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We report the first characterization of anl-arginine:glycine amidinotrans-ferase from a prokaryote. The enzyme, CyrA, is involved in the pathway for biosynthesis of the polyketide-derived hepatotoxin cylindrospermopsin from Cylindrospermopsis raciborskiiAWT205. CyrA is phylogenetically dis-tinct from other amidinotransferases, and structural alignment shows dif-ferences between the active site residues of CyrA and the well-characterized human l-arginine:glycine amidinotransferase (AGAT). | A novel prokaryotic L-arginine glycine amidinotransferase is involved in cylindrospermopsin biosynthesis Julia Muenchhoff1 Khawar S. Siddiqui1 Anne Poljak2 3 Mark J. Raftery2 Kevin D. Barrow1 and Brett A. Neilan1 1 Schoolof Biotechnology and Biomolecular Sciences University of New South Wales Sydney NSW Australia 2 BioanalyticalMass Spectrometry Facility University of New South Wales Sydney NSW Australia 3 Schoolof MedicalSciences University of New South Wales Sydney NSW Australia Keywords amidinotransferase cyanobacterialtoxin enzyme kinetics protein stability toxin biosynthesis Correspondence B. A. Neilan Schoolof Biotechnology and Biomolecular Sciences University of New South Wales Sydney NSW 2052 Australia Fax 61 2 9385 1591 Tel 61 2 9385 3235 E-mail b.neilan@unsw.edu.au Received 7 June 2010 revised 16 July 2010 accepted 22 July 2010 doi 10.1111 j.1742-4658.2010.07788.x We report the first characterization of an L-arginine glycine amidinotrans-ferase from a prokaryote. The enzyme CyrA is involved in the pathway for biosynthesis of the polyketide-derived hepatotoxin cylindrospermopsin from Cylindrospermopsis raciborskii AWT205. CyrA is phylogenetically distinct from other amidinotransferases and structural alignment shows differences between the active site residues of CyrA and the well-characterized human L-arginine glycine amidinotransferase AGAT . Overexpression of recombinant CyrA in Escherichia coli enabled biochemical characterization of the enzyme and we confirmed the predicted function of CyrA as an L-arginine glycine amidinotransferase by 1H NMR. As compared with AGAT CyrA showed narrow substrate specificity when presented with substrate analogs and deviated from regular Michaelis-Menten kinetics in the presence of the non-natural substrate hydroxylamine. Studies of initial reaction velocities and product inhibition and identification of intermediate reaction products were used to probe the kinetic mechanism of CyrA which is best described as a hybrid .
