGR, plays a crucial role in regulating cholesterol biosynthesis and represents the main pharmacological target of statins. In mammals, this enzyme localizes to the endoplasmic reticulum membrane. HMGR includes different regions, an integral N-terminal domain connected by a linker-region to a cytosolic C-terminal domain, the latter being responsible for enzymatic activity. The aim of this work was to design a simple strategy for cloning, expression, and purification of the catalytic C-terminal domain of the human HMGR (cf-HMGR), in order to spectrophotometrically test its enzymatic activity. The recombinant cf-HMGR protein was heterologously expressed in Escherichia coli, purified by Ni+-agarose affinity chromatography and reconstituted in its active form. MALDI mass spectrometry was adopted to monitor purification procedure as a technique orthogonal to the classical Western blot analysis. Protein identity was validated by MS and MS/MS analysis, confirming about 82% of the recombinant sequence. The specific activity of the purified and dialyzed cf-HMGR preparation was enriched about 85-fold with respect to the supernatant obtained from cell lysate. The effective, cheap, and easy method here described could be useful for screening statin-like molecules, so simplifying the search for new drugs with hypocholesterolemic effects.
Asunto(s)
Hidroximetilglutaril-CoA Reductasas/química , Hidroximetilglutaril-CoA Reductasas/genética , Inhibidores de Hidroximetilglutaril-CoA Reductasas/farmacología , Secuencia de Aminoácidos/genética , Dominio Catalítico , Cromatografía de Afinidad , Clonación Molecular , Evaluación Preclínica de Medicamentos/métodos , Pruebas de Enzimas/métodos , Escherichia coli/genética , Expresión Génica , Humanos , Hidroximetilglutaril-CoA Reductasas/aislamiento & purificación , Hidroximetilglutaril-CoA Reductasas/metabolismo , Inhibidores de Hidroximetilglutaril-CoA Reductasas/química , Inhibidores de Hidroximetilglutaril-CoA Reductasas/aislamiento & purificación , Inhibidores de Hidroximetilglutaril-CoA Reductasas/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Espectrometría de Masas en TándemRESUMEN
The enzyme 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase (HMGR), in most organisms, catalyzes the four-electron reduction of the thioester (S)-HMG-CoA to the primary alcohol (R)-mevalonate, utilizing NADPH as the hydride donor. In some organisms, including the opportunistic lung pathogen Burkholderia cenocepacia, it catalyzes the reverse reaction, utilizing NAD+ as a hydride acceptor in the oxidation of mevalonate. B. cenocepacia HMGR has been previously shown to exist as an ensemble of multiple non-additive oligomeric states, each with different levels of enzymatic activity, suggesting that the enzyme exhibits characteristics of the morpheein model of allostery. We have characterized a number of factors, including pH, substrate concentration, and enzyme concentration, that modulate the structural transitions that influence the interconversion among the multiple oligomers. We have also determined the crystal structure of B. cenocepacia HMGR in the hexameric state bound to coenzyme A and ADP. This hexameric assembly provides important clues about how the transition among oligomers might occur, and why B. cenocepacia HMGR, unique among characterized HMGRs, exhibits morpheein-like behavior.
Asunto(s)
Proteínas Bacterianas/metabolismo , Burkholderia cenocepacia/enzimología , Hidroximetilglutaril-CoA Reductasas/metabolismo , Estructura Cuaternaria de Proteína , Adenosina Trifosfato/química , Proteínas Bacterianas/química , Proteínas Bacterianas/aislamiento & purificación , Coenzima A/química , Cristalografía por Rayos X , Pruebas de Enzimas , Hidroximetilglutaril-CoA Reductasas/química , Hidroximetilglutaril-CoA Reductasas/aislamiento & purificación , Simulación de Dinámica Molecular , Multimerización de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismoRESUMEN
Although many aspects of the endoplasmic reticulum (ER)-associated degradation (ERAD) pathway have been elucidated, methods to detect and examine intermediate steps in the process are lacking. Here, we describe the use of a protease protection assay to study the metabolically regulated ERAD substrate HMG CoA reductase. Studies utilizing this assay reveal that ubiquitinated reductase becomes extracted across the ER membrane prior to its cytosolic release and proteasomal degradation through reactions mediated by distinct AAA-ATPases. A similar approach could be applied to other substrates to determine whether membrane extraction is an intermediate step in their ERAD.
