1vao Citations

Crystal structures and inhibitor binding in the octameric flavoenzyme vanillyl-alcohol oxidase: the shape of the active-site cavity controls substrate specificity.

Structure 5 907-20 (1997)
Related entries: 2vao, 1ahv, 1ahu, 1ahz

Cited: 64 times
EuropePMC logo PMID: 9261083



Lignin degradation leads to the formation of a broad spectrum of aromatic molecules that can be used by various fungal micro-organisms as their sole source of carbon. When grown on phenolic compounds, Penicillium simplicissimum induces the strong impression of a flavin-containing vanillyl-alcohol oxidase (VAO). The enzyme catalyses the oxidation of a vast array of substrates, ranging from aromatic amines to 4-alkyphenols. VAO is a member of a novel class of widely distributed oxidoreductases, which use flavin adenine dinucleotide (FAD) as a cofactor covalently bound to the protein. We have carried out the determination of the structure of VAO in order to shed light on the most interesting features of these novel oxidoreductases, such as the functional significance of covalent flavinylation and the mechanism of catalysis.


The crystal structure of VAO has been determined in the native state and in complexes with four inhibitors. The enzyme is an octamer with 42 symmetry; the inhibitors bind in a hydrophobic, elongated cavity on the si side of the flavin molecule. Three residues, Tyr108, Tyr503 and Arg504 form an anion-binding subsite, which stabilises the phenolate form of the substrate. The structure of VAO complexed with the inhibitor 4-(1-heptenyl)phenol shows that the catalytic cavity is completely filled by the inhibitor, explaining why alkylphenols bearing aliphatic substituents longer than seven carbon atoms do not bind to the enzyme.


The shape of the active-site cavity controls substrate specificity by providing a 'size exclusion mechanism'. Inside the cavity, the substrate aromatic ring is positioned at an angle of 18 degrees to the flavin ring. This arrangement is ideally suited for a hydride transfer reaction, which is further facilitated by substrate deprotonation. Burying the substrate beneath the protein surface is a recurrent strategy, common to many flavoenzymes that effect substrate oxidation or reduction via hydride transfer.

Reviews - 1vao mentioned but not cited (1)

  1. Structural perspective on enzymatic halogenation. Blasiak LC, Drennan CL. Acc. Chem. Res. 42 147-155 (2009)

Articles - 1vao mentioned but not cited (1)

  1. Molecular characterization and expression of a novel alcohol oxidase from Aspergillus terreus MTCC6324. Chakraborty M, Goel M, Chinnadayyala SR, Dahiya UR, Ghosh SS, Goswami P. PLoS ONE 9 e95368 (2014)

Reviews citing this publication (14)

