Time-resolved crystallographic and spectroscopic studies of the mononuclear non-haem iron oxygenases, IPNS and VioC

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Research within the field of structural biology aims to provide full understanding of the structure andfunction of biological macromolecules, information that ultimately facilitates rational drug design andprotein engineering. Application of structural biology to the class of mononuclear non-haem ironoxygenases has potential to help combat the growing problem of antibiotic resistance. These enzymesare responsible for the incorporation of molecular oxygen (O2) into a wide variety of substrates. Dueto the intrinsic properties of dioxygen, reactions are difficult but biologically rewarding, exemplifiedby the number of enzymes within the family and the range of functions they facilitate. Two structurallysimilar enzymes from this class are isopenicillin N synthase (IPNS) and viomycin biosynthetic proteinC. The former catalyses the of bicyclic ring closure forming the B-lactam core of penicillin andcephalosporin antibiotics. Viomycin biosynthetic protein C (VioC) catalyses hydroxylation of L-arginine(L-arg), which is subsequently cyclised forming the non-standard amino acid L-capreomycidine (L-cam)and incorporated into the peptide precursor of viomycin. While structurally similar, IPNS and VioC aremechanistically distinct. VioC catalysis requires the presence of a cofactor (a-ketoglutarate) while theIPNS reaction is cofactor-independent. Due to the importance of this class of enzymes, both proteinshave been subject to extensive research. Nevertheless, questions of their precise reactionmechanisms remain unanswered. The aim of this project is to fully elucidate the mechanisms of bothenzymes. The project will involve the use of time-resolved crystallographic studies made possible byX-ray free electron laser facilities. Due to structural similarity within the oxygenase family, theinformation collected from these enzymes will provide understanding of a wide range of biologicalrelevant oxygenase proteins.