Asunto(s)
Retículo Endoplásmico , Epítopos , Hidroximetilglutaril-CoA Reductasas , Membranas Intracelulares , Proteolisis , Animales , Línea Celular , Retículo Endoplásmico/química , Retículo Endoplásmico/enzimología , Epítopos/química , Epítopos/aislamiento & purificación , Humanos , Hidroximetilglutaril-CoA Reductasas/química , Hidroximetilglutaril-CoA Reductasas/aislamiento & purificación , Membranas Intracelulares/química , Membranas Intracelulares/enzimologíaRESUMEN
3-Hydroxy-3-methylglutaryl-CoA reductases (HMGR) plays an important role in catalyzing the first committed step of isoprenoid biosynthesis in the mevelonic (MVA) pathway (catalyzes the conversion of HMG-CoA to MVA) in plants. The present manuscript reports the full length cDNA cloning of HMGR (CaHMGR, GenBank accession number: KJ939450.2) and its characterization from Centella asiatica. Sequence analysis indicated that the cDNA was of 1965 bp, which had an open reading frame of 1617 bp and encoded a protein containing 539 amino-acids with a mol wt of 57.9 kDa. A BLASTp search against non-redundant (nr) protein sequence showed that C. asiatica HMGR (CaHMGR) has 65-81% identity with HMGRs from different plant species and multi-alignment comparison analysis showed the presence of two motif each corresponding to HMG-CoA-binding and NADP(H)-binding. The Conserved Domain Database analysis predicted that CaHMGR belongs to Class I hydroxymethylglutaryl-coenzyme A (HMG-CoA) reductase. Three-dimensional modeling confirmed the novelty of CaHMGR with a spatial structure similar to Homo sapiens (PDB id: 1IDQ8_A). Tissue Expression analysis indicates that CaHMGR is ubiquitous albeit differentially expressed among different tissues analysed, Strong expression was recorded in the nodes and leaves and low in the roots. The present investigation confirmed that nodes are vital to terpenoid synthesis in C. asiatica. Thus, the cloning of full length CDS, characterization and structure-function analysis of HMGR gene in Centella facilitate to understand the HMGR's functions and regulatory mechanisms involved in mevalonate pathway in C. asiatica at genetic level.
Asunto(s)
Centella/enzimología , Hidroximetilglutaril-CoA Reductasas/genética , Secuencia de Aminoácidos , Secuencia de Bases , Centella/genética , Clonación Molecular , Expresión Génica , Hidroximetilglutaril-CoA Reductasas/aislamiento & purificación , Hidroximetilglutaril-CoA Reductasas/metabolismo , Datos de Secuencia Molecular , Filogenia , Hojas de la Planta/enzimología , Hojas de la Planta/genética , Raíces de Plantas/enzimología , Raíces de Plantas/genética , Conformación Proteica , Alineación de SecuenciaRESUMEN
We isolated two full-length cDNAs encoding 3-hydroxy-3-methyl-glutaryl coenzyme A synthase (HMG-S) and 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-R) from the Chinese white pine beetle (Dendroctonus armandi), and carried out some bioinformatic analysis on the full-length nucleic acid sequences and deduced amino acid sequences. Differential expression of the DaHMG-S and DaHMG-R genes was observed between sexes (emerged adults), and within these significant differences among development stage, tissue distribution, fed on phloem of Pinus armandi and topically applied juvenile hormone (JH) III. Increase of DaHMG-S and DaHMG-R mRNA levels in males suggested that they may play a role in mevalonate pathway. Information from the present study might contribute to understanding the relationship between D. armandi and its semiochemical production.
