Proposed mechanism for reduction of deprotonated carboxylic acid by Oye1.

<p>Engineering of the enzyme by substitution of the native residues with putative X and Y residues could lead to the formation of hydrogen bonds between the enzyme binding pocket and both oxygens of the carboxylate group. Upon hydrogen bonding to the enzyme (hashed lines) electrons from the double bond are shifted towards the catalytic residues Asn194 and His191 for one of the oxygens, and to the residues X and Y for the other oxygen (dotted lines), thereby creating a partial positive charge on the β-carbon (δ⁺) of the substrate, which activates the double bond, making it prone to attack. When the double bond is activated the transfer of a hydride from the flavin N₅ to the β-carbon of the substrate and protonation from Tyr196 can occur, resulting in hexanoic acid as the final product. The movement of electrons involved in the hydride attack and protonation are indicated by the curved arrows. For simplicity, only the mechanism for Oye1 is shown.