Structural Insights Into Microcystin Synthetase From Microcystis Aeruginosa

Water blooms of cyanobacteria are of worldwide concern due to their production of a wide range of hepatotoxins and neurotoxins. Of the known toxins, hepatotoxic microcystins are produced by a diverse range of cyanobacteria, including species of the genera Microcystis, Anabaena, Nostoc and Oscillatoria. It is known that acute poisoning of microcystins leads to death due to hepatic hemorrhage, whereas ingestion of sub-lethal microcystins increases the incidence rate of liver cancer.Previous biochemical and genetic studies showed that microcystins are assembled by a mixed polyketide synthetase/nonribosomal peptide synthetase (PKS/NRPS) encoded by microcystins synthetase (mcy) gene cluster (mcyA-J). The PKS/NRPS belongs to a family of large multi-domain enzymes that use a modular architecture to produce important biological polyketides/nonribosomal peptides such as antibiotics. Comparing modular organization of the mcy gene cluster with other PKS/NPRS, a biosynthetic scheme containing48sequential catalytic reactions for microcystin was proposed. Of the48sequential catalytic reactions for microcytin biosynthesis,45reactions are carried out by six large multienzyme synthetases (McyA-E, G) which are belonging to PKS/NRPS.McyG, which is the start module for biosynthesis of microcystin in Microcystis aeruginosa, contains an adenylation-peptidyl carrier protein (A-PCP) didomain for loading the starter unit to assemble the side chain of Adda residue. However, the catalytic mechanism of McyG A-PCP remains unclear. Here we report the2.45A crystal structure of McyG A-PCP complexed with the catalytic intermediate L-phenylalanyl-adenylate (L-Phe-AMP). Each asymmetry unit contains two protein molecules, one of which consists of the A-PCP didomain and the other comprises only the A domain. Structural analyses suggest that Va1227is most likely critical for the selection of hydrophobic substrates. Moreover, we observed two distinct interfaces between PCP domain and A domain, demonstrating variable crosstalk within A-PCP didomain during reaction. Altogether, we propose a catalytic cycle driven by the A-PCP didomain.In addition, crystallization of McyC, which serves as termination module for the microcystin biosynthetic cluster, is in progress.