Identification and characterization of two families of F420 H2-dependent reductases from Mycobacteria that catalyse aflatoxin degradation

Abstract

Aflatoxins are polyaromatic mycotoxins that contaminate a range of food crops as a result of fungal growth and contribute to serious health problems in the developing world because of their toxicity and mutagenicity. Although relatively resistant to biotic degradation, aflatoxins can be metabolized by certain species of Actinomycetales. However, the enzymatic basis for their breakdown has not been reported until now. We have identified nine Mycobacterium smegmatis enzymes that utilize the deazaflavin cofactor F(420) H(2) to catalyse the reduction of the α,β-unsaturated ester moiety of aflatoxins, activating the molecules for spontaneous hydrolysis and detoxification. These enzymes belong to two previously uncharacterized F(420) H(2) dependent reductase (FDR-A and -B) families that are distantly related to the flavin mononucleotide (FMN) dependent pyridoxamine 5'-phosphate oxidases (PNPOxs). We have solved crystal structures of an enzyme from each FDR family and show that they, like the PNPOxs, adopt a split barrel protein fold, although the FDRs also possess an extended and highly charged F(420) H(2) binding groove. A general role for these enzymes in xenobiotic metabolism is discussed, including the observation that the nitro-reductase Rv3547 from Mycobacterium tuberculosis that is responsible for the activation of bicyclic nitroimidazole prodrugs belongs to the FDR-A family.

Fig. 1

Chemical structures of compounds used in this study.

Fig. 2

Purification protocol. Protein fractions that showed F₄₂₀H₂ dependent AFG1 degradation as measured by TLC were separated from M. smegmatis extracts. The ammonium sulphate precipitated proteins were first purified by hydrophobic interaction chromatography (HIC). Active fractions were further purified by anion exchange chromatography before separation by SDS-PAGE. Bands were cut from SDS-PAGE gels, digested with trypsin and analysed by LC/MS/MS. Peptides were identified from the annotated M. smegmatis genome sequence and corresponding results are shown for some of the excised bands. In the TLCs ‘-ve’ denotes no enzyme negative control and ‘sm’ denotes M. smegmatis cell extract positive control.

Fig. 3

Phylogenetic relationship between the PNPOx, FDR-A and FDR-B families. A condensed tree was constructed from 146 protein sequences of FDRs/PNPOxs from seven species of Actinomycetales (M. smegmatis, M. tuberculosis H37Rv, M. vanbaalenii, Rhodococcus sp. RH1, Arthrobacter sp. FB24, S. coelicolor, Frankia alni and Nocardioides sp. JS614), plus the known PNPOx enzymes from H. sapiens, E. coli, Saccharomyces cerevisiae, Caenorhabditis elegans and Mus musculus. Solid red circles denote the FDRs described in this paper, and open red circles denote other potential FDRs from M. smegmatis (TIGR locus number shown). For clarity, only enzymes from other species that have been previously characterized are labelled: blue triangles denote M. tuberculosis enzymes with known structures or previously described functions and black squares denote previously described PNPOxs.

Fig. 4

The structures of FDR-A and FDR-B enzymes. A. MSMEG_3356; from left to right, a topology diagram, showing the arrangement of the split barrel of anti-parallel β-sheets, surrounded by three large α-helices at the side and a short α-helix above, a cartoon diagram, and an electrostatic surface potential representation with F₄₂₀H₂ docked. B. MSMEG_3380; from left to right, a topology diagram of the structure, showing the arrangement of the split barrel of anti-parallel β-sheets, surrounded by one large α-helix at the side and three short α-helices above and below, a cartoon diagram of the 3380 dimer, and an electrostatic surface potential representation with F₄₂₀H₂ docked.

Fig. 5

F₄₂₀H₂ cofactor binding to enzymes 3356 and 3380. The apparent K_M's for F₄₂₀H₂ were calculated by measuring the velocity of the reaction with varying concentrations of F₄₂₀H₂ from 0.05 to 10 µM while maintaining all other components at 100 µM aflatoxin, 1 µM enzyme. For clarity the velocity is expressed as percent of maximum velocity. Empty circles, 3356; filled circles, 3380.

Fig. 6

The F₄₂₀H₂ binding grooves of an FDR-A, MSMEG_3356, and an FDR-B, MSMEG_3380. The deazaflavin pockets are shown to be rich in aromatic and hydrophobic residues, while the side-chain binding grooves are rich in basic residues.

Fig. 7

Proposed reaction mechanism of AFB1 and AFG1 reduction by reduced F₄₂₀. A. The proposed reduction mechanism of AFB1, with the mass spectrum of the substrate and product below the chemical structures. B. The mass spectrum and chemical structure of AFG1 and the reduced product.