RESUMEN
Penicillin-binding proteins (PBPs) are membrane proteins involved in the final stages of peptidoglycan synthesis and represent the targets of beta-lactam antibiotics. Enterococci are naturally resistant to these antibiotics because they produce a PBP, named PBP5fm in Enterococcus faecium, with low-level affinity for beta-lactams. We report here the crystal structure of the acyl-enzyme complex of PBP5fm with benzylpenicillin at a resolution of 2.4 A. A characteristic of the active site, which distinguishes PBP5fm from other PBPs of known structure, is the topology of the loop 451-465 defining the left edge of the cavity. The residue Arg464, involved in a salt bridge with the residue Asp481, confers a greater rigidity to the PBP5fm active site. In addition, the presence of the Val465 residue, which points into the active site, reducing its accessibility, could account for the low affinity of PBP5fm for beta-lactam. This loop is common to PBPs of low affinity, such as PBP2a from Staphylococcus aureus and PBP3 from Bacillus subtilis. Moreover, the insertion of a serine after residue 466 in the most resistant strains underlines even more the determining role of this loop in the recognition of the substrates.
Asunto(s)
Proteínas Bacterianas , Proteínas Portadoras/química , Proteínas Portadoras/metabolismo , Enterococcus faecium , Hexosiltransferasas , Muramoilpentapéptido Carboxipeptidasa/química , Muramoilpentapéptido Carboxipeptidasa/metabolismo , Penicilina G/metabolismo , Penicilinas/metabolismo , Peptidil Transferasas , Secuencia de Aminoácidos , Sitios de Unión , Proteínas Portadoras/genética , Cristalografía por Rayos X , Enterococcus faecium/efectos de los fármacos , Sustancias Macromoleculares , Modelos Moleculares , Datos de Secuencia Molecular , Muramoilpentapéptido Carboxipeptidasa/genética , Mutación , Penicilina G/química , Resistencia a las Penicilinas , Proteínas de Unión a las Penicilinas , Penicilinas/química , Unión Proteica , Estructura Terciaria de Proteína , Alineación de SecuenciaRESUMEN
The recently published human genome with its relatively modest number of genes has highlighted the importance of post-transcriptional and post-translational modifications, such as alternative splicing or glycosylation, in generating the complexities of human biology. The human UDP-N-acetylglucosamine (UDPGlcNAc) pyrophosphorylases AGX1 and AGX2, which differ in sequence by an alternatively spliced 17 residue peptide, are key enzymes synthesizing UDPGlcNAc, an essential precursor for protein glycosylation. To better understand the catalytic mechanism of these enzymes and the role of the alternatively spliced segment, we have solved the crystal structures of AGX1 and AGX2 in complexes with UDPGlcNAc (at 1.9 and 2.4 A resolution, respectively) and UDPGalNAc (at 2.2 and 2.3 A resolution, respectively). Comparison with known structures classifies AGX1 and AGX2 as two new members of the SpsA-GnT I Core superfamily and, together with mutagenesis analysis, helps identify residues critical for catalysis. Most importantly, our combined structural and biochemical data provide evidence for a change in the oligomeric assembly accompanied by a significant modification of the active site architecture, a result suggesting that the two isoforms generated by alternative splicing may have distinct catalytic properties.