Asunto(s)
Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Hidroximetilglutaril-CoA Reductasas/genética , Hidroximetilglutaril-CoA Sintasa/genética , Gorgojos/crecimiento & desarrollo , Gorgojos/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Biología Computacional , Ingestión de Alimentos , Femenino , Hidroximetilglutaril-CoA Reductasas/química , Hidroximetilglutaril-CoA Reductasas/aislamiento & purificación , Hidroximetilglutaril-CoA Reductasas/metabolismo , Hidroximetilglutaril-CoA Sintasa/química , Hidroximetilglutaril-CoA Sintasa/aislamiento & purificación , Hidroximetilglutaril-CoA Sintasa/metabolismo , Masculino , Ácido Mevalónico/metabolismo , Datos de Secuencia Molecular , Transporte de Proteínas/efectos de los fármacos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Sesquiterpenos/farmacología , Gorgojos/efectos de los fármacos , Gorgojos/fisiologíaRESUMEN
The enzyme 3-hydroxy-3-methylglutaryl CoA (HMG-CoA) reductase catalyzes the NADPH-mediated reductive deacylation of HMG-CoA to mevalonic acid, which is the first committed step of the mevalonate pathway for isoprenoid biosynthesis. In agreement with its key regulatory role in the pathway, plant HMG-CoA reductase is modulated by many diverse external stimuli and endogenous factors and can be detected to variable levels in every plant tissue. A fine determination of HMG-CoA reductase activity levels is required to understand its contribution to plant development and adaptation to changing environmental conditions. Here, we report a procedure to reliably determine HMG-CoA reductase activity in plants. The method includes the sample collection and homogenization strategies as well as the specific activity determination based on a classical radiochemical assay.
Asunto(s)
Arabidopsis/enzimología , Pruebas de Enzimas/métodos , Hidroximetilglutaril-CoA Reductasas/metabolismo , Cromatografía en Capa Delgada , Hidroximetilglutaril-CoA Reductasas/aislamiento & purificación , Especificidad por SustratoRESUMEN
3-Hydroxy-3-methylglutaryl-coenzyme A reductase (HMGR) is the first rate-limiting enzyme for sesquiterpene synthesis in the mevalonate (MVA) pathway. The specific primers were designed according to the transcript sequence of AsHMGR2 from the Aquilaria sinensis (Lour.) Gilg transcriptome database. The full-length cDNA of AsHMGR2 was cloned by RT-PCR and rapid amplification of cDNA ends (RACE) technology, and was analyzed at bioinformatics levels; AsHMGR2 expression profiles in different tissues and in responds to different treatments were analyzed by real-time PCR. The length of AsHMGR2 Open Reading Frame (ORF) was 1 749 bp, encoding 582 amino acids. The GenBank accession number is KC140287. Tissue expression analysis indicated that AsHMGR2 was mainly expressed in root and shoot tips, followed by stem, and was lowest in leaves. Inducible-experiments showed that the genes were induced by mechanical wound as well as chemical liquid induction, and reached the highest expression level at 6 h and 8 h, separately. The full-length cDNA of AsHMGR2 and its expression patterns will provide a foundation for further research on its function in agarwood sesquiterpene biosynthesis.
Asunto(s)
Hidroximetilglutaril-CoA Reductasas/genética , Sistemas de Lectura Abierta , Thymelaeaceae/enzimología , Secuencia de Aminoácidos , Clonación Molecular , ADN Complementario/genética , Amplificación de Genes , Hidroximetilglutaril-CoA Reductasas/aislamiento & purificación , Isoenzimas/genética , Isoenzimas/aislamiento & purificación , Filogenia , Hojas de la Planta/enzimología , Raíces de Plantas/enzimología , Brotes de la Planta/enzimología , Tallos de la Planta/enzimología , Plantas Medicinales/enzimología , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
Class II 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductases are potential targets for novel antibiotic development. In order to obtain a precise structural model for use in virtual screening and inhibitor design, HMG-CoA reductase of Streptococcus pneumoniae was cloned, overexpressed and purified to homogeneity using Ni-NTA affinity chromatography. Crystals were obtained using the hanging-drop vapour-diffusion method. A complete data set was collected from a single frozen crystal on a home X-ray source. The crystal diffracted to 2.3â Å resolution and belonged to the orthorhombic space group C222(1), with unit-cell parameters a = 773.4836, b = 90.3055, c = 160.5592â Å, α = ß = γ = 90°. Assuming the presence of two molecules in the asymmetric unit, the solvent content was estimated to be 54.1% (V(M) = 2.68â Å(3)â Da(-1)).
