Abstract
Terpenes make up the most abundant class of naturally occurring organic molecules and can be used in a wide variety of industries. In-depth understanding of their biosynthetic mechanisms allows scientists to reengineer them and manufacture useful chemicals that are not currently available in nature. Additionally, these enzymes make multiple stereocenters with a high degree of specificity, and currently we don’t understand how they achieve this. Limonene is the simplest monoterpene product requiring a simple cyclization reaction by limonene synthase (LS). This molecule is found in nature as two distinct (+) and (-) enantiomers which are each made by their own specific enzymes: (+)-limonene is made by (+)-LS found in navel oranges while (-)-limonene is made by (-)-LS found in spearmint. The goal of my research is to elucidate the determinants of (+)-LS enantioselectivity by swapping the active site residues found in (+)-LS to those found in (-)-LS and making (+)-LS more (-)-enantiomer-specific. Here we report that the single mutation F484L generates a promiscuous enzyme that is slower than the wildtype, but it results in a 50% production of (-)-limonene. Double mutations with a second-shell residue fail to enhance the enantioselectivity further. Future efforts will focus on determining the structure of the mutant enzyme bound to a substrate analogue and determining other mutations that could alter the enantiomeric outcome completely.