PNNL

Abstract

Plant plastidial acyl-acyl carrier protein (ACP) desaturases are a soluble class of diiron-containing enzymes that are distinct from the diiron-containing integral membrane desaturases found in plants and other organisms. The archetype of this class is the stearoyl-ACP desaturase which converts stearoyl-ACP into oleoyl (18:1D9cis)-ACP. Several variants expressing distinct regioselectivity have been described including a D6-16:0-ACP desaturase from Thunbergia alata. We solved a crystal structure of the T. alata desaturase at 2.05 Å resolution. Using molecular dynamics simulations, we identified a low-energy complex between 16:0-ACP and the desaturase that would position C6 and C7 of the acyl chain adjacent to the diiron active site. This model complex was used to identify mutant variants that could convert the T. alata D6 desaturase to D9 regioselectivity. Additional molecular dynamics simulations between ACP and the mutant variants confirmed the predicted regioselectivity. To validate our in-silico predictions, we synthesized two variants of the T. alata desaturase and analyzed their reaction products using gas chromatography-coupled mass spectrometry. Assay results confirmed that mutants designed to convert T. alata D6 to D9 selectivity exhibited the predicted changes. In complementary experiments, variants of the castor desaturase designed to convert D9 to D6 selectivity lost some of their D9 desaturation ability and gained the ability to desaturate at the D6 position. The computational approach 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.