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Determinants of Enantiospecificity in Limonene Synthases.

Monoterpene synthases catalyze the first committed step in the biosynthesis of monoterpenes and are in part responsible for the enormous structural diversity among this class of metabolites. Here, we explore the structure-function relationships underlying the formation of limonene enantiomers in limonene synthases that bind geranyl diphosphate as a common substrate. Based on analyses that consider both crystal structural data and amino acid sequence divergence, we identified candidate active site residues with potential roles in catalyzing reactions that involve accommodating reaction intermediates of opposite enantiomeric series. We demonstrate that spearmint (-)-limonene synthase (which generates > 99% (-)-limonene over (+)-limonene) can be converted into a mutant enzyme, by exchanging four residues (C321S, N345I, S453V, M458V), that produces (+)-limonene with reversed enantiospecificity (80% (+)-limonene and 3% (-)-limonene; remainder are mostly bicyclic monoterpenes). This study provides the foundation for a more in-depth understanding of the formation of enantiomeric series of monoterpenes, which can have vastly different olfactory properties.