Engineering the substrate specificity and regioselectivity of Burkholderia thailandensis lipoxygenase

Chrisnasari, Ruth and Hennebelle, Marie and van Berkel, Willem J. H. and Vincken, Jean-Paul and Ewing, Tom A. (2024) Engineering the substrate specificity and regioselectivity of Burkholderia thailandensis lipoxygenase. New Biotechnology, 84. pp. 64-76. ISSN 1871-6784; E-ISSN 1876-4347

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Abstract

Lipoxygenases (LOXs) catalyze the regioselective dioxygenation of polyunsaturated fatty acids (PUFAs), generating fatty acid hydroperoxides (FAHPs) with diverse industrial applications. Bacterial LOXs have garnered significant attention in recent years due to their broad activity towards PUFAs, yet knowledge about the structural factors influencing their substrate preferences remains limited. Here, we characterized a bacterial LOX from Burkholderia thailandensis (Bt-LOX), and identified key residues affecting its substrate preference and regioselectivity through site-directed mutagenesis. Bt-LOX preferred ω-6 PUFAs and exhibited regioselectivity at the ω-5 position. Mutations targeting the substrate binding pocket and the oxygen access channel led to the production of three active variants with distinct catalytic properties. The A431G variant bifurcated dioxygenation between the ω-5 and ω-9 positions, while F446V showed reduced regioselectivity with longer PUFAs. Interestingly, L445A displayed altered substrate specificity, favoring ω-3 over ω-6 PUFAs. Furthermore, L445A shifted the regioselectivity of dioxygenation to the ω-2 position in ω-3 PUFAs, and, for some substrates, facilitated dioxygenation closer to the carboxylic acid terminus, suggesting an altered substrate orientation. Among these variants, L445A represents a significant milestone in LOX research, as these alterations in substrate specificity, dioxygenation regioselectivity, and substrate orientation were achieved by a single mutation only. These findings illuminate key residues governing substrate preference and regioselectivity in Bt-LOX, offering opportunities for synthesizing diverse FAHPs and highlighting the potential of bacterial LOXs as biocatalysts with widespread applications.

Item Type:	Article
Uncontrolled Keywords:	Dioxygenation; Hydroperoxide; Lipoxygenase; Fatty acid oxidation
Subjects:	Q Science > QR Microbiology
Divisions:	Faculty of Technobiology > Department of Biology
Depositing User:	RUTH CHRISNASARI - 210001
Date Deposited:	09 Oct 2024 09:00
Last Modified:	09 Oct 2024 09:00
URI:	http://repository.ubaya.ac.id/id/eprint/47206

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