title: Stereoselective hydride transfer by aryl-alcohol oxidase, a member of the GMC superfamily.
abstract: Primary alcohol oxidation by aryl-alcohol oxidase (AAO), a flavoenzyme providing H(2) O(2)  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)-α-deuterated p-methoxybenzyl alcohol, a primary KIE (≈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 Nε, respectively) relative to the alcohol Cα-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 α-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.
authors.affiliation: Centro de Investigaciones Biológicas (CIB), Consejo Superior de Investigaciones Científicas (CSIC), Ramiro de Maeztu 9, 28040 Madrid, Spain.
authors.forename: Aitor
authors.initials: A
authors.lastname: Hernández-Ortega
authors.forename: Patricia
authors.initials: P
authors.lastname: Ferreira
authors.forename: Pedro
authors.initials: P
authors.lastname: Merino
authors.forename: Milagros
authors.initials: M
authors.lastname: Medina
authors.forename: Victor
authors.initials: V
authors.lastname: Guallar
authors.forename: Angel T
authors.initials: AT
authors.lastname: Martínez
mesh_terms: D000429:Alcohol Oxidoreductases / Q000737:chemistry / Q000378:metabolism*; D000840:Anisoles / Q000737:chemistry / Q000378:metabolism; D006639:Histidine / Q000378:metabolism; D006859:Hydrogen / Q000737:chemistry / Q000378:metabolism*; D008958:Models, Molecular; D010084:Oxidation-Reduction; D013237:Stereoisomerism
chemical_list: D000840:Anisoles; D006639:Histidine; C066943:anisyl alcohol; D006859:Hydrogen; D000429:Alcohol Oxidoreductases; C077759:aryl-alcohol oxidase