RESUMEN
Xylella fastidiosa is a xylem-restricted plant pathogen that causes a range of diseases in several and important crops. Through comparative genomic sequence analysis many genes were identified and, among them, several potentially involved in plant-pathogen interaction. The experimental determination of the primary sequence of some markedly expressed proteins for X. fastidiosa and the comparison with the nucleic acids sequence of genome identified one of them as being SCJ21.16 (XFa0032) gene product. The comparative analysis of this protein against SWISSPROT database, in special, resulted in similarity with alpha-hydroxynitrile lyase enzyme (HNL) from Arabidopsis thaliana, causing interest for being one of the most abundant proteins both in the whole cell extract as well as in the extracellular protein fraction. It is known that HNL enzyme are involved in a process termed "cyanogenesis", which catalyzes the dissociation of alpha-hydroxinitrile into carbonyle and HCN when plant tissue is damaged. Although the complete genome sequences of X. fastidiosa are available and the cyanogenesis process is well known, the biological role of this protein in this organism is not yet functionally characterized. In this study we presented the cloning, expression, characterization of recombinant HNL from X. fastidiosa, and its probable function in the cellular metabolism. The successful cloning and heterologous expression in Escherichia coli resulted in a satisfactory amount of the recombinant HNL expressed in a soluble, and active form giving convenient access to pure enzyme for biochemical and structural studies. Finally, our results confirmed that the product of the gene XFa0032 can be positively assigned as FAD-independent HNLs.
Asunto(s)
Aldehído-Liasas/química , Proteínas Bacterianas/química , Clonación Molecular , Expresión Génica , Xylella/enzimología , Aldehído-Liasas/genética , Aldehído-Liasas/aislamiento & purificación , Aldehído-Liasas/metabolismo , Secuencia de Aminoácidos , Proteínas Bacterianas/genética , Proteínas Bacterianas/aislamiento & purificación , Proteínas Bacterianas/metabolismo , Estabilidad de Enzimas , Escherichia coli/genética , Escherichia coli/metabolismo , Datos de Secuencia Molecular , Peso Molecular , Alineación de Secuencia , Xylella/química , Xylella/genéticaRESUMEN
Investigations of the (S)-selective hydroxynitrile lyase from Hevea brasiliensis were performed by electrospray mass spectroscopy, (1)H-NMR and with an enzyme activity assay. For the trans-cyanohydrin reaction (transcyanation) a two step reaction could be established. The results furthermore indicate a fast deactivation of the enzyme at low pH and a strong substrate dependence of its stability. They rule out an enzyme-HCN complex or a covalently bound carbonyl compound. Therefore the earlier postulated reaction intermediate as well as the proposed action of the catalytic triad have to be reevaluated. The calculated molecular mass could be confirmed by mass spectroscopy.