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Regioselectivity mechanism of the Thunbergia alata Δ6-16:0-acyl carrier protein desaturase.

Regioselectivity mechanism of the Thunbergia alata Δ6-16:0-acyl carrier protein desaturase.

Authors :
Guy JE
Cai Y
Baer MD
Whittle E
Chai J
Yu XH
Lindqvist Y
Raugei S
Shanklin J
Source :
Plant physiology [Plant Physiol] 2022 Mar 04; Vol. 188 (3), pp. 1537-1549.
Publication Year :
2022

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.<br /> (Published by Oxford University Press on behalf of American Society of Plant Biologists 2021. This work is written by US Government employees and is in the public domain in the US.)

Details

Language :
English
ISSN :
1532-2548
Volume :
188
Issue :
3
Database :
MEDLINE
Journal :
Plant physiology
Publication Type :
Academic Journal
Accession number :
34893899
Full Text :
https://doi.org/10.1093/plphys/kiab577