Regioselectivity of the Thunbergia alata Δ6-16:0-acyl carrier protein desaturase by molecular dynamics.

Abstract

The plant acyl-acyl carrier protein (ACP) desaturases are a family of soluble enzymes that convert saturated fatty-acyl ACPs into their cis-monounsaturated equivalents in an oxygen-dependent reaction. These enzymes play a key role in biosynthesis of mono-unsaturated fatty acids in plants. The archetype of this group is the Δ9-18:0-ACP desaturase that introduces a cis double bond between carbons 9 and 10 to produce oleoyl-ACP. Several variants expressing distinct regioselectivity have been described including a Δ6-16:0-ACP desaturase from black-eyed Susan vine (Thunbergia alata). ACPs are central proteins in the fatty acid biosynthesis that deliver the substrate to the desaturase. They have been reported to show a varying degree of local dynamics and structural variability depending on the substrate length. It has been suggested that the substrate specific change in structure and dynamics is crucial for the decreasing enzymatic activity in the case of the Δ9-Desaturase for chains length shorter than 18. Using molecular dynamics (MD) simulations, we identified a low-energy complex between 16:0-ACP and the desaturase that would position carbons 6 and 7 of the acyl chain adjacent to the diiron active site. The model complex was used to identify mutant variants that could convert the T. alata Δ6 desaturase to Δ9 regioselectivity. Additional modeling between ACP and the mutant variants confirmed the predicted regioselectivity. The computational workflow for revealing the mechanistic understanding of regioselectivity presented herein lays a foundation for designing acyl-ACP desaturases with novel selectivities to increase the diversity of monoenes available for bioproduct applications.