Asunto(s)
Hidroximetilglutaril-CoA Reductasas/química , Streptococcus pneumoniae/enzimología , Cristalización , Cristalografía por Rayos X , Hidroximetilglutaril-CoA Reductasas/aislamiento & purificaciónRESUMEN
(E)-N-ethyl-N-(6,6-dimethyl-2-hepten-4-ynyl)-3-[2-methyl-2-(3-thienylmethoxy)propyloxy]benzylamine hydrochloride (FR194738) inhibited squalene epoxidase activity in HepG2 cell homogenates with an IC50 value of 9.8 nM. In the study using intact HepG2 cells, FR194738 inhibited cholesterol synthesis from [14C]acetate with an IC50 value of 4.9 nM, and induced intracellular [14C]squalene accumulation. On the other hand, the 3-hydroxy-3-methylglutaryl (HMG)-CoA reductase inhibitor simvastatin reduced both cholesterol and squalene synthesis from [14C]acetate. Incubation with simvastatin for 18 h produced increases in HMG-CoA reductase activity in HepG2 cells, which was related to the degree of reduction in cholesterol synthesis. The HMG-CoA reductase activity increased by 13- and 19-fold at the concentrations of simvastatin that inhibited cholesterol synthesis by 65% and 82%, respectively. In contrast, FR194738 did not increase HMG-CoA reductase activity at the concentrations that inhibited cholesterol synthesis by 24% and 69%, and moderate increase (4.6-fold) was observed at the concentration that inhibited cholesterol synthesis by 90%. These results suggest that non-sterol metabolite(s) derived from mevalonate prior to the squalene epoxidation step in the cholesterol synthetic cascade have a regulatory role in the suppression of HMG-CoA reductase activity. We speculate that FR194738 inhibits cholesterol synthesis with a minimal change of the regulator(s) and would be highly effective in the treatment of hypercholesterolemia.
Asunto(s)
Bencilaminas/farmacología , Colesterol/metabolismo , Inhibidores Enzimáticos/farmacología , Oxigenasas/antagonistas & inhibidores , Simvastatina/análogos & derivados , Colesterol/biosíntesis , Humanos , Hidroximetilglutaril-CoA Reductasas/aislamiento & purificación , Hidroximetilglutaril-CoA Reductasas/metabolismo , Inhibidores de Hidroximetilglutaril-CoA Reductasas/farmacología , Estructura Molecular , Simvastatina/farmacología , Escualeno/metabolismo , Escualeno-Monooxigenasa , Células Tumorales CultivadasRESUMEN
At least three different subcellular compartments, including peroxisomes, are involved in cholesterol biosynthesis. Because proper CNS development depends on de novo cholesterol biosynthesis, peroxisomes must play a critical functional role in this process. Surprisingly, no information is available on the peroxisomal isoprenoid/cholesterol biosynthesis pathway in normal brain tissue or on the compartmentalization of isoprene metabolism in the CNS. This has been due mainly to the lack of a well-defined isolation procedure for brain tissue, and also to the presence of myelin in brain tissue, which results in significant contamination of subcellular fractions. As a first step in characterizing the peroxisomal isoprenoid pathway in the CNS, we have established a purification procedure to isolate peroxisomes and other cellular organelles from the brain stem, cerebellum and spinal cord of the mouse brain. We demonstrate by use of marker enzymes and immunoblotting with antibodies against organelle specific proteins that the isolated peroxisomes are highly purified and well separated from the ER and mitochondria, and are free of myelin contamination. The isolated peroxisomal fraction was purified at least 40-fold over the original homogenate. In addition, we show by analytical subcellular fractionation and immunoelectron microscopy that HMG-CoA reductase protein and activity are localized both in the ER and peroxisomes in the CNS.
