Membrane fatty acyl desaturases (mFAD) are ubiquitous enzymes in eukaryotes. They introduce double bonds into fatty acids (FAs), producing structurally diverse unsaturated FAs which serve as membrane lipid components or precursors of signaling molecules. The mechanisms controlling enzymatic specificity and selectivity of desaturation are, however, poorly understood. We found that the physicochemical properties, particularly side chain volume, of a single amino acid (aa) residue in insect mFADs (Lepidoptera: Bombyx mori and Manduca sexta) control the desaturation products. Molecular dynamics simulations of systems comprising wild-type or mutant mFADs with fatty acyl-CoA substrates revealed that the single aa substitution likely directs the outcome of the desaturation reaction by modulating the distance between substrate fatty acyl carbon atoms and active center metal ions. These findings, as well as our methodology combining mFAD mutational screening with molecular dynamics simulations, will facilitate prediction of desaturation products and facilitate engineering of mFADs for biotechnological applications.

Division of Organic Chemistry and Biochemistry Ruđer Bošković Institute Bijenička 54 HR 10000 Zagreb Croatia

Institute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences Flemingovo n 2 166 10 Prague 6 Czech Republic

Max Planck Institute for Chemical Ecology Hans Knöll Str 8 D 07745 Jena Germany

Okinawa Institute of Science and Technology Graduate University 1919 1 Tancha 904 0495 Okinawa Japan

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