RESUMEN
Isovaleryl-CoA dehydrogenase (IVD) is an intramitochondrial homotetrameric flavoenzyme that catalyzes the conversion of isovaleryl-CoA to 3-methylcrotonyl-CoA in the leucine catabolism pathway. Deficiency of IVD in humans causes isovaleric acidemia, which shows tremendous clinical variability for reasons that are unknown. To help better understand this disorder, we have cloned and sequenced the mouse IVD genomic and cDNAs. The mouse IVD gene spans approximately 17 kb and contains 12 coding exons organized identically to the human gene. It maps to mouse chromosome 2 in the area of band 2E4-E5, corresponding to the syntenic region of human chromosome 15. Mouse IVD predicted amino acid sequences are 95.8 and 89.6% identical to that of the rat and human sequences, respectively, with conservation of key functional residues. We have now identified IVD sequences from seven species. Comparison of these sequences shows that the rat and mouse proteins are the most closely related, both of which, in turn, share highest homology to human. All of the mammalian enzymes appear to be more closely related than any of the IVDs on other branches of the phylogram, while the fly and worm IVDs are the most divergent. The invertebrate IVDs are more closely related to the mammalian enzymes than to those from two plant species.
Asunto(s)
ADN Complementario/genética , ADN/genética , Evolución Molecular , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH , Oxidorreductasas/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Clonación Molecular , ADN/química , ADN Complementario/química , Exones , Genes/genética , Intrones , Isovaleril-CoA Deshidrogenasa , Ratones , Datos de Secuencia Molecular , Filogenia , Alineación de Secuencia , Análisis de Secuencia de ADN , Homología de Secuencia de AminoácidoRESUMEN
Isovaleryl-CoA dehydrogenase (IVD, EC ) catalyzes the third step in the catabolism of leucine in mammals. Deficiency of this enzyme leads to the clinical disorder isovaleric acidemia. IVD has been purified and characterized from human and rat liver, and the x-ray crystallographic structure of purified recombinant human IVD has been reported. Nothing is known about IVD activity in plants, although cDNA clones from Arabidopsis thaliana and partial sequences from Gossypium hirsutum and Oryza sativa have been identified as putative IVDs based on sequence homology and immuno cross-reactivity. In this report we describe the identification and characterization of an IVD from pea, purification of the enzyme using a novel and rapid auxin affinity chromatography matrix, and cloning of the corresponding gene. At the amino acid level, pea IVD is 60% similar to human and rat IVD. The specific activity and abundance of plant IVD was found to be significantly lower than for its human counterpart and exhibits developmental regulation. Substrate specificity of the plant enzyme is similar to the human IVD, and it cross-reacts to anti-human IVD antibodies. Molecular modeling of the pea enzyme based on the structure of human IVD indicates a high degree of structural similarity among these enzymes. Glu-244, shown to function as the catalytic base in human IVD along with most of the amino acids that make up the acyl CoA binding pocket, is conserved in pea IVD. The genomic structure of the plant IVD gene consists of 13 exons and 12 introns, spanning approximately 4 kilobases, and the predicted RNA splicing sites exhibit the extended consensus sequence described for other plant genes.
Asunto(s)
Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH , Oxidorreductasas/aislamiento & purificación , Pisum sativum/enzimología , Secuencia de Aminoácidos , Secuencia de Bases , Clonación Molecular , Isovaleril-CoA Deshidrogenasa , Modelos Moleculares , Datos de Secuencia Molecular , Oxidorreductasas/química , Oxidorreductasas/genética , Especificidad por SustratoRESUMEN
Several approaches were successfully performed to directly assign and characterize auxin binding of ABP44 in gel. The 44 kDa high affinity auxin binding protein ABP44 from pea was tested for its ability to bind 5-azido-[7-3H]-IAA in photoaffinity labeling experiments. Competition experiments with several auxin analogues confirm data published previously (Reinard and Jacobsen 1995). Critical reflections of the limitations of the method are also discussed. Immunostaining using the antibody D16 (Napier and Venis 1992), which is directed against the putative binding site of ABP1, revealed that ABP44's auxin binding site is at least partially related to the corresponding site of ABP1. Nevertheless, both proteins do not share any further immunological relationships. Our results with D16 recommend a careful reconsideration of data published by other authors. Furthermore, a 80 kDa, dimeric glutathione dependent formaldehyde dehydrogenase (FDH) from mung bean, described recently, was found to be different from ABP44. In contrast to the described FDH, ABP44 exhibited no FDH activity.
Asunto(s)
Ácidos Indolacéticos/metabolismo , Reguladores del Crecimiento de las Plantas , Proteínas de Plantas/metabolismo , Receptores de Superficie Celular/metabolismo , GelesRESUMEN
A simple and inexpensive system for equilibrium dialysis-based binding assays has been developed employing 1.5-ml microtubes. The main advantages are small volume of test buffers, no time-consuming assembly of the test vials, and inexpensive test cells. A comparison of the results derived from this equilibrium dialysis system with those of polyethylenimine-filtration assay and those of a commercially available equilibrium dialysis test system (Diachema from Dianorm) shows good agreement.