RESUMO
Prolidase isolated from the hyperthermophilic archaeon Pyrococcus furiosus has potential for application for decontamination of organophosphorus compounds in certain pesticides and chemical warfare agents under harsh conditions. However, current applications that use an enzyme-based cocktail are limited by poor long-term enzyme stability and low reactivity over a broad range of temperatures. To obtain a better enzyme for OP nerve agent decontamination and to investigate structural factors that influence protein thermostability and thermoactivity, randomly mutated P. furiosus prolidases were prepared by using XL1-red-based mutagenesis and error-prone PCR. An Escherichia coli strain JD1 (lambdaDE3) (auxotrophic for proline [DeltaproA] and having deletions in pepQ and pepP dipeptidases with specificity for proline-containing dipeptides) was constructed for screening mutant P. furiosus prolidase expression plasmids. JD1 (lambdaDE3) cells were transformed with mutated prolidase expression plasmids and plated on minimal media supplemented with 50 muM Leu-Pro as the only source of proline. By using this positive selection, Pyrococcus prolidase mutants with improved activity over a broader range of temperatures were isolated. The activities of the mutants over a broad temperature range were measured for both Xaa-Pro dipeptides and OP nerve agents, and the thermoactivity and thermostability of the mutants were determined.
Assuntos
Proteínas Arqueais/metabolismo , Substâncias para a Guerra Química/metabolismo , Dipeptidases/metabolismo , Compostos Organofosforados/metabolismo , Praguicidas/metabolismo , Pyrococcus furiosus/enzimologia , Substituição de Aminoácidos/genética , Proteínas Arqueais/química , Proteínas Arqueais/genética , Proteínas Arqueais/isolamento & purificação , Meios de Cultura/química , Dipeptidases/química , Dipeptidases/genética , Dipeptidases/isolamento & purificação , Estabilidade Enzimática , Escherichia coli/genética , Modelos Moleculares , Mutagênese , Plasmídeos , Reação em Cadeia da Polimerase/métodos , Estabilidade Proteica , Estrutura Terciária de Proteína , Pyrococcus furiosus/genética , Deleção de Sequência , TemperaturaRESUMO
Prolidases hydrolyze the unique bond between X-Pro dipeptides and can also cleave the P-F and P-O bonds found in organophosphorus compounds, including the nerve agents, soman and sarin. The advantages of using hyperthermophilic enzymes in biodetoxification strategies are based on their enzyme stability and efficiency. Therefore, it is advantageous to examine new thermostable prolidases for potential use in biotechnological applications. Two thermostable prolidase homologs, PH1149 and PH0974, were identified in the genome of Pyrococcus horikoshii based on their sequences having conserved metal binding and catalytic amino acid residues that are present in other known prolidases, such as the previously characterized Pyrococcus furiosus prolidase. These P. horikoshii prolidases were expressed recombinantly in the Escherichia coli strain BL21 (lambdaDE3), and both were shown to function as proline dipeptidases. Biochemical characterization of these prolidases shows they have higher catalytic activities over a broader pH range, higher affinity for metal and are more stable compared to P. furiosus prolidase. This study has important implications for the potential use of these enzymes in biotechnological applications and provides further information on the functional traits of hyperthermophilic proteins, specifically metalloenzymes.
Assuntos
Proteínas Arqueais , Biotecnologia/métodos , Dipeptidases , Temperatura Alta , Pyrococcus horikoshii/enzimologia , Sequência de Aminoácidos , Proteínas Arqueais/química , Proteínas Arqueais/genética , Proteínas Arqueais/metabolismo , Domínio Catalítico , Cobalto/metabolismo , Dipeptidases/química , Dipeptidases/genética , Dipeptidases/metabolismo , Estabilidade Enzimática , Concentração de Íons de Hidrogênio , Dados de Sequência Molecular , Pyrococcus horikoshii/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidade por SubstratoRESUMO
Prolidase is a metallopeptidase that is ubiquitous in nature and has been isolated from mammals, bacteria and archaea. Prolidase specifically hydrolyzes dipeptides with a prolyl residue in the carboxy terminus (NH(2)-X-/-Pro-COOH). Currently, the only solved structure of prolidase is from the hyperthermophilic archaeon Pyrococcus furiosus. This enzyme is of particular interest because it can be used in many biotechnological applications. Prolidase is able to degrade toxic organophosphorus (OP) compounds, namely, by cleaving the P-F and P-O bonds in the nerve agents, sarin and soman. Applications using prolidase to detoxify OP nerve agents include its incorporation into fire-fighting foams and as biosensors for OP compound detection. Prolidases are also employed in the cheese-ripening process to improve cheese taste and texture. In humans, prolidase deficiency (PD) is a rare autosomal recessive disorder that affects the connective tissue. Symptoms of PD include skin lesions, mental retardation and recurrent respiratory infections. Enzyme replacement therapies are currently being studied in an effort to optimize enzyme delivery and stability for this application. Previously, prolidase has been linked to collagen metabolism and more recently is being associated with melanoma. Increased prolidase activity in melanoma cell lines has lead investigators to create cancer prodrugs targeting this enzyme. Thus, there are many biotechnological applications using recombinant and native forms of prolidase and this review will describe the biochemical and structural properties of prolidases as well as discuss their most current applications.
