RESUMEN
Biofilm formation is a critical determinant in the pathopoiesis of Pseudomonas aeruginosa It could significantly increase bacterial resistance to drugs and host defense. Thus, inhibition of biofilm matrix production could be regarded as a promising attempt to prevent colonization of P. aeruginosa and the subsequent infection. PpgL, a periplasmic gluconolactonase, has been reported to be involved in P. aeruginosa quorum-sensing (QS) system regulation. However, the detailed function and catalysis mechanism remain elusive. Here, the crystal structure of PpgL is described in the current study, along with biochemical analysis, revealing that PpgL is a typical ß-propeller enzyme with unique metal-independent lactone hydrolysis activity. Consequently, comparative analysis of seven-bladed propeller lactone-catalyzing enzymes and mutagenesis studies identify the critical sites which contribute to the diverse catalytic and substrate recognition functions. In addition, the reduced biofilm formation and attenuated invasion phenotype resulting from deletion of ppgL confirm the importance of PpgL in P. aeruginosa pathogenesis. These results suggest that PpgL is a potential target for developing new agents against the diseases caused by P. aeruginosa.
Asunto(s)
Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Hidrolasas de Éster Carboxílico/química , Hidrolasas de Éster Carboxílico/metabolismo , Lactonas/metabolismo , Infecciones por Pseudomonas/microbiología , Pseudomonas aeruginosa/enzimología , Pseudomonas aeruginosa/patogenicidad , Proteínas Bacterianas/genética , Biocatálisis , Biopelículas , Hidrolasas de Éster Carboxílico/genética , Células HeLa , Humanos , Lactonas/química , Metales/química , Metales/metabolismo , Periplasma/química , Periplasma/enzimología , Periplasma/genética , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/fisiología , Especificidad por Sustrato , VirulenciaRESUMEN
In plants and microorganisms, aspartate kinase (AK) catalyzes an initial commitment step of the aspartate family amino acid biosynthesis. Owing to various structural organizations, AKs from different species show tremendous diversity and complex allosteric controls. We report the crystal structure of AK from Pseudomonas aeruginosa (PaAK), a typical α2ß2 hetero-tetrameric enzyme, in complex with inhibitory effectors. Distinctive features of PaAK are revealed by structural and biochemical analyses. Essentially, the open conformation of Lys-/Thr-bound PaAK structure clarifies the inhibitory mechanism of α2ß2-type AK. Moreover, the various inhibitory effectors of PaAK have been identified and a general amino acid effector motif of AK family is described.
Asunto(s)
Aspartato Quinasa/química , Aspartato Quinasa/metabolismo , Pseudomonas aeruginosa/enzimología , Regulación Alostérica/genética , Sitio Alostérico/genética , Secuencia de Aminoácidos , Aspartato Quinasa/genética , Catálisis , Modelos Moleculares , Organismos Modificados Genéticamente , Dominios y Motivos de Interacción de Proteínas/genética , Pseudomonas aeruginosa/genética , Alineación de SecuenciaRESUMEN
PepP is a virulence-associated gene in Pseudomonas aeruginosa, making it an attractive target for anti-P. aeruginosa drug development. The encoded protein, aminopeptidases P (Pa-PepP), is a type of X-prolyl peptidase that possesses diverse biological functions. The crystal structure verified its canonical pita-bread fold and functional tetrameric assembly, and the functional studies measured the influences of different metal ions on the activity. A trimetal manganese cluster was observed at the active site, elucidating the mechanism of inhibition by metal ions. Additionally, a loop extending from the active site appeared to be important for specific large-substrate binding. Based on the structural comparison and bacterial invasion assays, we showed that this non-conserved surface loop was critical for P. aeruginosa virulence. Taken together, these findings can extend our understanding of the catalytic mechanism and virulence-related functions of Pa-PepP and provide a solid foundation for the design of specific inhibitors against pathogenic-bacterial infections.