CYP116B5: a new class VII catalytically self‐sufficient cytochrome P450 from Acinetobacter radioresistens that enables growth on alkanes

Abstract

SummaryA gene coding for a class VII cytochrome P450 monooxygenase (CYP116B5) was identified from Acinetobacter radioresistens S13 growing on media with medium (C14, C16) and long (C24, C36) chain alkanes as the sole energy source. Phylogenetic analysis of its N-and C-terminal domains suggests an evolutionary model involving a plasmid-mediated horizontal gene transfer from the donor Rhodococcus jostii RHA1 to the receiving A. radioresistens S13. This event was followed by fusion and integration of the new gene in A. radioresistens chromosome. Heterologous expression of CYP116B5 in Escherichia coli BL21, together with the A. radioresistens Baeyer-Villiger monooxygenase, allowed the recombinant bacteria to grow on long-and medium-chain alkanes, showing that CYP116B5 is involved in the first step of terminal oxidation of medium-chain alkanes overlapping AlkB and in the first step of sub-terminal oxidation of long-chain alkanes. It was also demonstrated that CYP116B5 is a self-sufficient cytochrome P450 consisting of a heme domain (aa 1-392) involved in the oxidation step of n-alkanes degradation, and its reductase domain (aa 444-758) comprising the NADPH-, FMN-and [2Fe2S]-binding sites. To our knowledge, CYP116B5 is the first member of this class to have its natural substrate and function identified.

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].…”

“…Because the sequence of Ar-BVMO is more closely related to that of EtaA than other known BVMO enzymes [9], known substrates of EtaA from M. tuberculosis [12] were initially considered. Therefore, the EtaA substrates, 2-octanone, 2-decanone and 2-dodecanone, were selected and following incubation with purified Ar-BVMO they were found to be converted into hexyl acetate, octyl acetate and a mixture of methyl undecanoate (8 ± 1%) and decyl acetate (92 ± 1%), respectively.…”

“…This protein was found to share a 49.7% sequence identity and 71.0% sequence similarity with the ethionamide-activating (EtaA) BVMO from Mycobacterium tuberculosis [10][11][12]. 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].…”

“…Although the genus Acinetobacter has been shown to be a source of several BVMO proteins, many of these enzymes are not available in recombinant form and therefore have not yet been characterized. Using sequence homologies and the presence of the conserved Baeyer-Villiger sequence motif, we have recently discovered a novel BVMO, Ar-BVMO, in the genome of Acinetobacter radioresistens S13 [9]. This protein was found to share a 49.7% sequence identity and 71.0% sequence similarity with the ethionamide-activating (EtaA) BVMO from Mycobacterium tuberculosis [10][11][12].…”

Enhancing the Catalytic Performance of a CYP116B Monooxygenase by Transdomain Combination Mutagenesis

“…[4] Self-sufficient CYP116B subfamilym embers fuse the heme and redox domains into as ingle polypeptide chain, and the redox partners shuttlee lectrons from the reductant NADPH, throught he specific FMN and Fe 2 S 2 domains, to their acceptor, the heme domain. [5] Until now,t here have been af ew typical members in the CYP116B subfamily, including P450 RhF (CYP116B2), [6] CYP116B3, [7] CYP116B1, [8] CYP116B5, [9] CYP116B4, [10] P450 SMO , [11] P450 RpMO, P450 ArMO ,P 450 CtMO [12] as wella st he latest reported CYP116B29,C YP116B46, CYP116B63,C YP116B64 and CYP116B65. [13] Moreover,v arious reactiont ypes have been reported, such as aromatic hydroxylation, O-dealkylation, epoxidation anda symmetric sulfoxidation by P450 RhF ; N-dealkylation by CYP116B1;b enzene derivatived ealkylation by CYP116B3; medium-and long-chain alkane (C 14 ,C 16 ,C 24 and C 36 )h ydroxylation by CYP116B5.…”