Asunto(s)
Sistema Nervioso Central/citología , Sistema Nervioso Central/enzimología , Hidroximetilglutaril-CoA Reductasas/análisis , Hidroximetilglutaril-CoA Reductasas/aislamiento & purificación , Peroxisomas/enzimología , Hidrolasas Diéster Fosfóricas , 2',3'-Nucleótido Cíclico 3'-Fosfodiesterasa , 2',3'-Nucleótido Cíclico Fosfodiesterasas/metabolismo , Animales , Tronco Encefálico/citología , Tronco Encefálico/enzimología , Tronco Encefálico/ultraestructura , Catalasa/metabolismo , Sistema Nervioso Central/ultraestructura , Centrifugación por Gradiente de Densidad , Cerebelo/citología , Cerebelo/enzimología , Cerebelo/ultraestructura , Colesterol/metabolismo , Retículo Endoplásmico/enzimología , Retículo Endoplásmico/ultraestructura , Hidroximetilglutaril-CoA Reductasas/ultraestructura , Hidroximetilglutaril-CoA-Reductasas NADP-Dependientes , Ratones , Ratones Endogámicos ICR , Microscopía Inmunoelectrónica , Peroxisomas/ultraestructura , Médula Espinal/citología , Médula Espinal/enzimologíaRESUMEN
In eukaryotes the enzyme 3-hydroxy-3-methylglutaryl CoA (HMG-CoA) reductase catalyses the synthesis of mevalonic acid, a common precursor to all isoprenoid compounds. Here we report the isolation and overexpression of the gene coding for HMG-CoA reductase from Leishmania major. The protein from Leishmania lacks the membrane domain characteristic of eukaryotic cells but exhibits sequence similarity with eukaryotic reductases. Highly purified protein was achieved by ammonium sulphate precipitation followed by chromatography on hydroxyapatite. Kinetic parameters were determined for the protozoan reductase, obtaining K(m) values for the overall reaction of 40.3+/-5.8 microM for (R,S)-HMG-CoA and 81.4+/-5.3 microM for NADPH; V(max) was 33.55+/-1.8 units x mg(-1). Gel-filtration experiments suggested an apparent molecular mass of 184 kDa with subunits of 46 kDa. Finally, in order to achieve a better understanding of the role of this enzyme in trypanosomatids, the effect of possible regulators of isoprenoid biosynthesis in cultured promastigote cells was studied. Neither mevalonic acid nor serum sterols appear to modulate enzyme activity whereas incubation with lovastatin results in significant increases in the amount of reductase protein. Western- and Northern-blot analyses indicate that this activation is apparently performed via post-transcriptional control.
Asunto(s)
Regulación Enzimológica de la Expresión Génica , Hidroximetilglutaril-CoA Reductasas/química , Hidroximetilglutaril-CoA Reductasas/genética , Leishmania major/enzimología , Secuencia de Aminoácidos , Sulfato de Amonio/farmacología , Animales , Northern Blotting , Western Blotting , Membrana Celular/metabolismo , Células Cultivadas , Cromatografía , Cromatografía en Gel , Mapeo Cromosómico , Durapatita/farmacología , Electroforesis en Gel de Poliacrilamida , Hidroximetilglutaril-CoA Reductasas/aislamiento & purificación , Inhibidores de Hidroximetilglutaril-CoA Reductasas/farmacología , Cetoconazol/farmacología , Cinética , Lovastatina/farmacología , Ácido Mevalónico/farmacología , Datos de Secuencia Molecular , Estructura Terciaria de Proteína , Procesamiento Postranscripcional del ARN , Análisis de Secuencia de ADN , Homología de Secuencia de AminoácidoRESUMEN
The thermostable class I HMG-CoA reductase of Sulfolobus solfataricus offers potential for industrial applications and for the initiation of crystallization trials of a biosynthetic HMG-CoA reductase. However, of the 15 arginine codons of the hmgA gene that encodes S. solfataricus HMG-CoA reductase, 14 (93%) are AGA or AGG, the arginine codons used least frequently by Escherichia coli. The presence of these rare codons in tandem or in the first 20 codons of a gene can complicate expression of that gene in E. coli. Problems include premature chain termination and misincorporation of lysine for arginine. We therefore sought to improve the expression and subsequent yield of S. solfataricus HMG-CoA reductase by expanding the pool size of tRNA(AGA,AGG), the tRNA that recognizes these two rare codons. Coexpression of the S. solfataricus hmgA gene with the argU gene that encodes tRNA(AGA,AGG) resulted in an over 10-fold increase in enzyme yield. This has provided significantly greater quantities of purified enzyme for potential industrial applications and for crystallographic characterization of a stable class I HMG-CoA reductase. It has, in addition, facilitated determination of kinetic parameters and of pH optima for all four catalyzed reactions, for determination of the K(i) for inhibition by the statin drug mevinolin, and for comparison of the properties of the HMG-CoA reductase of this thermophilic archaeon to those of other class I HMG-CoA reductases.
