Mechanistic studies of sulfur-carbon bond formation by metal-dependent enzymes

Abstract

Sulfur is an essential element for all living organisms. In a large variety of relevant compounds like amino acids, cofactors and other natural products carbon-sulfur bonds are found. The understanding of the mechanistic details of carbon sulfur bond formation catalyzed by enzymes is from particular interest. The gained knowledge can be used for the discovery of new biosynthetic pathways of sulfur containing natural products as well as for the design of artificial enzymes able to perform carbon-sulfur bond formation. In this thesis we investigate bacterial and fungal enzymes that catalyze carbon-sulfur bond formation. First, we characterized the catalytic activity of an unusual class of glutathione-S-transferases. These enzymes require a bivalent metal ion to activate their thiol substrate for nucleophilic attack onto carbon electrophiles. In vitro reconstitution of these enzymes, variation of the electrophile, the catalytic metal and the reaction conditions, compounded with structural studies revealed important insight into the catalytic mechanism of enzyme-catalyzed metal-dependent C-S bond formation. Secondly, we examined a distantly related class of metalloenzymes that occur in bacteria and fungi. These metalloenzymes are oxygen-dependent and catalyze oxidative coupling of histidine derivatives with cysteine derivatives by forming a new C-S bond. This reaction is a key step in ergothioneine biosynthesis. Kinetic and structural examination of these enzymes were used to study the subtle but functionally significant differences between fungal and bacterial enzymes. Overall, we have broadened our knowledge of enzymes forming carbon-sulfur bonds and containing a DinB domain, along with discovering a new class of fungal cysteine dioxygenase.