Alternative Splicing of the Aflatoxin-Associated Baeyer⁻Villiger Monooxygenase from Aspergillus flavus: Characterisation of MoxY Isoforms

Abstract

Aflatoxins are carcinogenic mycotoxins that are produced by the filamentous fungus Aspergillus flavus, a contaminant of numerous food crops. Aflatoxins are synthesised via the aflatoxin biosynthesis pathway, with the enzymes involved encoded by the aflatoxin biosynthesis gene cluster. MoxY is a type I Baeyer⁻Villiger monooxygenase (BVMO), responsible for the conversion of hydroxyversicolorone (HVN) and versicolorone (VN) to versiconal hemiacetal acetate (VHA) and versiconol acetate (VOAc), respectively. Using mRNA data, an intron near the C-terminus was identified that is alternatively spliced, creating two possible MoxY isoforms which exist in vivo, while analysis of the genomic DNA suggests an alternative start codon leading to possible elongation of the N-terminus. These four variants of the moxY gene were recombinantly expressed in Escherichia coli, and their activity evaluated with respect to their natural substrates HVN and VN, as well as surrogate ketone substrates. Activity of the enzyme is absolutely dependent on the additional 22 amino acid residues at the N-terminus. Two MoxY isoforms with alternative C-termini, MoxYAltN and MoxYAltNC, converted HVN and VN, in addition to a range of ketone substrates. Stability and flavin-binding data suggest that MoxYAltN is, most likely, the dominant isoform. MoxYAltNC is generated by intron splicing, in contrast to intron retention, which is the most prevalent type of alternative splicing in ascomycetes. The alternative C-termini did not alter the substrate acceptance profile, or regio- or enantioselectivity of the enzyme, but did significantly affect the solubility and stability.

Keywords: Baeyer–Villiger monooxygenase; MoxY; aflatoxin biosynthesis; alternative splicing; hydroxyversicolorone; versicolorone.

Figure 1

The metabolic grid in the intermediate stages of aflatoxin biosynthesis. Parallel reactions are catalysed by the same enzymes, while the lateral reactions are catalysed by VrdA. Enzymes proven to catalyse reactions by recombinant expression, or by purification from the native host, are indicated in normal case, while genes involved in reactions, as proven by disruption experiments, are indicated in italics.

Figure 2

Structure of (A) moxY and (B) moxYAltNC genes. The exons are shown as boxes with the introns shown as lines. The common intron to moxY and moxYAltNC is located at positions 1413–1462, while the second intron in moxYAltNC is located at position 1689–1746, splicing out the stop codon and encoding a variant C-terminus, shown in orange. The alternative start codon, 66 bp upstream from the original start codon, leads to the alternative N-terminus, shown in green. (C) The four possible variants of the MoxY protein.

Figure 3

Alignment of the orthologues of MoxY from the aflatoxin- and sterigmatocystin-producing members of the aspergilli, in comparison to MoxYAltN and MoxY from A. flavus. NCBI protein accession numbers: A. oryzae (XP_001821532.3), A. parasiticus (Q6UEF3.1), A. nomius (XP_015410220.1), A. arachidicola (PIG79656.1), A. bombycis (XP_022391198.1), A. nidulans (StcW, Q00730.2), and A. ochraceoroseus (PTU23613.1).

Figure 4

SDS-PAGE analysis of the (A) total protein fraction and (B) soluble protein fraction of E. coli BL21-Gold (DE3) co-expressing the MoxY variants from the pET-28b(+) vector, and the GroES/EL chaperone from the pGro7 vector. M, PageRuler Prestained protein ladder; 1, pET-28b(+) empty vector control; 2, MoxY (66.5 kDa); 3, MoxYAltN (69.0 kDa); 4, MoxYAltNC (73.1 kDa); 5, MoxYAltC (70.7 kDa).

Figure 5

SDS-PAGE analysis of purified (A) MoxYAltN and (B) MoxYAltNC. M, PageRuler Prestained protein ladder; lane 1, purified MoxYAltN or MoxYAltNC. (C) Absorbance spectra of MoxYAltN before and after denaturation with 8 M urea. Extinction coefficient of free FAD at 450 nm: 11.3 mM⁻¹ cm⁻¹; enzyme-bound FAD at 454 nm: 12.5 mM⁻¹ cm⁻¹.

Figure 6

HPLC elution profiles showing the conversion of (A) hydroxyversicolorone (HVN) to versiconal hemiacetal acetate (VHA), and (B) versicolorone (VN) to versiconol acetate (VOAc). Mass spectra (negative ionisation) of (C) HVN, (D) VHA, (E) VN, and (F) VOAc.

Figure 7

(A) Enantio- and regioselective conversion of rac-bicyclo[3.2.0]hept-2-en-6-one (S) to the proximal (PP) and distal (DP) products by Baeyer–Villiger monooxygenases (BVMOs). Conversion of rac-bicyclo[3.2.0]hept-2-en-6-one by (B) MoxYAltN and (C) MoxYAltNC over time. Averages of duplicate experiments are shown.