Enantioselective hydroxylation of 4‐alkylphenols by vanillyl alcohol oxidase

Proc. Natl. Acad. Sci. U.S.A.

Ferrer

et al. 2000

Self Cite

Vanillyl-alcohol oxidase (VAO) is the prototype of a newly recognized family of structurally related oxidoreductases sharing a conserved FAD-binding domain. The active site of VAO is formed by a cavity where the enzyme is able to catalyze many reactions with phenolic substrates. Among these reactions is the stereospecific hydroxylation of 4-ethylphenol-forming (R)-1-(4 -hydroxyphenyl)ethanol. During this conversion, Asp-170 is probably critical for the hydration of the initially formed p-quinone methide intermediate. By site-directed mutagenesis, the putative active site base has been relocated to the opposite face of the active site cavity. In this way, a change in stereospecificity has been achieved. Like native VAO, the single mutants T457E, D170A, and D170S preferentially converted 4-ethylphenol to the (R)-enantiomer of 1-(4 -hydroxyphenyl)ethanol. The double mutants D170A͞T457E and D170S͞T457E exhibited an inverted stereospecificity with 4-ethylphenol. Particularly, D170S͞ T457E was strongly (S)-selective, with an enantiomeric excess of 80%. The crystal structure of D170S͞T457E, in complex with trifluoromethylphenol, showed a highly conserved mode of ligand binding and revealed that the distinctive catalytic properties of this mutant are not caused by major structural changes.

supporting

confidence: 62%

Inversion of stereospecificity of vanillyl-alcohol oxidase

Heuvel

Proc. Natl. Acad. Sci. U.S.A.

Ferrer

et al. 2000

Self Cite

“…tion is limiting the rate of overall catalysis. Vanillyl-alcohol oxidase converts both 4-ethylphenol and 4-propylphenol to a mixture of the corresponding benzylic alcohol and alkenylic phenol in a ratio of approximately 4 :1 [15]. gesting that the rate of enzyme reduction is partially limiting the HPLC product analysis revealed that deuteration of CA has no rate of overall catalysis.…”

Section: Synthesis Of 4-[b-2 H 3 ]Methylphenolmentioning

confidence: 99%

“…Furthermore, the extent of reduction also decreased indicating formed at pH 9.4. These results suggest that in the second relatively slow phase of the reductive half-reactions of vanillyl-alcothat the rate of reduction (k 2 ) decreased to a greater extent than vinylic phenols are formed [15]. It is not clear whether this competing rearrangement of the 4-alkylphenol p-quinone methide intermediates is related to the water accessibility of the active site.…”

Section: Synthesis Of 4-[b-2 H 3 ]Methylphenolmentioning

confidence: 99%

“…It is not clear whether this competing rearrangement of the 4-alkylphenol p-quinone methide intermediates is related to the water accessibility of the active site. In this respect it is interesting to note that, in the reaction of vanillyl-alcohol oxidase with 2-methoxy-4-propylphenol, hardly any vinylic product is formed [15].…”

Section: Synthesis Of 4-[b-2 H 3 ]Methylphenolmentioning

confidence: 99%

“…However, the results presented here indicate that with both 4-ethylphenol is shielded from solvent [9]. This corresponds to the observed enantioselective hydroxylation of 4-alkylphenols [15], which is and 4-propylphenol, an N5 flavin adduct is formed under anaerobic conditions. From this, it is reasonable to assume that E red~P indicative for an enzyme-mediated nucleophilic attack of water at the methide carbon.…”

Section: Synthesis Of 4-[b-2 H 3 ]Methylphenolmentioning

confidence: 99%

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

European Journal of Biochemistry

Heuvel

Roelofs

et al. 1998

Self Cite

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...

Flavoenzyme-Catalyzed Oxygenations and Oxidations of Phenolic Compounds

Moonen

Advanced Synthesis & Catalysis

Rietjens

et al. 2002

Flavin‐dependent monooxygenases and oxidases play an important role in the mineralization of phenolic compounds. Because of their exquisite regioselectivity and stereoselectivity, these enzymes are of interest for the biocatalytic production of fine chemicals and food ingredients. In our group, we have characterized several flavoenzymes that act on phenolic compounds, including 4‐hydroxybenzoate 3‐hydroxylase, 3‐hydroxyphenylacetate 6‐hydroxylase, 4‐hydroxybenzoate 1‐hydroxylase (decarboxylating), hydroquinone hydroxylase, 2‐hydroxybiphenyl 3‐monooxygenase, phenol hydroxylase, 4‐hydroxyacetophenone monooxygenase and vanillyl‐alcohol oxidase. The catalytic properties of these enzymes are reviewed here, together with insights obtained from site‐directed and random mutagenesis.