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.

Blue Light-Dependent Pre-mRNA Splicing Controls Pigment Biosynthesis in the Mushroom Terana caerulea

“…In the context of catabolism, the lignocellulose-degrading mushroom Phanerochaete chrysosporium showed substrate-dependent alternative splicing of a gene encoding exocellobiohydrolase, which hypothetically changes the enzyme’s substrate preferences ( 54 ). Alternative splicing has also been recognized as an important regulatory mechanism in the human-pathogenic basidiomycetous yeast Cryptococcus neoformans ( 55 ) and is well established in numerous ascomycetes, among them Sclerotinia sclerotiorum ( 56 ), Sordaria macrospora ( 57 ), and aspergilli ( 58 , 59 ). However, these and other reports on alternative splicing pertain to developmental or metabolic aspects, but none of them pertain to the regulation of natural product biosynthetic pathways, and none pertain to light as a prerequisite for splicing.…”

Baeyer–Villiger Monooxygenases: Tunable Oxidative Biocatalysts

“…These motifs are flanked by two Rossmann fold domains harboring a GxGxx­[G/A] motif required for tight binding of the two cofactors. In some cases, minor deviations from the consensus for the nucleotide binding sequence have been reported (MoxY, CPDMO). , Although the exact functional role of the fingerprint residues is not completely clear, the long consensus sequence entails the conserved active-site aspartate, while the short fingerprint is related to the linker connecting the FAD and NADP-binding domains. , As a common feature, type I BVMOs share the strict dependence on FAD as a tightly bound prosthetic group and NADPH as electron donor, with the exception of MekA from Pseudomonas veronii MEK700, which seems to accept either NADH or NADPH . The preferred host for producing recombinant BVMOs, has been Escherichia coli , which does not contain a native homologue itself.…”