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

结构生物学领域的研究旨在提供对生物大分子的结构和功能的充分理解，这些信息最终有助于合理的药物设计和蛋白质工程。结构生物学在单核非血红素铁加氧酶类中的应用有可能帮助解决日益严重的抗生素耐药性问题。这些酶负责将分子氧（O2）结合到多种底物中。由于分子氧的固有性质，反应是困难的，但生物学上是有益的，这取决于该家族中酶的数量和它们促进的功能范围。该类中两种结构相似的酶是异青霉素N合酶（IPNS）和紫霉素生物合成蛋白C。前者催化双环的闭合，形成青霉素和头孢菌素抗生素的β-内酰胺核心。紫霉素生物合成蛋白C（VioC）催化L-精氨酸（L-arg）的羟基化，随后环化形成非标准氨基酸L-卷曲霉素（L-cam）并掺入紫霉素的肽前体中。虽然结构相似，但IPNS和VioC在机理上不同。VioC催化需要辅因子（α-酮戊二酸）的存在，而IPNS反应是辅因子独立的。由于这类酶的重要性，这两种蛋白质都受到了广泛的研究。然而，关于它们的精确反应机制的问题仍然没有答案。本项目的目的是充分阐明这两种酶的作用机制。该项目将涉及利用X射线自由电子激光设备进行时间分辨晶体学研究。由于加氧酶家族内的结构相似性，从这些酶中收集的信息将提供对广泛的生物相关加氧酶蛋白的理解。

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.