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

Guy, Jodie E.; Cai, Yuanheng; Baer, Marcel D.; Whittle, Edward; Chai, Jin; Yu, Xiao-Hong; Lindqvist, Ylva; Raugei, Simone; Shanklin, John

doi:10.1093/plphys/kiab577

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

Journal Article · Fri Dec 10 00:00:00 EST 2021 · Plant Physiology

DOI:https://doi.org/10.1093/plphys/kiab577· OSTI ID:1834036

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Karolinska Inst., Stockholm (Sweden)
Stony Brook Univ., NY (United States)
Pacific Northwest National Lab. (PNNL), Richland, WA (United States)
Brookhaven National Lab. (BNL), Upton, NY (United States)ac
Brookhaven National Lab. (BNL), Upton, NY (United States)

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. Furthermore, 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.

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Research Organization:: Brookhaven National Laboratory (BNL), Upton, NY (United States); Pacific Northwest National Laboratory (PNNL), Richland, WA (United States)

Sponsoring Organization:: USDOE Office of Science (SC), Basic Energy Sciences (BES). Chemical Sciences, Geosciences & Biosciences Division; USDOE

Grant/Contract Number:: SC0012704; AC05-76RL01830; AC05-576 76RL01830; 2011-05917

OSTI ID:: 1834036

Alternate ID(s):: OSTI ID: 1837108; OSTI ID: 1862221

Report Number(s):: BNL-222444-2021-JAAM; PNNL-SA-166222

Journal Information:: Plant Physiology, Vol. 188, Issue 3; ISSN 1532-2548

Publisher:: American Society of Plant BiologistsCopyright Statement

Country of Publication:: United States

Language:: English

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