Abstract
Scientists have been fascinated by enzymatic catalysis for centuries and still marvel at the efficiency and selectivity with which these highly evolved molecular machines are able to catalyze complex reactions. For chemists, understanding the precise catalytic mechanisms can open up new possibilities in synthetic chemistry. From a medicinal standpoint, mechanistic knowledge provides valuable information on how enzymes can be targeted by drugs, as the molecular basis for many diseases lies in enzymatic malfunction. Among the different techniques used by researchers to investigate enzymatic catalysis, chemical probes are an indispensable tool. By synthetically inserting chemical changes into the substrate of a target enzyme, a wealth of information can be gained on structural and mechanistic features. In this thesis we investigate three enzymes with the help of chemical probes. The first and second chapters focus on the first two steps in the biosynthetic pathway of ergothioneine, an essential thiohistidine with antioxidant properties. The third chapter focuses on a key enzyme in the maturation of sulfatases, inserting a unique and crucial catalytic residue into the latter’s active site. In the first chapter, we shed light on the molecular toolbox of the SAM-dependent methyltransferase EgtD. This enzyme catalyzes the first step in the synthesis of ergothioneine, the processive methylation of histidine to Nα,Nα,Nα-trimethyl histidine. By labelling the substrates with stable isotopes, we have created probes enabling us to decipher the molecular strategies which allow this enzyme to carry out three consecutive methylation steps with nearly equal efficiency. In the second chapter we investigate the sulfoxide synthase EgtB, a non-heme iron dependent enzyme catalyzing the second step in ergothioneine biosynthesis. Interestingly, this system can potentially catalyze two distinct reactions, namely C-S bond formation and thiol dioxygenation. The factors governing this bifurcation in reactivity were investigated using substrate analogs with inhibitory properties. In the third and final chapter we examine the formylglycine generating enzyme (FGE), a copper-dependent enzyme catalyzing oxidative C-H bond cleavage of a cysteine residue in the active site of sulfatases. By synthesizing substrate peptides carrying stereoselective deuterium-labels we have identified the rate limiting step of this reaction and have gained valuable insight into the geometry of the enzyme active site.
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Disclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.