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

Magnesium ions (Mg²⁺) 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 Mg²⁺ within its active site. This finding has shed light on the previously elusive question of Mg²⁺ 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.