Asunto(s)
Hidroximetilglutaril-CoA Reductasas/biosíntesis , Sulfolobus/enzimología , Acilcoenzima A/metabolismo , Acilación , Cromatografía en Agarosa , Electroforesis en Gel de Poliacrilamida , Escherichia coli/enzimología , Concentración de Iones de Hidrógeno , Hidroximetilglutaril-CoA Reductasas/química , Hidroximetilglutaril-CoA Reductasas/aislamiento & purificación , Cinética , Lovastatina/farmacología , Ácido Mevalónico/metabolismo , ARN de Transferencia de Arginina/genética , ARN de Transferencia de Arginina/metabolismoRESUMEN
The eubacterial 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase (EC 1.1.1.34) was purified 3,000-fold from Streptomyces sp. strain CL190 to apparent homogeneity with an overall yield of 2.1%. The purification procedure consisted of (NH4)2SO4 precipitation, heat treatment and anion exchange, hydrophobic interaction, and affinity chromatographies. The molecular mass of the enzyme was estimated to be 41 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 100 to 105 kDa by gel filtration chromatography, suggesting that the enzyme is most likely to be a dimer. The enzyme showed a pH optimum of around 7.2, with apparent Km values of 62 microM for NADPH and 7.7 microM for HMG-CoA. A gene from CL190 responsible for HMG-CoA reductase was cloned by the colony hybridization method with an oligonucleotide probe synthesized on the basis of the N-terminal sequence of the purified enzyme. The amino acid sequence of the CL190 HMG-CoA reductase revealed several limited motifs which were highly conserved and common to the eucaryotic and archaebacterial enzymes. These sequence conservations suggest a strong evolutionary pressure to maintain amino acid residues at specific positions, indicating that the conserved motifs might play important roles in the structural conformation and/or catalytic properties of the enzyme.
Asunto(s)
Hidroximetilglutaril-CoA Reductasas/genética , Streptomyces/genética , Terpenos/metabolismo , Secuencia de Aminoácidos , Proteínas Bacterianas/genética , Proteínas Bacterianas/aislamiento & purificación , Proteínas Bacterianas/metabolismo , Clonación Molecular , Escherichia coli/genética , Hidroximetilglutaril-CoA Reductasas/aislamiento & purificación , Hidroximetilglutaril-CoA Reductasas/metabolismo , Datos de Secuencia Molecular , Proteínas Recombinantes/aislamiento & purificación , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido , Streptomyces/enzimologíaRESUMEN
The subcellular localization of 3-hydroxy-3-methyl-glutaryl-CoA (HMG-CoA) reductase, which catalyzes the first committed step of the mevalonate pathway, was investigated in Trypanosoma cruzi epimastigotes using well-established cell fractionation procedures. It was found that ca. 80% of the activity of the enzyme was associated with the glycosomes, microbody-like organelles unique to kinetoplastid protozoa which contain most of the enzymes of the glycolytic pathway, while the rest of the activity was found in the soluble (cytoplasmatic) fraction, with almost no activity associated with microsomes. The glycosome-associated enzyme is not membrane-bound as it was recovered quantitatively in the aqueous phase of the biphasic system formed by Triton X-114 at 30 degrees C. Studies with digitonin-permeabilized intact epimastigotes demonstrated the presence of two pools of soluble HMG-CoA reductase in these cells, associated to the cytoplasmic and glycosomal compartments. Steady-state kinetic studies of the glycosome-associated enzyme indicated classical Michaelis-Menten behavior with Km,app (HMG-CoA) 28 +/- 3 microM, Km,app (NADPH) 37 +/- 4 microM, and Vm,app 3.9 +/- 0.2 nmol/min mg protein; the transition-state analog lovastatin behaved as a competitive inhibitor with respect to HMG-CoA with Kis 23 nM and a noncompetitive inhibitor toward NADPH with Kii 29 nM. The results are in complete agreement with recent gene cloning and expression studies which showed that T. cruzi HMG-CoA reductase lacks the NH2-terminal membrane-spanning sequence. This is the first demonstration of a soluble eukaryotic HMG-CoA reductase and also the first report on the presence of an enzyme of the isoprenoid biosynthesis pathway in glycosomes.
