Functional and Structural Characterization of a (+)-Limonene Synthase from Citrus sinensis

Area of Science:

• Biochemistry
• Structural Biology
• Natural Product Synthesis

Background:

• Terpenes are a large, diverse class of natural compounds with significant commercial and medical value.
• Limonene, a cyclic monoterpene, exists as (+) and (-) enantiomers produced by distinct enzymes.
• Understanding (+)-limonene synthase is crucial for elucidating enantiomeric selectivity in terpene biosynthesis.

Purpose of the Study:

• To clone and biochemically characterize a (+)-limonene synthase from navel orange (Citrus sinensis).
• To determine the crystal structure of the (+)-limonene synthase.
• To compare the structure with (-)-limonene synthase to understand enantiomeric selectivity.

Main Methods:

• Cloning and expression of the (+)-limonene synthase gene.
• Biochemical characterization using Michaelis-Menten kinetics.
• X-ray crystallography to determine the enzyme's structure at 2.3 Å resolution.

Main Results:

• The cloned enzyme exclusively synthesizes the (+)-enantiomer of limonene.
• The crystal structure reveals an 'open' conformation of the apoprotein.
• Structural comparison indicates significant conformational changes upon substrate binding, including helix movement and tyrosine residue repositioning.

Conclusions:

• The first biochemical and structural characterization of a (+)-limonene synthase is reported.
• The findings provide mechanistic insights into how this enzyme achieves enantiomeric selectivity.
• This work contributes to understanding the diversity and function of terpene synthases.