7pp7

Publication Abstract from PubMed

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 (18:1Delta9cis)-ACP. Several variants expressing distinct regioselectivity have been described including a Delta6-16:0-ACP desaturase from black-eyed Susan vine (Thunbergia alata). We solved a crystal structure of the T. alata desaturase at 2.05 A 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 Delta6 desaturase to Delta9 regioselectivity. Additional modelling 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 Delta6 to Delta9 selectivity exhibited the predicted changes. In complementary experiments, variants of the castor desaturase designed to convert Delta9 to Delta6 selectivity lost some of their Delta9 desaturation ability and gained the ability to desaturate at the Delta6 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.

Regioselectivity mechanism of the Thunbergia alata Delta6-16:0-acyl carrier protein desaturase.,Guy JE, Cai Y, Baer MD, Whittle E, Chai J, Yu XH, Lindqvist Y, Raugei S, Shanklin J Plant Physiol. 2021 Dec 10. pii: 6459183. doi: 10.1093/plphys/kiab577. PMID:34893899^[3]

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.