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Title CHARACTERIZATION OF THE BAEYER-VILLIGER MONOOXYGENASE MOXY AND CLOSELY-RELATED HOMOLOGUES FROM ASPERGILLUS FLAVUS
AuthorTolmie, Carmien
SubjectMicrobial, Biochemical and Food Biotechnology
Date2014-08-19
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AbstractAflatoxins are carcinogenic mycotoxins produced by certain species of the Aspergilli, with the most prominent producers being Aspergillus flavus and A. parasiticus. Exposure to aflatoxins causes liver cancer, immune suppression, retardation in growth and in extreme cases, even death. A. parasiticus and A. flavus infect a wide range of crops, including corn, cotton, peanuts and tree nuts. Consequently, aflatoxin contamination has a severe impact on public health, as well as the agricultural sector. Several control strategies are currently in place to combat aflatoxin contamination. Although great progress has been made in developing innovative methods for aflatoxin control, no strategy is completely efficient in eliminating aflatoxin contamination. Also, the application of the strategies is often limited by practical and economic factors. This necessitates the development of further aflatoxin control strategies. A yet under-exploited resource is the aflatoxin biosynthesis pathway. Aflatoxins are synthesised in a complex polyketide-initiated pathway that requires multiple enzymatic steps. The genes encoding the aflatoxin biosynthetic enzymes are located in a cluster which contains both the regulatory and structural genes. A novel approach to aflatoxin control is the direct inhibition of a target enzyme in the aflatoxin biosynthesis pathway. An ideal candidate is the Baeyer-Villiger monooxygenase (BVMO), MoxY, that catalyses the conversion of hydroxyversicolorone to versiconal hemiacetal acetate. BVMOs are flavin-containing proteins that catalyse the oxidation of ketones or cyclic ketones to esters or lactones, respectively. BVMOs occur only in the genomes of bacteria and fungi, rendering the MoxY enzyme as a suitable candidate for targeted enzyme inhibition. In silico analysis of the moxY gene indicated that MoxY may exist with an elongated N-terminus or an alternative C-terminus, due to alternative splicing of the mRNA. In addition to cloning the moxY gene, variations of the moxY gene were created that encode a protein with both the alternative termini or either an alternative N-terminus or an alternative C-terminus. The MoxY variants were heterologously expressed in E. coli and MoxY with an elongated N-terminus (MoxYAltN) was found to be the only active recombinant form of the MoxY enzyme. MoxYAltN is promiscuous with regard to the substrate accepted, converting linear, aromatic, substituted aromatic and bicyclic ketones. MoxYAltN was purified and the kinetic parameters determined with respect to the reaction with a surrogate substrate, phenylacetone. Purified MoxYAltN was demonstrated to convert synthetic [1â-2H]hydroxyversicolorone to versiconal hemiacetal acetate, confirming the role of MoxYAltN in the aflatoxin biosynthesis pathway. Intragenomic complementation of an impaired aflatoxin biosynthetic step is a common theme that has emerged from the experimental study of the aflatoxin biosynthetic pathway. Therefore, the complementation of the activity of MoxY(AltN) by another BVMO in the A. flavus genome was investigated. The genome contains 26 putative BVMOs and phylogenetic analysis indicted that six of these are closely-related to MoxY, which were selected for heterologous expression in E. coli. Three of the homologues expressed as soluble proteins and activity was observed for two. The substrate profiles of the homologues differ significantly from that of MoxYAltN, with the only common substrate converted being (±)-cis-bicyclo[3.2.0]hept-2-en-6-one. The enantio- and regioselective conversion of (±)-cis-bicyclo[3.2.0]hept-2-en-6-one by BVMOs is a well-characterised reaction. Chiral analysis indicated divergent biocatalytic profiles of MoxYAltN and the two homologues with respect to the conversion of (±)-cis-bicyclo[3.2.0]hept-2-en-6-one, which suggests that the substrate binding pockets of the enzymes are likely to differ. The ability of the homologues to complement the activity of MoxY(AltN) by converting [1â-2H]hydroxyversicolorone remains uncertain. However, the differential substrate profiles demonstrate that phylogenetic clustering is not an absolute indication of overlapping biocatalytic abilities. Therefore, more distantly related BVMOs in A. flavus have to be considered as candidates for intragenomic complementation as well.
PublisherUniversity of the Free State
Identifierhttp://etd.uovs.ac.za//theses/available/etd-08192014-133455/restricted/