Stereoselective Hydride Transfer by Aryl-Alcohol Oxidase, a Member of the GMC Superfamily

Primary alcohol oxidation by aryl-alcohol oxidase (AAO), a flavoenzyme providing H2O2 to ligninolytic peroxidases, is produced by concerted proton and hydride transfers, as shown by substrate and solvent kinetic isotope effects (KIEs). Interestingly, when the reaction was investigated with synthesized (R)- and (S)-a-deuterated p-methoxybenzyl alcohol, a primary KIE (approximate to 6) was observed only for the R enantiomer, revealing that the hydride transfer is highly stereoselective. Docking of p-methoxybenzyl alcohol at the buried crystal active site, together with QM/MM calculations, showed that this stereoselectivity is due to the position of the hydride- and proton-receiving atoms (flavin N5 and His502 Ne, respectively) relative to the alcohol Ca-substituents, and to the concerted nature of transfer (the pro-S orientation corresponding to a 6 kcal mol-1 penalty with respect to the pro-R orientation). The role of His502 is supported by the lower activity (by three orders of magnitude) of the H502A variant. The above stereoselectivity was also observed, although activities were much lower, in AAO reactions with secondary aryl alcohols (over 98% excess of the R enantiomer after treatment of racemic 1-(p-methoxyphenyl)ethanol, as shown by chiral HPLC) and especially with use of the F501A variant. This variant has an enlarged active site that allow better accommodation of the a-substituents, resulting in higher stereoselectivity (S/R ratios) than is seen with AAO. High enantioselectivity in a member of the GMC oxidoreductase superfamily is reported for the first time, and shows the potential for engineering of AAO for deracemization purposes.