RESUMEN
Tryptophanase (Trpase) is a pyridoxal 5'-phosphate (PLP)-dependent homotetrameric enzyme which catalyzes the degradation of L-tryptophan. Trpase is also known for its cold lability, which is a reversible loss of activity at low temperature (2°C) that is associated with the dissociation of the tetramer. Escherichia coli Trpase dissociates into dimers, while Proteus vulgaris Trpase dissociates into monomers. As such, this enzyme is an appropriate model to study the protein-protein interactions and quaternary structure of proteins. The aim of the present study was to understand the differences in the mode of dissociation between the E. coli and P. vulgaris Trpases. In particular, the effect of mutations along the molecular axes of homotetrameric Trpase on its dissociation was studied. To answer this question, two groups of mutants of the E. coli enzyme were created to resemble the amino-acid sequence of P. vulgaris Trpase. In one group, residues 15 and 59 that are located along the molecular axis R (also termed the noncatalytic axis) were mutated. The second group included a mutation at position 298, located along the molecular axis Q (also termed the catalytic axis). Replacing amino-acid residues along the R axis resulted in dissociation of the tetramers into monomers, similar to the P. vulgaris Trpase, while replacing amino-acid residues along the Q axis resulted in dissociation into dimers only. The crystal structure of the V59M mutant of E. coli Trpase was also determined in its apo form and was found to be similar to that of the wild type. This study suggests that in E. coli Trpase hydrophobic interactions along the R axis hold the two monomers together more strongly, preventing the dissociation of the dimers into monomers. Mutation of position 298 along the Q axis to a charged residue resulted in tetramers that are less susceptible to dissociation. Thus, the results indicate that dissociation of E. coli Trpase into dimers occurs along the molecular Q axis.
Asunto(s)
Proteínas Bacterianas/química , Escherichia coli/química , Subunidades de Proteína/química , Proteus vulgaris/química , Triptófano/química , Triptofanasa/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Sitios de Unión , Biocatálisis , Cristalografía por Rayos X , Escherichia coli/enzimología , Escherichia coli/genética , Expresión Génica , Cinética , Modelos Moleculares , Mutación , Unión Proteica , Multimerización de Proteína , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , Proteus vulgaris/enzimología , Proteus vulgaris/genética , Fosfato de Piridoxal/química , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidad de la Especie , Homología Estructural de Proteína , Triptófano/metabolismo , Triptofanasa/genética , Triptofanasa/metabolismoRESUMEN
The cuticle, covering the surface of all primary plant organs, plays important roles in plant development and protection against the biotic and abiotic environment. In contrast to vegetative organs, very little molecular information has been obtained regarding the surfaces of reproductive organs such as fleshy fruit. To broaden our knowledge related to fruit surface, comparative transcriptome and metabolome analyses were carried out on peel and flesh tissues during tomato (Solanum lycopersicum) fruit development. Out of 574 peel-associated transcripts, 17% were classified as putatively belonging to metabolic pathways generating cuticular components, such as wax, cutin, and phenylpropanoids. Orthologs of the Arabidopsis (Arabidopsis thaliana) SHINE2 and MIXTA-LIKE regulatory factors, activating cutin and wax biosynthesis and fruit epidermal cell differentiation, respectively, were also predominantly expressed in the peel. Ultra-performance liquid chromatography coupled to a quadrupole time-of-flight mass spectrometer and gas chromatography-mass spectrometry using a flame ionization detector identified 100 metabolites that are enriched in the peel tissue during development. These included flavonoids, glycoalkaloids, and amyrin-type pentacyclic triterpenoids as well as polar metabolites associated with cuticle and cell wall metabolism and protection against photooxidative stress. Combined results at both transcript and metabolite levels revealed that the formation of cuticular lipids precedes phenylpropanoid and flavonoid biosynthesis. Expression patterns of reporter genes driven by the upstream region of the wax-associated SlCER6 gene indicated progressive activity of this wax biosynthetic gene in both fruit exocarp and endocarp. Peel-associated genes identified in our study, together with comparative analysis of genes enriched in surface tissues of various other plant species, establish a springboard for future investigations of plant surface biology.
Asunto(s)
Perfilación de la Expresión Génica , Genes de Plantas , Solanum lycopersicum/metabolismo , Secuencia de Bases , Cromatografía Liquida , Cartilla de ADN , Cromatografía de Gases y Espectrometría de Masas , Solanum lycopersicum/genética , Espectrometría de Masas , Familia de Multigenes , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
The gene encoding the wild type Integrase protein of coliphage HK022 was integrated chromosomally and expressed in Arabidopsis thaliana plants. Double-transgenic plants cloned with the int gene as well as with a T-DNA fragment carrying the proper att sites in a tandem orientation showed that Int catalyzed a site-specific integration reaction (attP x attB) as well as a site-specific excision reaction (attL x attR). The reactions took place without the need to provide any of the accessory proteins that are required by Int in the bacterial host. When expressed in tobacco plants a GFP-Int fusion exhibits a predominant nuclear localization.