Regioselectivity mechanism of the Thunbergia alata Delta(6)-16:0-acyl carrier protein desaturase

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 (18:1 Delta(9)cis)-ACP. Several variants expressing distinct regioselectivity have been described including a Delta(6)-16:0-ACP desaturase from black-eyed Susan vine (Thunbergia alata). We solved a crystal structure of the T. alata desaturase at 2.05 angstrom resolution. Using molecular dynamics (MD) 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. The model complex was used to identify mutant variants that could convert the T. alata Delta(6) desaturase to Delta(9) regioselectivity. Additional modeling between ACP and the mutant variants confirmed the predicted regioselectivity. To validate the 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 Delta(6) to Delta(9) selectivity exhibited the predicted changes. In complementary experiments, variants of the castor desaturase designed to convert Delta(9) to Delta(6) selectivity lost some of their Delta 9 desaturation ability and gained the ability to desaturate at the Delta(6) position. 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. Predictions regarding the mechanism of Delta(6) regioselectivity of the Thunbergia alata desaturase based on X-ray crystallography and molecular dynamics simulations are confirmed by experiment.