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  2. A 30-angstrom-long U-shaped catalytic tunnel in the crystal structure of polyamine oxidase. Binda C, Coda A, Angelini R, Federico R, Ascenzi P, Mattevi A. Structure 7 265-276 (1999)
  3. Crystal structure and mechanism of CO dehydrogenase, a molybdo iron-sulfur flavoprotein containing S-selanylcysteine. Dobbek H, Gremer L, Meyer O, Huber R. Proc. Natl. Acad. Sci. U.S.A. 96 8884-8889 (1999)
  4. Monomeric sarcosine oxidase: structure of a covalently flavinylated amine oxidizing enzyme. Trickey P, Wagner MA, Jorns MS, Mathews FS. Structure 7 331-345 (1999)
  5. Methionine 286 in transmembrane domain 3 of the GABAA receptor beta subunit controls a binding cavity for propofol and other alkylphenol general anesthetics. Krasowski MD, Nishikawa K, Nikolaeva N, Lin A, Harrison NL. Neuropharmacology 41 952-964 (2001)
  6. Crystal structures of the active and alloxanthine-inhibited forms of xanthine dehydrogenase from Rhodobacter capsulatus. Truglio JJ, Theis K, Leimkühler S, Rappa R, Rajagopalan KV, Kisker C. Structure 10 115-125 (2002)
  7. Detection of intact megaDalton protein assemblies of vanillyl-alcohol oxidase by mass spectrometry. van Berkel WJ, van den Heuvel RH, Versluis C, Heck AJ. Protein Sci. 9 435-439 (2000)
  8. ThermoFAD, a Thermofluor-adapted flavin ad hoc detection system for protein folding and ligand binding. Forneris F, Orru R, Bonivento D, Chiarelli LR, Mattevi A. FEBS J. 276 2833-2840 (2009)
  9. The crystal structure of D-lactate dehydrogenase, a peripheral membrane respiratory enzyme. Dym O, Pratt EA, Ho C, Eisenberg D. Proc. Natl. Acad. Sci. U.S.A. 97 9413-9418 (2000)
  10. Structural basis of inhibition of Mycobacterium tuberculosis DprE1 by benzothiazinone inhibitors. Batt SM, Jabeen T, Bhowruth V, Quill L, Lund PA, Eggeling L, Alderwick LJ, Fütterer K, Besra GS. Proc. Natl. Acad. Sci. U.S.A. 109 11354-11359 (2012)
  11. Structure of L-aspartate oxidase: implications for the succinate dehydrogenase/fumarate reductase oxidoreductase family. Mattevi A, Tedeschi G, Bacchella L, Coda A, Negri A, Ronchi S. Structure 7 745-756 (1999)
  12. Structures of the flavocytochrome p-cresol methylhydroxylase and its enzyme-substrate complex: gated substrate entry and proton relays support the proposed catalytic mechanism. Cunane LM, Chen ZW, Shamala N, Mathews FS, Cronin CN, McIntire WS. J. Mol. Biol. 295 357-374 (2000)
  13. X-ray structure of 12-oxophytodienoate reductase 1 provides structural insight into substrate binding and specificity within the family of OYE. Breithaupt C, Strassner J, Breitinger U, Huber R, Macheroux P, Schaller A, Clausen T. Structure 9 419-429 (2001)
  14. Identification of the oxygen activation site in monomeric sarcosine oxidase: role of Lys265 in catalysis. Zhao G, Bruckner RC, Jorns MS. Biochemistry 47 9124-9135 (2008)
  15. The PHBH fold: not only flavoenzymes. Mattevi A. Biophys. Chem. 70 217-222 (1998)
  16. Aryl-alcohol oxidase protein sequence: a comparison with glucose oxidase and other FAD oxidoreductases. Varela E, Jesús Martínez M, Martínez AT. Biochim. Biophys. Acta 1481 202-208 (2000)
  17. Regio- and stereospecific conversion of 4-alkylphenols by the covalent flavoprotein vanillyl-alcohol oxidase. van den Heuvel RH, Fraaije MW, Laane C, van Berkel WJ. J. Bacteriol. 180 5646-5651 (1998)
  18. Crystal structure of 6-hydroxy-D-nicotine oxidase from Arthrobacter nicotinovorans. Koetter JW, Schulz GE. J. Mol. Biol. 352 418-428 (2005)
  19. Structure and function of ∆1-tetrahydrocannabinolic acid (THCA) synthase, the enzyme controlling the psychoactivity of Cannabis sativa. Shoyama Y, Tamada T, Kurihara K, Takeuchi A, Taura F, Arai S, Blaber M, Shoyama Y, Morimoto S, Kuroki R. J. Mol. Biol. 423 96-105 (2012)
  20. Probing oxygen activation sites in two flavoprotein oxidases using chloride as an oxygen surrogate. Kommoju PR, Chen ZW, Bruckner RC, Mathews FS, Jorns MS. Biochemistry 50 5521-5534 (2011)
  21. Covalent flavinylation of vanillyl-alcohol oxidase is an autocatalytic process. Jin J, Mazon H, van den Heuvel RH, Heck AJ, Janssen DB, Fraaije MW. FEBS J. 275 5191-5200 (2008)
  22. Discovery of a eugenol oxidase from Rhodococcus sp. strain RHA1. Jin J, Mazon H, van den Heuvel RH, Janssen DB, Fraaije MW. FEBS J. 274 2311-2321 (2007)
  23. Structural and kinetic analyses of the H121A mutant of cholesterol oxidase. Lim L, Molla G, Guinn N, Ghisla S, Pollegioni L, Vrielink A. Biochem. J. 400 13-22 (2006)
