Available online 5 September 2022, 110120

Potential activity-enhancing mutation sites on the lid structure were identified.

The kcat/Km of mutants increased by 1.4–9.2 times over wild-type LOX and the half-life (t1/2) at 50°C increased by 4.6 min.

Increased flexibility of bound substrate was beneficial for catalytic activity

Lipoxygenase (LOX) catalyzes the peroxidation of unsaturated fatty acids to produce hydroperoxides, which had been widely used in food, medicine and chemical industries due to its decoloration of food and conversion of renewable oils. Thus, higher catalytic activity and stability is desired for low-cost and expanded industrial applications of LOX. To improve the catalytic activity of LOX, a mutant library of Pseudomonas aeruginosa lipoxygenase (PaLOX) was firstly built via semi-rational design. The kcat/Km of mutant increased by 9.2-fold and the half-life (t1/2) at 50°C increased by 4.6 min. Molecular dynamics (MD) simulation indicated that mutation reduced steric hindrance to substrate binding and increased the flexibility of the lid domain that covered the bound unsaturated fatty acid substrate. In addition, van der Waals interactions between the substrate and amino acid residues of the binding pocket increased and alkyl and Pi-alkyl interactions decreased, which might improve the flexibility and substrate binding affinity. These findings promoted understanding of the structure-function relationship of LOX and increase its catalytic efficiency and stability for further industrial application.

Lipoxygenase

semi-rational design

molecular dynamics simulation

substrate flexibility

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