Functional studies of enzymes in the biosynthesis of the cyanobacterial toxin cylindrospermopsin

Abstract

Characterisation of the enzymes responsible for biosynthesis of the polyketide-derived cyanotoxin cylindrospermopsin in Cylindrospermopsis raciborskii AWT205 is vital to understand the unique enzymatic mechanisms involved and to unravel the novel biochemistry of this pathway. The first enzyme in the pathway, CyrA, was shown to utilise L-arginine and glycine for the formation of guanidinoacetate and ornithine using proton nuclear magnetic resonance. CyrA was phylogenetically distinct from other L-arginine:glycine amidinotransferases and displayed narrow substrate specificity. Studies of initial reaction velocities and product inhibition, as well as identification of intermediate reaction products by mass spectrometry were used to probe the kinetic mechanism of CyrA, which is best described as a hybrid of ping-pong and sequential. Investigations into the stability of CyrA using circular dichroism and fluorescence spectrophotometry revealed the thermolabile, psychrophilic character of this protein. Site-directed mutagenesis was employed to replace amino acids in the active site of CyrA (F245N and S247M), in order to elucidate the structure-function-stability relationship of the enzyme. Both mutations abolished the stringent substrate specificity of wild-type CyrA. Experiments with substrate analogues indicated that donor substrates require a carboxylate group for binding and that two distinct binding sites for donor and acceptor substrates are present, confirming the hybrid kinetic mechanism. Evidence from initial velocity studies and homology modelling implied that CyrA undergoes ligand-induced structural changes, which are initiated by displacement of the side chain of F245 by large substrates. The amidohydrolases CyrG and CyrH are proposed to form the uracil moiety of CYLN. The use of a multiple sequence alignment and homology models allowed deduction of potential metal-binding and catalytic residues, revealing significant differences in CyrG and CyrH despite their high sequence identity to each other. When the activity of recombinant CyrG and CyrH was probed with various substrate analogues, both recombinant proteins were inactive. The adenylation domain of the non-ribosomal peptide synthetase/polyketide synthase hybrid CyrB and the putative sulfotransferase CyrJ were cloned, heterologously expressed and purified, however, enzymatic activity was not detected for either protein. Problems related to the structural integrity of these proteins were identified and potential solutions to overcome these problems are proposed.