Asunto(s)
Hidroximetilglutaril-CoA Reductasas/metabolismo , Trypanosoma cruzi/enzimología , Animales , Fraccionamiento Celular , Digitonina , Glucólisis , Hidroximetilglutaril-CoA Reductasas/aislamiento & purificación , Cinética , Orgánulos/enzimología , Solubilidad , Fracciones Subcelulares/enzimología , Trypanosoma cruzi/metabolismoRESUMEN
We report the isolation and characterization of a genomic clone containing the open reading frame sequence for 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase from Trypanosoma cruzi, the causative agent of Chagas' disease. The protozoan gene encoded for a smaller polypeptide than the rest of the genes described from eukaryotic organisms and the deduced amino acid sequence could be aligned with the C-terminal half of animal and plant reductases exhibiting pronounced similarity to other eukaryotic counterparts. Further examination of the 5' flanking region by cDNA analysis and establishment of the splice acceptor sites clearly indicated that the corresponding mRNA apparently lacks sequences encoding a membrane N-terminal domain. The reductase gene is a single copy and is located on a chromosome of 1.36 Mb as determined by contour-clamped homogeneous electric field electrophoresis. The overall cellular distribution of enzymic activity was investigated after differential centrifugation of Trypanosoma cell extracts. Reductase activity was primarily associated with the cellular soluble fraction because 95% of the total cellular activity was recovered in the supernatant and was particularly sensitive to proteolytic inactivation. Furthermore the enzyme can be efficiently overexpressed in a highly active form by using the expression vector pET-11c. Thus Trypanosoma cruzi HMG-CoA reductase is unique in the sense that it totally lacks the membrane-spanning sequences present in all eukaryotic HMG-CoA reductases so far characterized.
Asunto(s)
Genes Protozoarios , Hidroximetilglutaril-CoA Reductasas/aislamiento & purificación , Proteínas Protozoarias/aislamiento & purificación , Trypanosoma cruzi/enzimología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Mapeo Cromosómico , Clonación Molecular , ADN Complementario/genética , ADN Protozoario/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Humanos , Hidroximetilglutaril-CoA Reductasas/genética , Hidroximetilglutaril-CoA Reductasas/metabolismo , Datos de Secuencia Molecular , Sistemas de Lectura Abierta , Proteínas Protozoarias/genética , Proteínas Protozoarias/metabolismo , Empalme del ARN , Mapeo Restrictivo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Trypanosoma cruzi/genéticaRESUMEN
We isolated two full-length cDNA clones encoding 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) from potato (Solanum tuberosum) L. tubers. The clones, designated hmg2.2 and hmg3.3, are members of previously described gene subfamilies. In addition to being induced by arachidonic acid in tubers, hmg2.2 transcript accumulates developmentally in young flowers, and in mature sepals and ovaries, whereas transcript for hmg3.3 accumulates in mature petals and anthers. Our data suggest that members of specific HMGR-encoding gene subfamilies might be involved in both defense responses and flower development. Accumulation of different HMGR transcripts could provide some control of isoprenoid biosynthesis by producing isoforms specific for classes of end-products produced in particular tissues.