  24. Inversion of stereospecificity of vanillyl-alcohol oxidase. van Den Heuvel RH, Fraaije MW, Ferrer M, Mattevi A, van Berkel WJ. Proc. Natl. Acad. Sci. U.S.A. 97 9455-9460 (2000)
  25. Elucidating the biosynthetic pathway for the polyketide-nonribosomal peptide collismycin A: mechanism for formation of the 2,2'-bipyridyl ring. Garcia I, Vior NM, Braña AF, González-Sabin J, Rohr J, Moris F, Méndez C, Salas JA. Chem. Biol. 19 399-413 (2012)
  26. Farnesol oxidation in insects: evidence that the biosynthesis of insect juvenile hormone is mediated by a specific alcohol oxidase. Sperry AE, Sen SE. Insect Biochem. Mol. Biol. 31 171-178 (2001)
  27. Enantioselective hydroxylation of 4-alkylphenols by vanillyl alcohol oxidase Drijfhout FP, Fraaije MW, Jongejan H, van Berkel WJ, Franssen MC. Biotechnol. Bioeng. 59 171-177 (1998)
  28. Site-directed mutagenesis of selected residues at the active site of aryl-alcohol oxidase, an H2O2-producing ligninolytic enzyme. Ferreira P, Ruiz-Dueñas FJ, Martínez MJ, van Berkel WJ, Martínez AT. FEBS J. 273 4878-4888 (2006)
  29. Geometric restraint drives on- and off-pathway catalysis by the Escherichia coli menaquinol:fumarate reductase. Tomasiak TM, Archuleta TL, Andréll J, Luna-Chávez C, Davis TA, Sarwar M, Ham AJ, McDonald WH, Yankovskaya V, Stern HA, Johnston JN, Maklashina E, Cecchini G, Iverson TM. J. Biol. Chem. 286 3047-3056 (2011)
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  32. Rationally engineered flavin-dependent oxidase reveals steric control of dioxygen reduction. Zafred D, Steiner B, Teufelberger AR, Hromic A, Karplus PA, Schofield CJ, Wallner S, Macheroux P. FEBS J. 282 3060-3074 (2015)
  33. The crystal structure and mechanism of an unusual oxidoreductase, GilR, involved in gilvocarcin V biosynthesis. Noinaj N, Bosserman MA, Schickli MA, Piszczek G, Kharel MK, Pahari P, Buchanan SK, Rohr J. J. Biol. Chem. 286 23533-23543 (2011)
  34. Searching distant homologs of the regulatory ACT domain in phenylalanine hydroxylase. Siltberg-Liberles J, Martinez A. Amino Acids 36 235-249 (2009)
  35. Crystal Structure of Alcohol Oxidase from Pichia pastoris. Koch C, Neumann P, Valerius O, Feussner I, Ficner R. PLoS ONE 11 e0149846 (2016)
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  37. Crystal structure and immunologic characterization of the major grass pollen allergen Phl p 4. Zafred D, Nandy A, Pump L, Kahlert H, Keller W. J. Allergy Clin. Immunol. 132 696-703.e10 (2013)
  38. Structure and catalytic mechanism of 3-ketosteroid-Delta4-(5α)-dehydrogenase from Rhodococcus jostii RHA1 genome. van Oosterwijk N, Knol J, Dijkhuizen L, van der Geize R, Dijkstra BW. J. Biol. Chem. 287 30975-30983 (2012)
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  40. Precursor of ether phospholipids is synthesized by a flavoenzyme through covalent catalysis. Nenci S, Piano V, Rosati S, Aliverti A, Pandini V, Fraaije MW, Heck AJ, Edmondson DE, Mattevi A. Proc. Natl. Acad. Sci. U.S.A. 109 18791-18796 (2012)
  41. Subcellular localization of vanillyl-alcohol oxidase in Penicillium simplicissimum. Fraaije MW, Sjollema KA, Veenhuis M, van Berkel WJ. FEBS Lett. 422 65-68 (1998)
  42. Biocatalytic Properties and Structural Analysis of Eugenol Oxidase from Rhodococcus jostii RHA1: A Versatile Oxidative Biocatalyst. Nguyen QT, de Gonzalo G, Binda C, Rioz-Martínez A, Mattevi A, Fraaije MW. Chembiochem 17 1359-1366 (2016)
  43. A single loop is essential for the octamerization of vanillyl alcohol oxidase. Ewing TA, Gygli G, van Berkel WJ. FEBS J. 283 2546-2559 (2016)
  44. First partial three-dimensional model of human monoamine oxidase A. Wouters J, Baudoux G. Proteins 32 97-110 (1998)
  45. Two tyrosine residues, Tyr-108 and Tyr-503, are responsible for the deprotonation of phenolic substrates in vanillyl-alcohol oxidase. Ewing TA, Nguyen QT, Allan RC, Gygli G, Romero E, Binda C, Fraaije MW, Mattevi A, van Berkel WJH. J. Biol. Chem. 292 14668-14679 (2017)
  46. Form, symmetry and packing of biomacromolecules. I. Concepts and tutorial examples. Janner A. Acta Crystallogr., A, Found. Crystallogr. 66 301-311 (2010)
  47. Substrate Channel Flexibility in Pseudomonas aeruginosa MurB Accommodates Two Distinct Substrates. Chen MW, Lohkamp B, Schnell R, Lescar J, Schneider G. PLoS ONE 8 e66936 (2013)
  48. The ins and outs of vanillyl alcohol oxidase: Identification of ligand migration paths. Gygli G, Lucas MF, Guallar V, van Berkel WJH. PLoS Comput. Biol. 13 e1005787 (2017)