RESUMEN
The production of experimental molecular vaccines against foot-and-mouth disease virus utilizes the viral encoded 3C protease for processing of the P1 polyprotein. Expression of wild type 3C protease is detrimental to host cells. The molecular vaccine constructs containing the 3C protease L127P mutant significantly reduce adverse effects associated with protease expression while retaining the ability to process and assemble virus-like particles. In published 3C protease crystal structures, the L127 residue is contained within the B2 ß-strand as part of the A2-B2 ß-sheet. To provide insight into the mechanism by which the L127P mutant alters the properties of the 3C protease, we performed scanning proline mutagenesis of residues 123-128 of the B2 ß-strand and monitored expression and P1 processing. Simultaneously, we utilized random mutagenesis of the full 3C sequence to identify additional mutations presenting a phenotype similar to the L127P mutation. Six of the tested mutants enhanced expression over wild type, and the I22P, T100P and V124P mutations surpassed the L127P mutation in certain cell lines. These data areinterpreted in conjunction with published 3C protease crystal structures to provide insight into the mechanism by which these mutations enhance expression.
Asunto(s)
Cisteína Endopeptidasas/química , Cisteína Endopeptidasas/genética , Virus de la Fiebre Aftosa/enzimología , Fiebre Aftosa/virología , Péptidos/genética , Proteínas Virales/química , Proteínas Virales/genética , Proteasas Virales 3C , Animales , Cisteína Endopeptidasas/metabolismo , Virus de la Fiebre Aftosa/genética , Virus de la Fiebre Aftosa/metabolismo , Regulación Viral de la Expresión Génica , Vectores Genéticos/genética , Vectores Genéticos/metabolismo , Mutagénesis , Péptidos/metabolismo , Plásmidos/genética , Plásmidos/metabolismo , Prolina/genética , Prolina/metabolismo , Conformación Proteica en Lámina beta , Procesamiento Postranscripcional del ARN , Proteínas Virales/metabolismoRESUMEN
Chlorite dismutases (Clds) convert chlorite to O2 and Cl(-), stabilizing heme in the presence of strong oxidants and forming the OâO bond with high efficiency. The enzyme from the pathogen Klebsiella pneumoniae (KpCld) represents a subfamily of Clds that share most of their active site structure with efficient O2-producing Clds, even though they have a truncated monomeric structure, exist as a dimer rather than a pentamer, and come from Gram-negative bacteria without a known need to degrade chlorite. We hypothesized that KpCld, like others in its subfamily, should be able to make O2 and may serve an in vivo antioxidant function. Here, it is demonstrated that it degrades chlorite with limited turnovers relative to the respiratory Clds, in part because of the loss of hypochlorous acid from the active site and destruction of the heme. The observation of hypochlorous acid, the expected leaving group accompanying transfer of an oxygen atom to the ferric heme, is consistent with the more open, solvent-exposed heme environment predicted by spectroscopic measurements and inferred from the crystal structures of related proteins. KpCld is more susceptible to oxidative degradation under turnover conditions than the well-characterized Clds associated with perchlorate respiration. However, wild-type K. pneumoniae has a significant growth advantage in the presence of chlorate relative to a Δcld knockout strain, specifically under nitrate-respiring conditions. This suggests that a physiological function of KpCld may be detoxification of endogenously produced chlorite.
Asunto(s)
Antioxidantes/metabolismo , Cloruros/metabolismo , Klebsiella pneumoniae/enzimología , Oxidorreductasas/metabolismo , Oxígeno/metabolismo , Humanos , Infecciones por Klebsiella/microbiología , Klebsiella pneumoniae/química , Klebsiella pneumoniae/metabolismo , Modelos Moleculares , Oxidorreductasas/química , Multimerización de ProteínaRESUMEN
The chlorite dismutases (C-family proteins) are a widespread family of heme-binding proteins for which chemical and biological roles remain unclear. An association of the gene with heme biosynthesis in Gram-positive bacteria was previously demonstrated by experiments involving introduction of genes from two Gram-positive species into heme biosynthesis mutant strains of Escherichia coli, leading to the gene being renamed hemQ. To assess the gene product's biological role more directly, a Staphylococcus aureus strain with an inactivated hemQ gene was generated and shown to be a slow growing small colony variant under aerobic but not anaerobic conditions. The small colony variant phenotype is rescued by the addition of exogenous heme despite an otherwise wild type heme biosynthetic pathway. The ΔhemQ mutant accumulates coproporphyrin specifically under aerobic conditions. Although its sequence is highly similar to functional chlorite dismutases, the HemQ protein has no steady state reactivity with chlorite, very modest reactivity with H2O2 or peracetic acid, and no observable transient intermediates. HemQ's equilibrium affinity for heme is in the low micromolar range. Holo-HemQ reconstituted with heme exhibits heme lysis after <50 turnovers with peroxide and <10 turnovers with chlorite. The heme-free apoprotein aggregates or unfolds over time. IsdG-like proteins and antibiotic biosynthesis monooxygenases are close sequence and structural relatives of HemQ that use heme or porphyrin-like organic molecules as substrates. The genetic and biochemical data suggest a similar substrate role for heme or porphyrin, with possible sensor-regulator functions for the protein. HemQ heme could serve as the means by which S. aureus reversibly adopts an SCV phenotype in response to redox stress.
Asunto(s)
Proteínas Bacterianas/metabolismo , Hemo/metabolismo , Estrés Oxidativo/fisiología , Oxidorreductasas/metabolismo , Staphylococcus aureus/enzimología , Proteínas Bacterianas/genética , Coproporfirinas/genética , Coproporfirinas/metabolismo , Escherichia coli/enzimología , Escherichia coli/genética , Eliminación de Gen , Hemo/genética , Oxidorreductasas/genética , Fenotipo , Staphylococcus aureus/genéticaRESUMEN
Heme proteins are extremely diverse, widespread, and versatile biocatalysts, sensors, and molecular transporters. The chlorite dismutase family of hemoproteins received its name due to the ability of the first-isolated members to detoxify anthropogenic ClO(2)(-), a function believed to have evolved only in the last few decades. Family members have since been found in 15 bacterial and archaeal genera, suggesting ancient roots. A structure- and sequence-based examination of the family is presented, in which key sequence and structural motifs are identified, and possible functions for family proteins are proposed. Newly identified structural homologies moreover demonstrate clear connections to two other large, ancient, and functionally mysterious protein families. We propose calling them collectively the CDE superfamily of heme proteins.