Laboratory-evolved vanillyl-alcohol oxidase produces natural vanillin

R.H.H. van den Heuvel, W.A.M. van den Berg, S. Rovida, W.J.H. van Berkel

Research output: Contribution to journalArticleAcademicpeer-review

39 Citations (Scopus)

Abstract

The flavoenzyme vanillyl-alcohol oxidase was subjected to random mutagenesis to generate mutants with enhanced reactivity to creosol (2-methoxy-4-methylphenol). The vanillyl-alcohol oxidase-mediated conversion of creosol proceeds via a two-step process in which the initially formed vanillyl alcohol (4-hydroxy-3-methoxybenzyl alcohol) is oxidized to the widely used flavor compound vanillin (4-hydroxy-3-methoxybenzaldehyde). The first step of this reaction is extremely slow due to the formation of a covalent FAD N-5-creosol adduct. After a single round of error-prone PCR, seven mutants were generated with increased reactivity to creosol. The single-point mutants I238T, F454Y, E502G, and T505S showed an up to 40-fold increase in catalytic efficiency (k(cat)/K-m) with creosol compared with the wild-type enzyme. This enhanced reactivity was due to a lower stability of the covalent flavin-substrate adduct, thereby promoting vanillin formation. The catalytic efficiencies of the mutants were also enhanced for other ortho-substituted 4-methylphenols, but not for p-cresol (4-methylphenol). The replaced amino acid residues are not located within a distance of direct interaction with the substrate, and the determined three-dimensional structures of the mutant enzymes are highly similar to that of the wild-type enzyme. These results clearly show the importance of remote residues, not readily predicted by rational design, for the substrate specificity of enzymes.
Original languageEnglish
Pages (from-to)33492-33500
JournalJournal of Biological Chemistry
Volume279
Issue number32
DOIs
Publication statusPublished - 2004

Keywords

  • directed evolution
  • penicillium-simplicissimum
  • dihydrofolate-reductase
  • substrate-specificity
  • cresol methylhydroxylase
  • catalytic mechanism
  • crystal-structures
  • enzyme-substrate
  • 4-alkylphenols
  • hydroxylation

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