Role of Active Site Histidines in the Two Half-Reactions of the Aryl-Alcohol Oxidase Catalytic Cycle

Abstract

The crystal structure of aryl-alcohol oxidase (AAO), a flavoenzyme involved in lignin degradation, reveals two active-site histidines, whose role in the two enzyme half-reactions was investigated. The redox state of flavin during turnover of the variants obtained show a stronger histidine involvement in the reductive than in the oxidative half-reaction. This was confirmed by the k cat/K m(Al) and reduction constants that are 2–3 orders of magnitude decreased for the His546 variants and up to 5 orders for the His502 variants, while the corresponding O2 constants only decreased up to 1 order of magnitude. These results confirm His502 as the catalytic base in the AAO reductive half-reaction. The solvent kinetic isotope effect (KIE) revealed that hydroxyl proton abstraction is partially limiting the reaction, while the α-deuterated alcohol KIE showed a stereoselective hydride transfer. Concerning the oxidative half-reaction, directed mutagenesis and computational simulations indicate that only His502 is involved. Quantum mechanical/molecular mechanical (QM/MM) reveals an initial partial electron transfer from the reduced FADH– to O2, without formation of a flavin-hydroperoxide intermediate. Reaction follows with a nearly barrierless His502H+ proton transfer that decreases the triplet/singlet gap. Spin inversion and second electron transfer, concomitant with a slower proton transfer from flavin N5, yields H2O2. No solvent KIE was found for O2 reduction confirming that the His502 proton transfer does not limit the oxidative half-reaction. However, the small KIE on k cat/K m(Ox), during steady-state oxidation of α-deuterated alcohol, suggests that the second proton transfer from N5H is partially limiting, as predicted by the QM/MM simulations.

Discovery and Characterization of a 5-Hydroxymethylfurfural Oxidase from Methylovorus sp. Strain MP688

“…In studies on AAO from P. eryngii, His502 was replaced with alanine, resulting in a decrease in the k cat value of almost 3,000-fold, similar to the effect seen in the HMFO mutant enzyme. The mutation also increased the K m value 80-fold, an effect much more severe than that observed in HMFO (21). Studies on CholO and AAO from P. eryngii suggest that this histidine is the active-site base (28).…”

“…One key feature of GMC-type oxidases is the presence of an absolutely conserved histidine in the C-terminal domain. The role of this histidine has been investigated in well-studied GMC family members such as glucose oxidase, choline oxidase (CholO), and aryl alcohol oxidase (AAO) (13,20,21). Replacement of this histidine with alanine resulted in a drastic reduction of the catalysis of all of these oxidases.…”

5‐hydroxymethylfurfural conversion by fungal aryl‐alcohol oxidase and unspecific peroxygenase

“…Recently, AAO structurefunction relationships and mechanistic aspects have been studied in-depth [15][16][17][18][19][20], and its ability to oxidize aromatic (and some aliphatic polyunsaturated) primary alcohols, as well as related aldehydes, has been demonstrated [21,22]. Comparison of the AAO activity for these substrates reveals much lower catalytic efficiency for oxidizing aldehydes than alcohols, due to both lower k cat and higher K m values (Table S1).…”

Focused Directed Evolution of Aryl-Alcohol Oxidase in Saccharomyces cerevisiae by Using Chimeric Signal Peptides

“…The past few years have witnessed an intense effort to discern the basis and mechanism of action underlying AAO catalysis (3)(4)(5)(6)(7)(8)(9)(10). The AAO catalytic cycle involves dehydrogenative oxidation mediated by two half-reactions: (i) the reductive half-reaction in which the donor alcohol is two-electron oxidized by the FAD cofactor, the latter receiving one of the alcohol's ␣-Hs through a hydride transfer process that yields the aldehyde product and the reduced flavin, and (ii) the oxidative half-reaction, in which O 2 is two-electron reduced by the FAD, releasing H 2 O 2 and the reoxidized flavin (5).…”