Escherichia coli Overexpressing a Baeyer-Villiger Monooxygenase from Acinetobacter radioresistens Becomes Resistant to Imipenem

Abstract

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 ␤-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.

Characterization of a new Baeyer-Villiger monooxygenase and conversion to a solely N-or S-oxidizing enzyme by a single R292 mutation

“…HPLC analysis was performed with an Agilent 1200 quaternary pump HPLC System (Agilent Technologies, Italy) with a C18 reverse phase column (LiChroCART 250-4 column packed with Lichrosphere RP C18 5 µM). Previously published methods to separate Ethionamide, Ethionamide-SO, Tozasertib and Tozasertib NO were used [2,9].…”

“…These proteins have great value in the conversion of ketones into corresponding esters and lactones, with wide applications in bioremediation and sustainable biocatalysis. In addition to BaeyerVilliger oxidations, BVMO enzymes can also catalyze heteroatom oxidations (nitrogen and sulfur) [1] that can have interesting applications to the pharmaceutical industry, as shown by our recent results on the oxidation of the antibiotic imipenem [2].…”

“…Furthermore, phylogenetic analysis of the Ar-BVMO not only placed this protein in the same branch as EtaA but also showed it to be closely related to the phase-1 drug metabolizing enzyme, human flavin-containing monooxygenase 3 (FMO3), but only distantly related to other canonical bacterial BVMO proteins [9,13]. Considering these relationships, initially we studied Ar-BVMO for its possible activities with "unusual/ non-typical" substrates and demonstrated that it is not only capable of oxidizing two anticancer drugs metabolized by human FMO3, Danusertib and Tozasertib [14,15], but it can also oxidize other synthetic drugs such as Imipenem [2]. Imipenem is a carbapenem, which is a clinically important antibiotic family used in the treatment of MultiDrug-Resistant (MDR) bacterial infections.…”

“…Imipenem is a carbapenem, which is a clinically important antibiotic family used in the treatment of MultiDrug-Resistant (MDR) bacterial infections. Interestingly, an identical gene to that of Ar-BVMO also exists in Acinetobacter Baumannii, one of the so-called superbugs [2]. In these cases, Ar-BVMO seems to be responsible for the de-activation of this antibiotic, and therefore it acts more like a drug metabolizing enzyme.…”

Chemical applications of Class B flavoprotein monooxygenases

“…BVMO enzymes are abundant in bacterial, fungal and plant genomes but nearly absent in animal or human genomes. They are involved in metabolic pathways (Chen et al, 1988;Sheng et al, 2001;Minerdi et al, 2015) and in some cases have medical relevance (Fraaije et al, 2004;Minerdi et al, 2012;Catucci et al, 2016;Minerdi et al, 2016). The first BVMO enzyme that was isolated and purified was a cyclohexanone monooxygenase (CHMO) from Acinetobacter NCIMB 9871 (Donoghue et al, 1975).…”