RESUMEN
Proline is a common compatible osmolyte in higher plants. Proline accumulation in response to water stress and salinity is preceded by a rapid increase of the mRNA level of delta 1-pyrroline-5-carboxylate synthase (P5CS) controlling the rate-limiting step of glutamate-derived proline biosynthesis. P5CS is encoded by two differentially regulated genes in Arabidopsis. Gene AtP5CS1 mapped to chromosome 2-78.5 is expressed in most plant organs, but silent in dividing cells. Gene AtP5CS2 located close to marker m457 on chromosome 3-101.3 contributes 20-40% of total P5CS mRNA in plant tissues, but is solely responsible for the synthesis of abundant P5CS mRNA in rapidly dividing cell cultures. Accumulation of AtP5CS transcripts is regulated in a tissue specific manner and inducible by drought, salinity, ABA, and to a lesser extent by auxin. Induction of AtP5CS1 mRNA accumulation in salt-treated seedlings involves an immediate early transcriptional response regulated by ABA signalling that is not inhibited by cycloheximide, but abolished by the deficiency of ABA biosynthesis in the aba1 Arabidopsis mutant. However, inhibition of protein synthesis by cycloheximide prevents the induction of AtP5CS2 mRNA accumulation, and blocks further increase of AtP5CS1 mRNA levels during the second, slow phase of salt-induction. Mutations abi1 and axr2, affecting ABA-perception in Arabidopsis, reduce the accumulation of both AtP5CS mRNAs during salt-stress, whereas ABA-signalling functions defined by the abi2 and abi3 mutations have no effect on salt-induction of the AtP5CS genes.
Asunto(s)
Ácido Abscísico/fisiología , Proteínas de Arabidopsis , Arabidopsis/genética , Regulación de la Expresión Génica de las Plantas , Sustancias de Crecimiento , Ornitina-Oxo-Ácido Transaminasa/genética , Fosfoproteínas Fosfatasas/fisiología , Proteínas de Plantas/fisiología , Prolina/metabolismo , Equilibrio Hidroelectrolítico/fisiología , Secuencia de Aminoácidos , Arabidopsis/enzimología , Arabidopsis/fisiología , Mapeo Cromosómico , ADN de Plantas/química , Meristema/enzimología , Meristema/fisiología , Datos de Secuencia Molecular , Concentración Osmolar , Proteínas de Plantas/genética , ARN Mensajero/metabolismo , ARN de Planta/metabolismo , Alineación de SecuenciaRESUMEN
Dihydrodipicolinate synthase (DHDPS) is the first enzyme unique to the lysine biosynthetic pathway and is feedback regulated by L-lysine in plants and some bacteria. The allosteric binding site has been localized by X-ray crystallography and is in agreement with reported mutations of plant DHDPS enzymes, which confer insensitivity to feedback inhibition. Three possible elements of the mechanism of lysine inhibition are discussed.
Asunto(s)
Hidroliasas/antagonistas & inhibidores , Isoenzimas/antagonistas & inhibidores , Lisina/fisiología , Secuencia de Aminoácidos , Bacterias/enzimología , Retroalimentación/fisiología , Hidroliasas/metabolismo , Isoenzimas/metabolismo , Lisina/metabolismo , Lisina/farmacología , Datos de Secuencia Molecular , Plantas/enzimologíaRESUMEN
Dihydrodipicolinate synthase (DHDPS) catalyzes the condensation of pyruvate with L-aspartate beta-semialdehyde. It is the first enzyme unique to the diaminopimelate pathway of lysine biosynthesis. Here we present the crystal structures of five complexes of Escherichia coli DHDPS with substrates, substrate analogs, and inhibitors. These include the complexes of DHDPS with (1) pyruvate, (2) pyruvate and the L-aspartate beta-semialdehyde analog succinate beta-semialdehyde, (3) the inhibitor alpha-ketopimelic acid, (4) dipicolinic acid, and (5) the natural feedback inhibitor L-lysine. The kinetics of inhibition were determined, and the binding site of the L-lysine was identified. NMR experiments were conducted in order to elucidate the nature of the product of the reaction catalyzed by DHDPS. By this method, (4S)-4-hydroxy-2,3,4,5-tetrahydro-(2S)-dipicolinic acid is identified as the only product. A reaction mechanism for DHDPS is proposed, and important features for inhibition are identified.
Asunto(s)
Escherichia coli/enzimología , Hidroliasas/química , Proteínas Bacterianas/química , Sitios de Unión , Cristalización , Cristalografía por Rayos X , Hidroliasas/metabolismo , Lisina/química , Lisina/metabolismo , Espectroscopía de Resonancia Magnética , Modelos Químicos , Modelos Moleculares , Estructura Molecular , Ácidos Picolínicos/química , Ácidos Picolínicos/metabolismo , Conformación Proteica , Piruvatos/química , Piruvatos/metabolismo , Bases de Schiff/metabolismo , Succinatos/química , Succinatos/metabolismoRESUMEN
DHDPS is the first enzyme unique to the lysine biosynthetic pathway in plants and bacteria and catalyses the formation of (4S)-4-hydroxy-2,3,4,5-tetrahydro-(2S)-dipicolinic acid. It is feedback-regulated in plants by L-lysine. The crystal structure of Nicotiana sylvestris DHDPS with and without inhibitory lysine bound to the enzyme has been solved to a resolution of 2.8 A. The molecule is a homotetramer composed of a dimer of dimers. Comparison with the structure of Escherichia coli DHDPS showed a novel quaternary structure by a profound rearrangement of the dimers forming the tetramer. The crystal structure of the enzyme in the presence of L-lysine revealed substantial changes. These changes together with the novel quaternary structure provide a structural basis for the strong inhibition of plant DHDPS enzymes by L-lysine.