RESUMEN
BACKGROUND: Ar-BVMO is a recently discovered Baeyer-Villiger monooxygenase from the genome of Acinetobacter radioresistens S13 closely related to medically relevant ethionamide monooxygenase EtaA (prodrug activator) and capable of inactivating the imipenem antibiotic. METHODS: The co-substrate preference as well as steady-state and rapid kinetics studies of the recombinant purified protein were carried out using stopped-flow spectroscopy under anaerobic and aerobic conditions. Kd values were measured by isothermal calorimetry. Enzymatic activity was determined by measuring the amount of product formed using high pressure liquid chromatography or gas chromatography. Site-directed mutagenesis experiments were performed to decipher the role of the active site arginine-292. RESULTS: Ar-BVMO was found to oxidize ethionamide as well as linear ketones. Mechanistic studies on the wild type enzyme using stopped-flow spectroscopy allowed for the detection of the characteristic oxygenating C4a-(hydro)peroxyflavin intermediate, which decayed rapidly in the presence of the substrate. Replacement of arginine 292 in Ar-BVMO by glycine or alanine resulted in greatly reduced or no Baeyer-Villiger activity, respectively, demonstrating the crucial role of this residue in catalysis of ketone substrates. However, both the R292A and R292G mutants are capable of carrying out N- and S-oxidation reactions. CONCLUSIONS: Substrate profiling of Ar-BVMO confirms its close relationship to EtaA; ethionamide is one of its substrates. The active site Arginine 292 is required for its Baeyer-Villiger activity but not for heteroatom oxidation. GENERAL SIGNIFICANCE: A single mutation converts Ar-BVMO to a unique S- or N-monooxygenase, a useful biocatalyst for the production of oxidized metabolites of human drug metabolizing enzymes.
Asunto(s)
Acinetobacter/enzimología , Proteínas Bacterianas/química , Etionamida/química , Flavinas/química , Cetonas/química , Oxigenasas de Función Mixta/química , Microbiología del Suelo , Acinetobacter/genética , Alanina/química , Alanina/metabolismo , Secuencia de Aminoácidos , Arginina/química , Arginina/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Biocatálisis , Dominio Catalítico , Clonación Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Etionamida/metabolismo , Flavinas/metabolismo , Expresión Génica , Glicina/química , Glicina/metabolismo , Cetonas/metabolismo , Cinética , Oxigenasas de Función Mixta/genética , Oxigenasas de Función Mixta/metabolismo , Mutagénesis Sitio-Dirigida , Mutación , Oxidación-Reducción , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Especificidad por SustratoRESUMEN
Antimicrobial resistance is a global issue currently resulting in the deaths of hundreds of thousands of people a year worldwide. Data present in the literature illustrate the emergence of many bacterial species that display resistance to known antibiotics; Acinetobacter spp. are a good example of this. We report here that Acinetobacter radioresistens has a Baeyer-Villiger monooxygenase (Ar-BVMO) with 100% amino acid sequence identity to the ethionamide monooxygenase of multidrug-resistant (MDR) Acinetobacter baumannii. Both enzymes are only distantly phylogenetically related to other canonical bacterial BVMO proteins. Ar-BVMO not only is capable of oxidizing two anticancer drugs metabolized by human FMO3, danusertib and tozasertib, but also can oxidize other synthetic drugs, such as imipenem. The latter is a member of the carbapenems, a clinically important antibiotic family used in the treatment of MDR bacterial infections. Susceptibility tests performed by the Kirby-Bauer disk diffusion method demonstrate that imipenem-sensitive Escherichia coli BL21 cells overexpressing Ar-BVMO become resistant to this antibiotic. An agar disk diffusion assay proved that when imipenem reacts with Ar-BVMO, it loses its antibiotic property. Moreover, an NADPH consumption assay with the purified Ar-BVMO demonstrates that this antibiotic is indeed a substrate, and its product is identified by liquid chromatography-mass spectrometry to be a Baeyer-Villiger (BV) oxidation product of the carbonyl moiety of the ß-lactam ring. This is the first report of an antibiotic-inactivating BVMO enzyme that, while mediating its usual BV oxidation, also operates by an unprecedented mechanism of carbapenem resistance.
Asunto(s)
Acinetobacter/enzimología , Antibacterianos/metabolismo , Farmacorresistencia Bacteriana Múltiple/genética , Escherichia coli/genética , Imipenem/metabolismo , Acinetobacter/clasificación , Acinetobacter/efectos de los fármacos , Acinetobacter/genética , Antibacterianos/farmacología , Antineoplásicos/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Benzamidas/metabolismo , Biotransformación , Clonación Molecular , Pruebas Antimicrobianas de Difusión por Disco , Escherichia coli/clasificación , Escherichia coli/efectos de los fármacos , Escherichia coli/enzimología , Expresión Génica , Imipenem/farmacología , Ingeniería Metabólica , Oxigenasas de Función Mixta/genética , Oxigenasas de Función Mixta/metabolismo , NADP/metabolismo , Oxidación-Reducción , Filogenia , Piperazinas/metabolismo , Pirazoles/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMEN
This work demonstrates that Acinetobacter radioresistens strain S13 during the growth on medium supplemented with long-chain alkanes as the sole energy source expresses almA gene coding for a Baeyer-Villiger monooxygenase (BVMO) involved in alkanes subterminal oxidation. Phylogenetic analysis placed the sequence of this novel BVMO in the same clade of the prodrug activator ethionamide monooxygenase (EtaA) and it bears only a distant relation to the other known class I BVMO proteins. In silico analysis of the 3D model of the S13 BVMO generated by homology modelling also supports the similarities with EtaA by binding ethionamide to the active site. In vitro experiments carried out with the purified enzyme confirm that this novel BVMO is indeed capable of typical Baeyer-Villiger reactions as well as oxidation of the prodrug ethionamide.
