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.