RESUMEN
Uracil-DNA glycosylase (UNG), a repair enzyme involved in the excision of uracil from DNA, from mycobacteria differs from UNGs from other sources, particularly in the sequence in the catalytically important loops. The structure of the enzyme from Mycobacterium tuberculosis (MtUng) in complex with a proteinaceous inhibitor (Ugi) has been determined by X-ray analysis of a crystal containing seven crystallographically independent copies of the complex. This structure provides the first geometric characterization of a mycobacterial UNG. A comparison of the structure with those of other UNG proteins of known structure shows that a central core region of the molecule is relatively invariant in structure and sequence, while the N- and C-terminal tails exhibit high variability. The tails are probably important in folding and stability. The mycobacterial enzyme exhibits differences in UNG-Ugi interactions compared with those involving UNG from other sources. The MtUng-DNA complex modelled on the basis of the known structure of the complex involving the human enzyme indicates a domain closure in the enzyme when binding to DNA. The binding involves a larger burial of surface area than is observed in binding by human UNG. The DNA-binding site of MtUng is characterized by the presence of a higher proportion of arginyl residues than is found in the binding site of any other UNG of known structure. In addition to the electrostatic effects produced by the arginyl residues, the hydrogen bonds in which they are involved compensate for the loss of some interactions arising from changes in amino-acid residues, particularly in the catalytic loops. The results arising from the present investigation represent unique features of the structure and interaction of mycobacterial Ungs.
Asunto(s)
Proteínas Bacterianas/química , Mycobacterium tuberculosis/enzimología , Uracil-ADN Glicosidasa/química , Secuencia de Aminoácidos , Animales , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Sitios de Unión , ADN/química , ADN/metabolismo , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Unión Proteica , Estructura Secundaria de Proteína , Homología de Secuencia de Aminoácido , Uracil-ADN Glicosidasa/genética , Uracil-ADN Glicosidasa/metabolismoRESUMEN
The mechanism of translation in eubacteria and organelles is thought to be similar. In eubacteria, the three initiation factors IF1, IF2, and IF3 are vital. Although the homologs of IF2 and IF3 are found in mammalian mitochondria, an IF1 homolog has never been detected. Here, we show that bovine mitochondrial IF2 (IF2(mt)) complements E. coli containing a deletion of the IF2 gene (E. coli DeltainfB). We find that IF1 is no longer essential in an IF2(mt)-supported E. coli DeltainfB strain. Furthermore, biochemical and molecular modeling data show that a conserved insertion of 37 amino acids in the IF2(mt) substitutes for the function of IF1. Deletion of this insertion from IF2(mt) supports E. coli for the essential function of IF2. However, in this background, IF1 remains essential. These observations provide strong evidence that a single factor (IF2(mt)) in mammalian mitochondria performs the functions of two eubacterial factors, IF1 and IF2.
Asunto(s)
Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Factores Eucarióticos de Iniciación/metabolismo , Proteínas Mitocondriales/metabolismo , Factor 1 Procariótico de Iniciación/metabolismo , Factor 2 Procariótico de Iniciación/metabolismo , Animales , Bovinos , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Factores Eucarióticos de Iniciación/genética , Eliminación de Gen , Prueba de Complementación Genética , Proteínas Mitocondriales/genética , Modelos Moleculares , Factor 1 Procariótico de Iniciación/genética , Factor 2 Procariótico de Iniciación/genética , Homología de Secuencia de AminoácidoRESUMEN
Uracil N-glycosylase is an enzyme which initiates the pathway of uracil-excision repair of DNA. The enzyme from Mycobacterium tuberculosis was co-expressed with a proteinaceous inhibitor from Bacillus subtilis phage and was crystallized in monoclinic space group C2, with unit-cell parameters a = 201.14, b = 64.27, c = 203.68 A, beta = 109.7 degrees. X-ray data from the crystal have been collected for structure analysis.