Elucidating the biochemical properties of limonene synthase from Agastache rugosa

Limonene, a monocyclic monoterpene abundant in citrus essential oils, is extensively utilized in the fragrance, flavor, and pharmaceutical industries. Limonene synthase (LS) is a key enzyme in limonene biosynthesis by converting geranyl pyrophosphate to limonene. Despite its industrial significance, microbial limonene production is limited to a few LSs due to expression challenges. There is a growing demand to discover and characterize new LS enzymes with enhanced catalytic efficiency and diverse functional properties. Although LS from Mentha spicata (MsLS) is commonly used for microbial limonene production, its industrial applicability is hindered by relatively low catalytic efficiency. Therefore, this study focuses on the characterization of LS from Agastache rugosa (ArLS), which has shown higher catalytic efficiency than MsLS under the tested conditions and may provide a valuable biochemical foundation for microbial limonene production. This study provides a comprehensive kinetic analysis and identifies putative active-site residues of ArLS using homology modeling and site-directed mutagenesis, techniques rarely applied to LSs.

ArLS was cloned and heterologously expressed in Escherichia coli, leading to high limonene production, approximately 60% of the theoretical maximum based on the provided substrate, which was 1.15 times higher than that achieved with MsLS. The enzyme exhibited optimal activity at 30 °C and pH 8.0 in the presence of 0.1 mM Mn²⁺, aligning with the typical catalytic conditions of plant terpene synthases. Kinetic analysis indicated that ArLS displayed the highest catalytic efficiency among reported LSs, with a significantly lower K_M (5.5 ± 0.8 µM) and a higher k_cat/K_M (0.4 ± 0.06 min^-1·µM^-1). Structural insights into the putative active site of ArLS were obtained through homology modeling using AlphaFold. To validate these predictions, site-directed alanine scanning mutagenesis was performed on the putative active-site residues. Comparative analysis with previously characterized LSs revealed higher substrate-to-product conversion rate.

Characterization of ArLS highlights its potential for biotechnological limonene production. Its high catalytic efficiency and substrate affinity indicate that it is a promising candidate for industrial applications. This study offers the initial comprehensive kinetic characterization and putative active-site analysis of ArLS, enhancing our understanding of its catalytic mechanism. Enzyme assays, kinetic assessments, and structural insights from this study could establish a basis for future enzyme engineering to improve enzyme performance. Additionally, this study lays the groundwork for further investigation of monoterpene synthases and the sustainable production of valuable terpenes.