Mechanistic and Structural Insights into the Regioselectivity of an Acyl-CoA Fatty Acid Desaturase via Directed Molecular Evolution
作者： Thomas Vanhercke , Pushkar Shrestha , Allan G. Green , Surinder P. Singh
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摘要: Membrane-bound fatty acid desaturases and related enzymes play a pivotal role in the biosynthesis of unsaturated various unusual acids. Structural insights into remarkable catalytic diversity wide range substrate specificities this class remain limited due to lack crystal structure. To investigate structural basis double bond positioning (regioselectivity) desaturation reaction more detail, we relied on combination directed evolution vitro powerful yeast complementation assay screen for Δx regioselectivity. After two selection rounds, variants bifunctional Δ12/Δ9-desaturase from house cricket (Acheta domesticus) exhibited increased Δ9-desaturation activity shorter chain This change specificity was result as few three mutations, some them near putative active site. Subsequent analysis individual substitutions revealed an important residue Phe-52 facilitating acyl substrates allowed redesign 16:0-specific Δ9-desaturase. Our results demonstrate that minimal number mutations can have profound impact regioselectivity acyl-CoA include first biochemical data supporting carrier desaturase able carry out Δ12-desaturation.
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