Covalent flavinylation of vanillyl-alcohol oxidase is an autocatalytic process

Jianfeng Jin; Hortense Mazon; Robert H. H. van den Heuvel; Albert J. Heck; Dick B. Janssen; Marco W. Fraaije

doi:10.1111/j.1742-4658.2008.06649.x

Covalent flavinylation of vanillyl-alcohol oxidase is an autocatalytic process

Jianfeng Jin
, Hortense Mazon
, Robert H. H. van den Heuvel
, Albert J. Heck
, Dick B. Janssen
, Marco W. Fraaije^*

^*Corresponding author for this work

Research output: Contribution to journal › Article › Academic › peer-review

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Abstract

Vanillyl-alcohol oxidase (VAO; EC 1.1.3.38) contains a covalently 8 alpha-histidyl bound FAD, which represents the most frequently encountered covalent flavin-protein linkage. To elucidate the mechanism by which VAO covalently incorporates the FAD cofactor, apo VAO was produced by using a riboflavin auxotrophic Escherichia coli strain. Incubation of apo VAO with FAD resulted in full restoration of enzyme activity. The rate of activity restoration was dependent on FAD concentration, displaying a hyperbolic relationship (K(FAD) = 2.3 mu M, k(activation) = 0.13 min(-1)). The time-dependent increase in enzyme activity was accompanied by full covalent incorporation of FAD, as determined by SDS/PAGE and ESI-MS analysis. The results obtained show that formation of the covalent flavin-protein bond is an autocatalytic process, which proceeds via a reduced flavin intermediate. Furthermore, ESI-MS experiments revealed that, although apo VAO mainly exists as monomers and dimers, FAD binding promotes the formation of VAO dimers and octamers. Tandem ESI-MS experiments revealed that octamerization is not dependent on full covalent flavinylation.

Original language	English
Pages (from-to)	5191-5200
Number of pages	10
Journal	Febs Journal
Volume	275
Issue number	20
DOIs	https://doi.org/10.1111/j.1742-4658.2008.06649.x
Publication status	Published - Oct-2008

Keywords

covalent flavinylation
FAD
post-translational modification
tandem ESI-MS
vanilly-lalcohol oxidase
MONOMERIC SARCOSINE OXIDASE
P-CRESOL METHYLHYDROXYLASE
OF-FLIGHT INSTRUMENT
PENICILLIUM-SIMPLICISSIMUM
MASS-SPECTROMETRY
SUBSTRATE-SPECIFICITY
CHOLESTEROL OXIDASE
PROTEIN ASSEMBLIES
ESCHERICHIA-COLI
REDOX PROPERTIES

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@article{55f39f6d5fe445e9974662d90a4172da,

title = "Covalent flavinylation of vanillyl-alcohol oxidase is an autocatalytic process",

abstract = "Vanillyl-alcohol oxidase (VAO; EC 1.1.3.38) contains a covalently 8 alpha-histidyl bound FAD, which represents the most frequently encountered covalent flavin-protein linkage. To elucidate the mechanism by which VAO covalently incorporates the FAD cofactor, apo VAO was produced by using a riboflavin auxotrophic Escherichia coli strain. Incubation of apo VAO with FAD resulted in full restoration of enzyme activity. The rate of activity restoration was dependent on FAD concentration, displaying a hyperbolic relationship (K(FAD) = 2.3 mu M, k(activation) = 0.13 min(-1)). The time-dependent increase in enzyme activity was accompanied by full covalent incorporation of FAD, as determined by SDS/PAGE and ESI-MS analysis. The results obtained show that formation of the covalent flavin-protein bond is an autocatalytic process, which proceeds via a reduced flavin intermediate. Furthermore, ESI-MS experiments revealed that, although apo VAO mainly exists as monomers and dimers, FAD binding promotes the formation of VAO dimers and octamers. Tandem ESI-MS experiments revealed that octamerization is not dependent on full covalent flavinylation.",

keywords = "covalent flavinylation, FAD, post-translational modification, tandem ESI-MS, vanilly-lalcohol oxidase, MONOMERIC SARCOSINE OXIDASE, P-CRESOL METHYLHYDROXYLASE, OF-FLIGHT INSTRUMENT, PENICILLIUM-SIMPLICISSIMUM, MASS-SPECTROMETRY, SUBSTRATE-SPECIFICITY, CHOLESTEROL OXIDASE, PROTEIN ASSEMBLIES, ESCHERICHIA-COLI, REDOX PROPERTIES",

