Abstract
Monoterpene synthases are enzymes that convert geranyl diphosphate to various monoterpenes. Pinene synthase is a monoterpene synthase found in grand fir (Abies grandis) that catalyzes the conversion of geranyl diphosphate to the monoterpenes α- and β- pinene. Most monoterpene synthases require a divalent metal cation for the reaction, such as Mg2+ or Mn2+. Pinene synthase is unique in that it cannot use Mg2+ for the catalysis. Previous work done by our research group used homology modeling to identify amino acid residues that may cause the metal ion specificity of pinene synthase. Similar monoterpene synthases - limonene synthases from Mentha spicata and Citrus sinensis which can use both Mg2+ and Mn2+ for catalysis - were analyzed and amino acid residues which interact with the metal ion co-factors were identified. By performing sequence alignment with the limonene synthases and pinene synthase, we identified two residues which differ between pinene synthase and the two limonene synthases, R522 and G523. We designed two clones encoding pinene synthase in which these residues have been mutated to those found in the limonene synthase from Mentha spicata, to see whether these mutations will allow pinene synthase to use Mg2+ for catalysis. The first clone has the single mutation R522A, whereas the second clone has the double mutation R522A/ G523D. We successfully expressed the two mutant enzymes in E. coli, purified them, and analyzed their metal-ion specificity. The two mutants showed no significant activity in the presence of either Mn2+ or Mg2+ ions. This indicates that the native residue R522 is necessary for the catalysis reaction of pinene synthase. The information obtained could be used toward future enzymology studies, which could lead to possible industrial applications.