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

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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

Fingerprint

vanillyl-alcohol oxidase
Enzymes
Substrates
Flavor compounds
Mutagenesis
Flavin-Adenine Dinucleotide
Substrate Specificity
creosol
vanillin
Alcohols

Keywords

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

Cite this

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title = "Laboratory-evolved vanillyl-alcohol oxidase produces natural vanillin",
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.",
keywords = "directed evolution, penicillium-simplicissimum, dihydrofolate-reductase, substrate-specificity, cresol methylhydroxylase, catalytic mechanism, crystal-structures, enzyme-substrate, 4-alkylphenols, hydroxylation",
author = "{van den Heuvel}, R.H.H. and {van den Berg}, W.A.M. and S. Rovida and {van Berkel}, W.J.H.",
year = "2004",
doi = "10.1074/jbc.M312968200",
language = "English",
volume = "279",
pages = "33492--33500",
journal = "Journal of Biological Chemistry",
issn = "0021-9258",
publisher = "American Society for Biochemistry and Molecular Biology",
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}

Laboratory-evolved vanillyl-alcohol oxidase produces natural vanillin. / van den Heuvel, R.H.H.; van den Berg, W.A.M.; Rovida, S.; van Berkel, W.J.H.

In: Journal of Biological Chemistry, Vol. 279, No. 32, 2004, p. 33492-33500.

Research output: Contribution to journalArticleAcademicpeer-review

TY - JOUR

T1 - Laboratory-evolved vanillyl-alcohol oxidase produces natural vanillin

AU - van den Heuvel, R.H.H.

AU - van den Berg, W.A.M.

AU - Rovida, S.

AU - van Berkel, W.J.H.

PY - 2004

Y1 - 2004

N2 - 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.

AB - 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.

KW - directed evolution

KW - penicillium-simplicissimum

KW - dihydrofolate-reductase

KW - substrate-specificity

KW - cresol methylhydroxylase

KW - catalytic mechanism

KW - crystal-structures

KW - enzyme-substrate

KW - 4-alkylphenols

KW - hydroxylation

U2 - 10.1074/jbc.M312968200

DO - 10.1074/jbc.M312968200

M3 - Article

VL - 279

SP - 33492

EP - 33500

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

SN - 0021-9258

IS - 32

ER -