Asunto(s)
Regulación de la Expresión Génica de las Plantas , Hidroximetilglutaril-CoA Reductasas/genética , Familia de Multigenes , Proteínas de Plantas/genética , Solanum tuberosum/enzimología , Solanum tuberosum/genética , Transcripción Genética , Secuencia de Aminoácidos , Ácido Araquidónico/farmacología , Clonación Molecular , ADN Complementario/biosíntesis , Regulación del Desarrollo de la Expresión Génica , Hidroximetilglutaril-CoA Reductasas/biosíntesis , Hidroximetilglutaril-CoA Reductasas/aislamiento & purificación , Datos de Secuencia Molecular , Proteínas de Plantas/biosíntesis , Proteínas de Plantas/aislamiento & purificación , Tallos de la Planta/enzimología , Tallos de la Planta/genéticaRESUMEN
The main molecules of hepatic cholesterol homeostasis are HMGCoA reductase, the key enzyme of the biosynthetic pathway, and LDL receptor, responsible for the uptake of plasma lipoproteins. Estrogens are reported to cause hypolipidemia in mammalians inducing hepatic LDL receptor. The effect of such hormones on HMGCoA reductase is very ambiguous. The mechanism and the time-dependence of the effects of these hormones on HMGCoA reductase and LDL receptor in rat liver have been investigated at mRNA and protein levels, at different times after estrogen administration. Estrogens cause an early increase of LDLr, at both mRNA and protein level, and an increase of HMGCoA reductase, just at protein level, detectable only after 5 days. The independent behavior of LDLr and HMGCoA reductase under estrogen treatment suggests a not coordinate regulation by these hormones.
Asunto(s)
Etinilestradiol/farmacología , Hidroximetilglutaril-CoA Reductasas/metabolismo , Hígado/metabolismo , Receptores de LDL/metabolismo , Transcripción Genética/efectos de los fármacos , Animales , Western Blotting , Electroforesis en Gel de Poliacrilamida , Femenino , Hidroximetilglutaril-CoA Reductasas/biosíntesis , Hidroximetilglutaril-CoA Reductasas/aislamiento & purificación , Cinética , Hígado/efectos de los fármacos , ARN Mensajero/biosíntesis , Ratas , Ratas Sprague-Dawley , Receptores de LDL/biosíntesis , Receptores de LDL/aislamiento & purificación , Factores de Tiempo , Regulación hacia ArribaRESUMEN
Prior work from this laboratory characterized eukaryotic (hamster) and eubacterial (Pseudomonas mevalonii) 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductases. We report here the characterization of an HMG-CoA reductase from the third domain, the archaea. HMG-CoA reductase of the halobacterium Haloferax volcanii was initially partially purified from extracts of H. volcanii. Subsequently, a portion of the H. volcanii lovastatin (formerly called mevinolin) resistance marker mev was subcloned into the Escherichia coli expression vector pT7-7. While no HMG-CoA reductase activity was detectable following expression in E. coli, activity could be recovered after extracts were exposed to 3 M KCl. Following purification to electrophoretic homogeneity, the specific activity of the expressed enzyme, 24 microU/mg, equaled that of homogeneous hamster or P. mevalonii HMG-CoA reductase. Activity was optimal at pH 7.3. Kms were 66 microM (NADPH) and 60 microM [(S)-HMG-CoA]. (R)-HMG-CoA and lovastatin inhibited competitively with (S)-HMG-CoA. H. volcanii HMG-CoA reductase also catalyzed the reduction of mevaldehyde [optimal activity at pH 6.0; Vmax 11 microU/mg; Kms 32 microM (NADPH), 550 microM [(R,S)-mevaldehyde]] and the oxidative acylation of mevaldehyde [optimal activity at pH 8.0; Vmax 2.1 microU/mg; Kms 350 microM (NADP+), 300 microM (CoA), 470 microM [(R,S)-mevaldehyde]]. These properties are comparable to those of hamster and P. mevalonii HMG-CoA reductases, suggesting a similar catalytic mechanism.