Deciphering magnesium binding site and structure-function insights in a class II sesquiterpene cyclase

Magnesium ions (Mg 2+ ) are crucial in class II terpene cyclases that utilize substrates with diphosphate groups. Interestingly, these enzymes catalyze reactions without cleaving the diphosphate group, instead initiating the reaction through protonation . In our recent research, we discovered a novel class II sesquiterpene cyclase in Streptomyces showdoensis . Notably, we determined its crystal structure and identified Mg 2+ within its active site. This finding has shed light on the previously elusive question of Mg 2+ binding in class II terpene cyclases. In this chapter, we outline our methods for discovering this novel enzyme , including steps for its purification , crystallization , and kinetic analysis.