RESUMEN
Penicillin V acylases (PVAs) and bile salt hydrolases (BSHs) have considerable sequence and structural similarity; however, they vary significantly in their substrate specificity. We have identified a PVA from a Gram-negative organism, Pectobacterium atrosepticum (PaPVA) that turned out to be a remote homolog of the PVAs and BSHs reported earlier. Even though the active site residues were conserved in PaPVA it showed high specificity towards penV and interestingly the penV acylase activity was inhibited by bile salts. Comparative modelling and docking studies were carried out to understand the structural differences of the binding site that confer this characteristic property. We show that PaPVA exhibits significant differences in structure, which are in contrast to those of known PVAs and such enzymes from Gram-negative bacteria require further investigation.
Asunto(s)
Proteínas Bacterianas/química , Pectobacterium/enzimología , Penicilina Amidasa/química , Amidohidrolasas/química , Ácidos y Sales Biliares/química , Dominio Catalítico , Relación Dosis-Respuesta a Droga , Enlace de Hidrógeno , Hidrólisis , Simulación del Acoplamiento Molecular , Unión Proteica , Estructura Secundaria de Proteína , Alineación de Secuencia , Especificidad por SustratoRESUMEN
The paper reports the purification and characterization of the first penicillin acylase from Bacillus subtilis. YxeI, the protein annotated as hypothetical, coded by the gene yxeI in the open reading frame between iol and hut operons in B. subtilis was cloned and expressed in Eshcherichia coli, purified and characterized. The purified protein showed measurable penicillin acylase activity with penicillin V. The enzyme was a homotetramer of 148 kDa. The apparent K(m) of the enzyme for penicillin V and the synthetic substrate 2-nitro-5-(phenoxyacetamido)-benzoic acid was 40 mM and 0.63 mM, respectively, and the association constants were 8.93×10(2) M(-1) and 2.51×10(5) M(-1), respectively. It was inhibited by cephalosporins and conjugated bile salts, substrates of the closely related bile acid hydrolases. It had good sequence homology with other penicillin V acylases and conjugated bile acid hydrolases, members of the Ntn hydrolase family. The N-terminal nucleophile was a cysteine which is revealed by a simple removal of N-formyl-methionine. The activity of the protein was affected by high temperature, acidic pH and the presence of the denaturant guanidine hydrochloride.
Asunto(s)
Bacillus subtilis/enzimología , Proteínas Bacterianas/química , Regulación Bacteriana de la Expresión Génica , Penicilina Amidasa/química , Secuencia de Aminoácidos , Bacillus subtilis/metabolismo , Proteínas Bacterianas/metabolismo , Ácidos y Sales Biliares/farmacología , Cefalosporinas/farmacología , Clonación Molecular , Escherichia coli/metabolismo , Guanidina/química , Concentración de Iones de Hidrógeno , Cinética , Datos de Secuencia Molecular , Peso Molecular , Especificidad por SustratoRESUMEN
Bile salt hydrolase (BSH) is an enzyme produced by the intestinal microflora that catalyzes the deconjugation of glycine- or taurine-linked bile salts. The crystal structure of BSH reported here from Bifidobacterium longum reveals that it is a member of N-terminal nucleophil hydrolase structural superfamily possessing the characteristic alphabetabetaalpha tetra-lamellar tertiary structure arrangement. Site-directed mutagenesis of the catalytic nucleophil residue, however, shows that it has no role in zymogen processing into its corresponding active form. Substrate specificity was studied using Michaelis-Menten and inhibition kinetics and fluorescence spectroscopy. These data were compared with the specificity profile of BSH from Clostridium perfrigens and pencillin V acylase from Bacillus sphaericus, for both of which the three-dimensional structures are available. Comparative analysis shows a gradation in activity toward common substrates, throwing light on a possible common route toward the evolution of pencillin V acylase and BSH.
Asunto(s)
Amidohidrolasas/química , Amidohidrolasas/metabolismo , Bifidobacterium/enzimología , Evolución Molecular , Penicilina Amidasa/genética , Amidohidrolasas/genética , Secuencia de Aminoácidos , Sitios de Unión , Clostridium perfringens/enzimología , Cristalografía por Rayos X , Dimerización , Enlace de Hidrógeno , Interacciones Hidrofóbicas e Hidrofílicas , Cinética , Modelos Moleculares , Datos de Secuencia Molecular , Estructura Molecular , Mutagénesis Sitio-Dirigida , Penicilina Amidasa/química , Penicilina Amidasa/metabolismo , Unión Proteica , Conformación Proteica , Estructura Cuaternaria de Proteína , Estructura Secundaria de Proteína , Homología de Secuencia de Aminoácido , Espectrometría de Fluorescencia , Electricidad Estática , Especificidad por SustratoRESUMEN
Penicillin acylase proteins are amidohydrolase enzymes that cleave penicillins at the amide bond connecting the side chain to their beta-lactam nucleus. An unannotated protein from Bacillus subtilis has been expressed in Escherichia coli, purified and confirmed to possess penicillin V acylase activity. The protein was crystallized using the hanging-drop vapour-diffusion method from a solution containing 4 M sodium formate in 100 mM Tris-HCl buffer pH 8.2. Diffraction data were collected under cryogenic conditions to a spacing of 2.5 A. The crystals belonged to the orthorhombic space group C222(1), with unit-cell parameters a = 111.0, b = 308.0, c = 56.0 A. The estimated Matthews coefficient was 3.23 A3 Da(-1), corresponding to 62% solvent content. The structure has been solved using molecular-replacement methods with B. sphaericus penicillin V acylase (PDB code 2pva) as the search model.