author = "Jianfeng Jin and Hortense Mazon and \{van den Heuvel\}, \{Robert H. H.\} and Heck, \{Albert J.\} and Janssen, \{Dick B.\} and Fraaije, \{Marco W.\}",

year = "2008",

month = oct,

doi = "10.1111/j.1742-4658.2008.06649.x",

language = "English",

volume = "275",

pages = "5191--5200",

journal = "Febs Journal",

issn = "1742-464X",

publisher = "NLM (Medline)",

number = "20",

}

TY - JOUR

T1 - Covalent flavinylation of vanillyl-alcohol oxidase is an autocatalytic process

AU - Jin, Jianfeng

AU - Mazon, Hortense

AU - van den Heuvel, Robert H. H.

AU - Heck, Albert J.

AU - Janssen, Dick B.

AU - Fraaije, Marco W.

PY - 2008/10

Y1 - 2008/10

N2 - Vanillyl-alcohol oxidase (VAO; EC 1.1.3.38) contains a covalently 8 alpha-histidyl bound FAD, which represents the most frequently encountered covalent flavin-protein linkage. To elucidate the mechanism by which VAO covalently incorporates the FAD cofactor, apo VAO was produced by using a riboflavin auxotrophic Escherichia coli strain. Incubation of apo VAO with FAD resulted in full restoration of enzyme activity. The rate of activity restoration was dependent on FAD concentration, displaying a hyperbolic relationship (K(FAD) = 2.3 mu M, k(activation) = 0.13 min(-1)). The time-dependent increase in enzyme activity was accompanied by full covalent incorporation of FAD, as determined by SDS/PAGE and ESI-MS analysis. The results obtained show that formation of the covalent flavin-protein bond is an autocatalytic process, which proceeds via a reduced flavin intermediate. Furthermore, ESI-MS experiments revealed that, although apo VAO mainly exists as monomers and dimers, FAD binding promotes the formation of VAO dimers and octamers. Tandem ESI-MS experiments revealed that octamerization is not dependent on full covalent flavinylation.

AB - Vanillyl-alcohol oxidase (VAO; EC 1.1.3.38) contains a covalently 8 alpha-histidyl bound FAD, which represents the most frequently encountered covalent flavin-protein linkage. To elucidate the mechanism by which VAO covalently incorporates the FAD cofactor, apo VAO was produced by using a riboflavin auxotrophic Escherichia coli strain. Incubation of apo VAO with FAD resulted in full restoration of enzyme activity. The rate of activity restoration was dependent on FAD concentration, displaying a hyperbolic relationship (K(FAD) = 2.3 mu M, k(activation) = 0.13 min(-1)). The time-dependent increase in enzyme activity was accompanied by full covalent incorporation of FAD, as determined by SDS/PAGE and ESI-MS analysis. The results obtained show that formation of the covalent flavin-protein bond is an autocatalytic process, which proceeds via a reduced flavin intermediate. Furthermore, ESI-MS experiments revealed that, although apo VAO mainly exists as monomers and dimers, FAD binding promotes the formation of VAO dimers and octamers. Tandem ESI-MS experiments revealed that octamerization is not dependent on full covalent flavinylation.

KW - covalent flavinylation

KW - FAD

KW - post-translational modification

KW - tandem ESI-MS

KW - vanilly-lalcohol oxidase

KW - MONOMERIC SARCOSINE OXIDASE

KW - P-CRESOL METHYLHYDROXYLASE

KW - OF-FLIGHT INSTRUMENT

KW - PENICILLIUM-SIMPLICISSIMUM

KW - MASS-SPECTROMETRY

KW - SUBSTRATE-SPECIFICITY

KW - CHOLESTEROL OXIDASE

KW - PROTEIN ASSEMBLIES

KW - ESCHERICHIA-COLI

KW - REDOX PROPERTIES

U2 - 10.1111/j.1742-4658.2008.06649.x

DO - 10.1111/j.1742-4658.2008.06649.x

M3 - Article

SN - 1742-464X

VL - 275

SP - 5191

EP - 5200

JO - Febs Journal

JF - Febs Journal

IS - 20

ER -

Covalent flavinylation of vanillyl-alcohol oxidase is an autocatalytic process

Abstract

Keywords

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