Kinetic mechanism of vanillyl‐alcohol oxidase with short‐chain 4‐alkylphenols

Abstract

The kinetic mechanism of vanillyl-alcohol oxidase with 4-methylphenol, 4-ethylphenol, 4-propylphenol and their CA-deuterated analogs has been studied at pH 7.5 and 25°C. Conversion of 4-methylphenol is extremely slow (0.005 s Ϫ1 ) while the enzyme is largely in the reduced form during turnover. 4-Ethylphenol and 4-propylphenol are readily converted while the enzyme is mainly in the oxidized form during turnover. The deuterium kinetic isotope effect for overall catalysis ranges between 7Ϫ10 whereas the intrinsic deuterium kinetic isotope effect for flavin reduction ranges over 9Ϫ10. With all three 4-alkylphenols, flavin reduction appeared to be a reversible process with the rate of reduction being in the same range as the rate for the reverse reaction. Keywords : alkylphenol; flavoprotein ; p-quinone methide ; kinetic isotope effect ; vanillyl-alcohol oxidase.Vanillyl-alcohol oxidase is a covalent flavoprotein isolated from Penicillium simplicissimum, a filamentous fungus capable of growing on a wide variety of aromatic compounds [1,2]. The enzyme is a rather stable homooctamer with each 65-kDa sub-The reaction mechanism of vanillyl-alcohol oxidase has unit containing an 8A-(N 3 -histidyl)-FAD molecule [3,4]. Vanilproperties in common with that of p-cresol methylhydroxylase lyl-alcohol oxidase is active with a wide range of para-substi- [7]. This bacterial flavocytochrome converts a wide range of tuted phenolic compounds [5], but the physiological function of 4-alkylphenols first into 4-hydroxybenzyl alcohols and subsethe enzyme is not fully understood. Based on induction experiquently into 4-hydroxybenzaldehydes. In contrast to vanillyl-alments, 4-(methoxymethyl)phenol has been proposed to represent cohol oxidase, flavin reoxidation in p-cresol methylhydroxylase the physiological substrate [2]. A detailed kinetic study with this involves the transfer of electrons to a tightly bound cytochrome phenolic methylether has pointed to a ternary complex mechasubunit [8]. nism in which flavin reduction is the rate-limiting step in cataly-The crystal structure of vanillyl-alcohol oxidase has recently sis [6]. The reaction of vanillyl-alcohol oxidase with 4-(methbeen solved at 2.5 Å resolution [9]. Each vanillyl-alcohol oxioxymethyl)phenol involves the initial formation of a binary dase monomer consists of two domains : one creates a binding complex of reduced enzyme and the p-quinone methide of site for the ADP moiety of the FAD molecule, while the other 4-(methoxymethyl)phenol. This complex then reacts with modomain covers the active center which is located between the lecular oxygen, reoxidizing the flavin, and after water addition two domains. The structure shows that the isoalloxazine ring of of the p-quinone methide, the products 4-hydroxybenzaldehyde the flavin makes a covalent bond with His422. Furthermore, the and methanol are formed (Eqn 1).active site is located in the interior of the protein and contains an Correspondence to W. J. H. van Berkel, Department of Biochemis-anion-binding pocket facilitating the depr...

Laboratory-evolved Vanillyl-alcohol Oxidase Produces Natural Vanillin

“…Before protein staining, gels were incubated in 5% (v/v) acetic acid for fluorescence detection of covalently bound FAD (38). Analytical size-exclusion chromatography was performed with a Superdex 200 PG 10/30 column (Amersham Biosciences) (27). Absorption spectra were recorded using a Hewlett-Packard HP 8453 diode array spectrophotometer.…”

“…On the other hand, 2-chloro-p-cresol was a substrate neither for the mutants nor for wild-type VAO. p-Cresol, the physiological substrate for PCMH, is a very poor substrate for VAO (27). Intriguingly, the mutant enzymes performed worse than wild-type VAO with this substrate (turnover rates Ͻ 0.001 s Ϫ1 ).…”

“…The catalytic cycle of VAO with 4-alkylphenols consists of two half-reactions (see Fig. 1) (26,27). In the reductive half-reaction, FAD is reduced by the aromatic substrate with the concomitant formation of a p-quinone methide product intermediate.…”

“…Superposition of the crystallographic models of VAO (Protein Data Bank code 1VAO) (23) and PCMH (code 1DIQ) (29) has revealed a root mean square deviation of 1.0 Å for 470 C-␣ atoms. The two enzymes catalyze similar reactions, but p-cresol (4-methylphenol), the physiological substrate of PCMH (30), is an extremely poor substrate for VAO (27). Kinetic studies have shown that the low activity of VAO with p-cresol and also with creosol is due to the formation of an air-stable flavin N-5 substrate adduct (21,27,31).…”

“…The two enzymes catalyze similar reactions, but p-cresol (4-methylphenol), the physiological substrate of PCMH (30), is an extremely poor substrate for VAO (27). Kinetic studies have shown that the low activity of VAO with p-cresol and also with creosol is due to the formation of an air-stable flavin N-5 substrate adduct (21,27,31). From the available crystallographic data, it is not obvious which structural features are involved in determining the different reactivity of both enzymes to 4-methylphenols.…”