Characterisation of the Tailoring and Transport Enzymes involved in the Microcystin Biosynthesis Pathway

Abstract

The cyanobacterium Microcystis aeruginosa is widely known for its production of the potent hepatotoxin microcystin. This cyclic heptapeptide is synthesised non-ribosomally by the thiotemplate function of a large, modular enzyme complex encoded within the 55 kb microcystin synthetase (mcy) gene cluster. The mcy gene cluster also encodes several stand-alone enzymes, putatively involved in the tailoring and export of microcystin. This thesis describes the characterisation of the Adda O-methyltransferase, McyJ, the 2-hydroxy-3-methylsuccinic acid dehydrogenase, McyI, and the ABC transporter, McyH. A combination of bioinformatic, molecular, and biochemical approaches have been used to elucidate the structure, function, regulation and evolution of these microcystin synthetase gene cluster encoded enzymes. Extensive sequence analyses are reported, including phylogenetic and structural studies. The distribution of mcyH, mcyI and mcyJ orthologues in different species of cyanobacteria has been investigated via genetic screening with M. aeruginosa specific, and degenerate oligonucleotide primers. McyH, McyI and McyJ have been heterologosly over-expressed in E. coli and enzymatically assayed. Finally, an McyH antibody has been engineered and used to investigate the regulation of the McyH ABC transporter in wild-type (WT) M. aeruginosa, and in various non-toxic engineered mutant strains. The results of these experiments are discussed with respect to the roles of McyH, McyI and McyJ in microcystin biosynthesis, and their relevance to the fields of water quality management and rational drug design and production.