RESUMO
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.
Assuntos
Aldeído Liases/química , Proteínas de Bactérias/química , Clonagem Molecular , Expressão Gênica , Xylella/enzimologia , Aldeído Liases/genética , Aldeído Liases/isolamento & purificação , Aldeído Liases/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Proteínas de Bactérias/metabolismo , Estabilidade Enzimática , Escherichia coli/genética , Escherichia coli/metabolismo , Dados de Sequência Molecular , Peso Molecular , Alinhamento de Sequência , Xylella/química , Xylella/genéticaRESUMO
Cu,Zn superoxide dismutase (Cu,Zn SOD) is an essential enzyme for protecting cells from the toxic effects of reactive oxygen species. In humans, two distinct Cu,Zn SOD genes are located on chromosomes 4 and 21 and mutations in the latter have been associated with familial amyotrophic lateral sclerosis. Similarly, schistosomes (trematode parasites responsible for the chronically debilitating disease schistosomiasis) also produce two distinct Cu,Zn SODs, in this case one cytosolic and one bearing a signal peptide. The crystal structure of the cytosolic form of the enzyme from the human trematode Schistosoma mansoni (SmCtSOD) was solved and refined to a resolution of 2.2 A (space group P2(1)2(1)2(1), R = 17.6% and R(free) = 24.1%) and 1.55 A (space group P2(1), R = 15.7% and R(free) = 17.1%). This is the first report of a crystal structure of a Cu,Zn superoxide dismutase derived from a human parasite. Alternate positions for the catalytic copper and its water ligand were refined for the 1.55 A SmCtSOD model, but the most interesting structural differences between SmCtSOD and the human homologue reside in the loops used for electrostatic guidance of the substrate to the enzyme active site.