Functional and evolutionary analysis of DXL1, a non-essential gene encoding a 1-deoxy-D-xylulose 5-phosphate synthase like protein in Arabidopsis thaliana

The synthesis of 1-deoxy-D-xylulose 5-phosphate (DXP), catalyzed by the enzyme DXP synthase (DXS), represents a key regulatory step of the 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway for isoprenoid biosynthesis. In plants DXS is encoded by small multigene families that can be classified into, at least, three specialized subfamilies. Arabidopsis thaliana contains three genes encoding proteins with similarity to DXS, including the well-known DXS1/CLA1 gene, which clusters within subfamily I. The remaining proteins, initially named DXS2 and DXS3, have not yet been characterized. Here we report the expression and functional analysis of A. thaliana DXS2. Unexpectedly, the expression of DXS2 failed to rescue Escherichia coli and A. thaliana mutants defective in DXS activity. Coherently, we found that DXS activity was negligible in vitro, being renamed as DXL1 following recent nomenclature recommendation. DXL1 is targeted to plastids as DXS1, but shows a distinct expression pattern. The phenotypic analysis of a DXL1 defective mutant revealed that the function of the encoded protein is not essential for growth and development. Evolutionary analyses indicated that DXL1 emerged from DXS1 through a recent duplication apparently specific of the Brassicaceae lineage. Divergent selective constraints would have affected a significant fraction of sites after diversification of the paralogues. Furthermore, amino acids subjected to divergent selection and likely critical for functional divergence through the acquisition of a novel, although not yet known, biochemical function, were identified. Our results provide with the first evidences of functional specialization at both the regulatory and biochemical level within the plant DXS family

Anàlisi molecular de la biosíntesi d'isoprenoides a "Arabidopsis".

[cat] La present tesi doctoral ha aprofunfitzat en el coneixement de la regulació de la biosíntesi d'isoprenoides a Arabidopsis. Els isoprenoides es sintetitzen a partir dels precursors isopentenil difosfat i del seu isòmer al·lilic, el dimetilalildifosfat. En plantes existeixen dues rutes de síntesi d'aquests precursors, la ruta del metileritritol fosfat (MEP), localitzada en els plàstids, i la ruta del mevalonat (MVA), localitzada en el citosol. L'enzim 1-desoxi-D-xilulosa 5-fosfat sintasa (DXS) catalitza la primera reacció de la ruta del MEP, i a Arabidopsis existeixen tres gens que codifiquen per possibles isoformes d'aquest enzim, DXS1, DXS2 i DXS3. Pel que fa a DXS1, la seva activitat ja habia estat demostrada, mentre que DXS3 presenta una seqüència aminoacídica molt divergida i, per tant, s'ha descartat que pugui tenir activitat DXS. En aquest treball s'ha demostrat que la DXS2 tampoc té activitat DXS que possibement està evolucionant (o ha evolucionat) cap a una nova funcionalitat. D'altra banda, s'ha demostrat que la molècula 1-desoxi-D-xilulosa (DX), després de fosforilar-se a DX5P resulta tòxica pel creixement d'Arabidopsis i E. coli. Aquesta toxicitat és produïda en el citosol de la cèl·lula, i probablement és deguda a una alteració del metabolisme glucídic. Finalment, s'han aïllat 16 mutants resistents a la inhibició per fosmidomicina (FSM), un inhibidor específic de l'enzim DXR de la ruta del MEP, anomenats rif. L'anàlisi bioquímic d'aquests mutants rif ha mostrat que en la majoria de casos els nivells de proteïna DXR es troben incrementats permetent la resistència a la FSM. La identificació del lloc d'inserció del T-DNA en els mutants rif i l'estudi dels possibles gens implicats en la resistència a la FSM, ha permès concloure que existeixen multitud de processos cel·lulars que regulen la homeostàsi de la biosíntesi d'isoprenoides a Arabidopsis.[eng] This thesis has depht in knowledge of the regulation of isoprenoid biosynthesis in Arabidopsis. All isoprenoids are synthesized from the precursors isopentenyl diphosphate and its isomer dimethylallyl pyrophosphate. In plants there are two routes of synthesis of these precursors, the metileritritol phosphate pathway (MEP), located in plastids, and the mevalonate pathway (MVA), located in the cytosol. The enzyme 1-deoxy-D-xilulose 5-phosphate synthase (DXS) catalyzes the first reaction of the MEP pathway, and there are three Arabidopsis genes encoding potential isoforms of this enzyme, DXS1, DXS2 and DXS3. Regarding DXS1, its activity was already been shown, while DXS3 presents a highly diverged amino acid sequence and therefore might have been dismissed DXS activity. This work has shown that DXS2 has not DXS activity, and that this gene is evolving (or has evolved) to a new functionality. Moreover, it has been shown that the molecule 1-deoxy-D-xilulose (DX), after being phosphorilated to DX5P is toxic to the growth of Arabidopsis and E. coli. This toxicity is produced in the cytosol of the cell, and it's probably due to an alteration of the glucose metabolism. Finally, we have isolated 16 mutants resistant to the inhibition by fosmidomycin (FSM), an specific inhibitor of the enzyme DXR of the MEP pathway, and they have been called rif mutants. The biochemical analysis of these rif mutants has shown that in most cases DXR protein levels are increased, allowing to FSM resistance. Site identification of the T-DNA insertion in the rif mutants and the study of the possible genes involved in the resistance to FSM reveals that there are a multitude of cellular processes that regulate the homeostasys of the biosynthesis of isoprenoids in Arabidopsis

Functional and evolutionary analysis of DXL1, a non-essential gene encoding a 1-deoxy-D-xylulose 5-phosphate synthase like protein in Arabidopsis thaliana

The synthesis of 1-deoxy-D-xylulose 5-phosphate (DXP), catalyzed by the enzyme DXP synthase (DXS), represents a key regulatory step of the 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway for isoprenoid biosynthesis. In plants DXS is encoded by small multigene families that can be classified into, at least, three specialized subfamilies. Arabidopsis thaliana contains three genes encoding proteins with similarity to DXS, including the well-known DXS1/CLA1 gene, which clusters within subfamily I. The remaining proteins, initially named DXS2 and DXS3, have not yet been characterized. Here we report the expression and functional analysis of A. thaliana DXS2. Unexpectedly, the expression of DXS2 failed to rescue Escherichia coli and A. thaliana mutants defective in DXS activity. Coherently, we found that DXS activity was negligible in vitro, being renamed as DXL1 following recent nomenclature recommendation. DXL1 is targeted to plastids as DXS1, but shows a distinct expression pattern. The phenotypic analysis of a DXL1 defective mutant revealed that the function of the encoded protein is not essential for growth and development. Evolutionary analyses indicated that DXL1 emerged from DXS1 through a recent duplication apparently specific of the Brassicaceae lineage. Divergent selective constraints would have affected a significant fraction of sites after diversification of the paralogues. Furthermore, amino acids subjected to divergent selection and likely critical for functional divergence through the acquisition of a novel, although not yet known, biochemical function, were identified. Our results provide with the first evidences of functional specialization at both the regulatory and biochemical level within the plant DXS family