7PP7

Thunberia alata 16:0-ACP desaturase

Summary for 7PP7

• Entry DOI: 10.2210/pdb7pp7/pdb
• Descriptor: Acyl-[acyl-carrier-protein] 6-desaturase, FE (III) ION (3 entities in total)
• Functional Keywords: desaturase, di-iron protein, oxidoreductase
• Biological source: Thunbergia alata (Black-eyed Susan vine)
• Total number of polymer chains: 1
• Total formula weight: 39565.57

Authors

Guy, J.E.,Whittle, E.,Cai, Y.,Chai, J.,Lindqvist, Y.,Shanklin, J. (deposition date: 2021-09-13, release date: 2021-12-22, Last modification date: 2024-01-31)

Primary citation

Guy, J.E.,Cai, Y.,Baer, M.D.,Whittle, E.,Chai, J.,Yu, X.H.,Lindqvist, Y.,Raugei, S.,Shanklin, J.
Regioselectivity mechanism of the Thunbergia alata Delta 6-16:0-acyl carrier protein desaturase.
Plant Physiol., 188:1537-1549, 2022

PubMed 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 (18:1Δ9cis)-ACP. Several variants expressing distinct regioselectivity have been described including a Δ6-16:0-ACP desaturase from black-eyed Susan vine (Thunbergia alata). We solved a crystal structure of the T. alata desaturase at 2.05 Å 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 Δ6 desaturase to Δ9 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 Δ6 to Δ9 selectivity exhibited the predicted changes. In complementary experiments, variants of the castor desaturase designed to convert Δ9 to Δ6 selectivity lost some of their Δ9 desaturation ability and gained the ability to desaturate at the Δ6 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.

PubMed: 34893899
DOI: 10.1093/plphys/kiab577

Experimental method

X-RAY DIFFRACTION (2.05 Å)