Grant: Discovery Grants Program - Individual

The objectives of this program at the interface of chemistry and biology are: a) to understand the enzyme structures and mechanisms of assembly of fungal polyketides and b) to obtain structural insight into antimicrobial peptides and their interactions with bacterial membranes and target molecules (e.g. Lipid II). In the first area, the detailed mode of fungal polyketide assembly will be examined. The targets include 5 reduced polycyclic metabolites, namely the cholesterol-lowering agent lovastatin, cladosporin (anti-malarial), dehydrocurvularin (immunomodulatory phytotoxin), cytochalasin E ( anti-angiogenesis anticancer) and hypothemycin (kinase inhibitor & anticancer). In contrast to bacterial polyketide synthase (PKS) enzymes that employ multiple proteins in an assembly line fashion, the fungal iterative PKS enzymes use the same active sites in a single protein repeatedly. Ultimately their function is to polymerize acetate and other precursors into chains having specific length, functionality & stereochemistry. We have already identified and expressed the fungal PKS proteins for all 5 metabolites. The function of fungal iterative PKS enzymes is controlled by both the protein sequence/structure as well as by the length and functionality of the growing substrate chain that is attached covalently as a thioester to the protein. We will use fully purified constituent enzymes of the iterative type I PKS, to examine the rates and mechanism of detailed assembly at each stage of chain elongation. This will be accomplished by loading labelled putative intermediates. We propose to identify the domain that catalyzes the Diels Alder reaction during lovastatin formation by LovB. We propose to convert CoA-SH to CoA-NH2 and use this to create acyl carrier protein (ACP) domain derivatives that will cross link to other domains (e.g. ketosynthase (KS)) to ""freeze"" the mobile enzymes at a single discrete stage for x-ray crystallographic analysis. In a second area, we propose to examine by NMR structures of antimicrobial peptides bound to analogs of Lipid II (bacterial peptidoglycan precursor) in dodecyl phosphocholine (DPC) micelles.We will first look at 4 target peptides, tridecaptin, lacticin 3147 A1 and A2, and paenicidin. The resulting 3D structures should help elucidate the mechanism of action and assist design of simpler potent antimicrobial agents. Antimicrobial peptides are already extensively used to preserve food and have great potential in human and veterinary medicine for treatment of infections resistant to current therapy. The structures of micelle lipids bound to antimicrobials and complexes with Lipid II analogs will be examined by NMR. The approach involves synthesis of specifically labelled 13C dodecyl phosphocholine (DPC) and isotope-edited NOESY. The targets include tridecaptin, lacticin 3147 A1 & A2, carnocyclin and potentially paenicidin and enterocin 7A / 7B.