Vanillyl alcohol oxidase

Fraaije MW, WJH van Berkel (1997) Catalytic mechanism of the oxidative demethylation of 4-(methoxymethyl)phenol by vanillyl-alcohol oxidase. Evidence for formation of a /7-quinone intermediate. /Sio/ Chem 272 18111-18116. 

Aryl alcohol oxidase from the ligninolytic fungus Pleurotus eryngii had a strong preference for benzylic and allylic alcohols, showing activity on phenyl-substituted benzyl, cinnamyl, naphthyl and 2,4-hexadien-l-ol [103,104]. Another aryl alcohol oxidase, vanillyl alcohol oxidase (VAO) from the ascomycete Penicillium simplicissimum catalyzed the oxidation of vanillyl alcohol and the demethylation of 4-(methoxymethyl)phenol to vanillin and 4-hydro-xybenzaldehyde. In addition, VAO also catalyzed deamination of vanillyl amine to vanillin, and hydroxylation and dehydrogenation of 4-alkylphenols. For the oxidation of 4-alkylphenol, the ratio between the alcohol and alkene product depended on the length and bulkiness of the alkyl side-chain [105,106]. 4-Ethylphenol and 4-propylphenol, were mainly converted to (R)-l-(4 -hydroxyphenyl) alcohols, whereas medium-chain 4-alkylphenols such as 4-butylphenol were converted to l-(4 -hydroxyphenyl)alkenes. 

Van den Heuvel, R.H.H., Laane, C. and van Berkel, W.J.H. (2001) Exploring the biocatalytic potential of vanillyl-alcohol oxidase by site-directed mutagenesis. Advanced Synthesis and Catalysis, 343 (6-7), 515-520. 

In flavin-dependent monooxygenases, a flavin-oxygen intermediate reacts with the substrate, also producing water in a second step, and requiring cofactors for regeneration of the flavin moiety. The unusual flavoprotein vanillyl-alcohol oxidase (EC 1.1.3.38), in which the flavin moiety is covalently bound, catalyzes the oxidation of p-substituted phenols as well as deamination, hydroxylation and dehydrogenation reactions [10]. 

Vanillyl alcohol oxidase (VAO) is a flavoenzyme from the ascomycete Penicil-lium simplicissimum that converts a broad range of 4-hydroxybenzyl alcohols and 4-hydroxybenzylamines into the corresponding aldehydes. This large substrate specificity makes it possible to obtain vanillin from two major pathways. 

Scheme 22.6 Oxidative deamination of capsaicin-derived vanillyl amine and formation of vanillin [83] VAO vanillyl alcohol oxidase...

In another case, 4-alkylphenols can be transformed with the help of the flavoenzyme vanillyl alcohol oxidase (VAO) to either (R)-l-(4 -hydroxyphenyl) alcohols or to 1 -(4 -hydroxyphenyl)alkenes. Both products pass through a common intermediate, the p-quinone methide, which then either is attacked by water or rearranges. The product spectrum can be controlled by medium engineering in organic solvents such as acetonitrile and toluene, more ris-alkene but not trans-alkene, and less alcohol, are produced (van den Heuvel, 2001). A similar shift in cis/trans-alkene was achieved by the addition of monovalent anions that bind specifically close to the active site. 

C. Laane, P. J. Halling, and W. J. H. van Berkel, Tuning of the product spectrum of vanillyl-alcohol oxidase by medium engineering, FEES Lett. 2001, 503, 213-216. 

The ability of this ionization method for the determination of very high molecular weights is illustrated in Figure 1.26 [68], The spectrum displayed is obtained from assemblies of vanillyl alcohol oxidase containing respectively 16 and 24 proteins. The spectrum was obtained with a hybrid quadrupole TOF instrument, Q-TOF Micromass, equiped with a micro-ESI source. To obtain such a spectrum one needs not only a mass spectrometer with sufficient mass range and resolution, but also high skill in protein purification. 

MicroESI Q-TOF spectrum of assemblies of vanillyl alcohol oxidase obtained by W.J.H. van Berkel and co-workers [68], At the left, an octamer-dimer of 1.02 MDa molecular weight, and at the right, the octamer-trimer of 1.53 MDa. Reproduced courtesy of Dr. A.J.R. Heck. 

Enzymatic syntheses of CPS have also been reported, using various lipase-catalyzed transacylations [69]. Interestingly, a biotechnological process to obtain natural vanillin from CPS has been developed, capitalizing on the enzymatic hydrolysis of CPS and the oxidation of vanillamine with a flavoprotein vanillyl alcohol oxidase [70]. 

Fraaije MW, van Berkel WJH. Catalytic mechanism of the oxidative demethylation of 4-(methoxymethyl)phenol by vanillyl-alcohol oxidase. Evidence for formation of a -quinone methide intermediate. J. Biol. Chem. 1997 272 18111-18116. van den Heuvel RHH, Fraaije MW, Ferrer M, Mattevi A, van Berkel WJH. Inversion of stereospecificity of vanillyl-alcohol oxidase. Proc. Natl. Acad. Sci. U.S.A. 2000 97 9455-9460. 

Fig. 7.13. Reactions catalyzed by vanillyl alcohol oxidase (VAO) (from Refs. [37,38]).

Vanillyl-alcohol oxidase with 4-alkylphenols. FEBS Lett 2000, 481 (2), 109-112. 

The enzyme vanillyl-alcohol oxidase (VAO, E.C. 1.1.3.38) was examined in detail with respect to mechanism, structural properties, and biotechnological applications by van Berkel and coworkers, giving an excellent example of how detailed biochemical studies provide a basis for preparative biocatalytic applications (for recent reviews see[1, 21). The homooctamer with a monomer mass of 65 kDa was isolated and purified from Penicillium simplicissimum. The catalytic mechanism of VAO-catalyzed oxidation of para-alkyl phenols was studied in detail[3-5 After initial hydride abstraction from the Ca atom, a binary complex of the intermediate para-quinone methide and reduced FAD reacts with molecular oxygen, regenerating the... 

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