RESUMO
Xanthomonas axonopodis pv. citri (Xac) SufE (XAC2355) is a member of a family of bacterial proteins that are conserved in several pathogens and phytopathogens. The Escherichia coli suf operon is involved in iron-sulfur cluster biosynthesis under iron-limitation and stress conditions. It has recently been demonstrated that SufE and SufS form a novel two-component cysteine desulfarase in which SufS catalyses the conversion of L-cysteine to L-alanine, forming a protein-bound persulfide intermediate. The S atom is then transferred to SufE, from which it is subsequently transferred to target molecules or reduced to sulfide in solution. Here, the cloning, expression, crystallization and phase determination of Xac SufE crystals are described. Recombinant SufE was crystallized in space group P2(1)2(1)2(1) and diffracted to 1.9 A resolution at a synchrotron source. The unit-cell parameters are a = 45.837, b = 58.507, c = 98.951 A, alpha = beta = gamma = 90 degrees. The calculated Matthews coefficient indicated the presence of two molecules in the asymmetric unit. Phasing was performed by molecular-replacement using E. coli SufE as a model (PDB code 1mzg) and an interpretable map was obtained.
Assuntos
Proteínas de Bactérias/química , Xanthomonas/química , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/isolamento & purificação , Liases de Carbono-Enxofre/biossíntese , Liases de Carbono-Enxofre/química , Liases de Carbono-Enxofre/isolamento & purificação , Clonagem Molecular , Cristalização/métodos , Cristalografia por Raios XRESUMO
Xanthomonas axonopodis pv. citri YaeQ (XAC2396) is a member of a family of bacterial proteins conserved in several Gram-negative pathogens. Here, the cloning, expression, purification and crystallization of the 182-residue (20.6 kDa) YaeQ protein are described. Recombinant YaeQ containing selenomethionine was crystallized in space group P2(1) and crystals diffracted to 1.9 A resolution at a synchrotron source. The unit-cell parameters are a = 39.75, b = 91.88, c = 48.03 A, beta = 108.37 degrees. The calculated Matthews coefficient suggests the presence of two YaeQ molecules in the asymmetric unit. Initial experimental phases were calculated by the multiple-wavelength anomalous dispersion technique and an interpretable electron-density map was obtained.
Assuntos
Proteínas de Bactérias/química , Xanthomonas/enzimologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Proteínas de Bactérias/metabolismo , Clonagem Molecular , Cristalização , Cristalografia por Raios X , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Selenometionina/química , Selenometionina/metabolismo , Xanthomonas/genética , Xanthomonas/metabolismoRESUMO
We applied a high-throughput strategy for the screening of targets for structural proteomics of Xanthomonas axonopodis pv citri. This strategy is based on the rapid (1)H-(15)N HSQC NMR analysis of bacterial lysates containing selectively (15)N-labelled heterologous proteins. Our analysis permitted us to classify the 19 soluble candidates in terms of 'foldedness', that is, the extent to which they present a well-folded solution structure, as reflected by the quality of their NMR spectra. This classification allowed us to define a priority list to be used as a guide to select protein candidates for further structural studies.
