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Structure of the Y108F mutant of vanillyl alcohol oxidase
Structural highlights
FunctionVAOX_PENSI Catalyzes the conversion of vanillin alcohol to vanillin, and also the conversion of a wide range of phenolic compounds bearing side chains of variable size at the para position of the aromatic ring. Crucial for the degradation of the secondary metabolites derived from the degradation of the lignin. Catalyzes besides the oxidation of 4-hydroxybenzyl alcohols, the oxidative deamination of 4-hydroxybenzylamines, the oxidative demethylation of 4-(methoxy-methyl)phenols and the oxidative hydration of 4-allylphenols. Most active with 4-allylphenols. Publication Abstract from PubMedA number of oxidoreductases from the VAO/para-cresol methylhydroxylase flavoprotein family catalyze the oxidation of para-substituted phenols. One of the best-studied is vanillyl-alcohol oxidase (VAO) from the fungus Penicillium simplicissimum For oxidation of phenols by VAO to occur, they must first be bound in the active site of the enzyme in their phenolate anion form. The crystal structure of VAO reveals that two tyrosine residues, Tyr-108 and Tyr-503, are positioned to facilitate this deprotonation. To investigate their role in catalysis, we created three VAO variants, Y108F, Y503F, and Y108F/Y503F, and studied their biochemical properties. Steady-state kinetics indicated that the presence of at least one of the tyrosine residues is essential for efficient catalysis by VAO. Stopped-flow kinetics revealed that the reduction of VAO by chavicol or vanillyl alcohol occurs at two different rates: kobs1, which corresponds to its reaction with the deprotonated form of the substrate, and kobs2, which corresponds to its reaction with the protonated form of the substrate. In Y108F, Y503F, and Y108F/Y503F, the relative contribution of kobs2 to the reduction is larger than in wild-type VAO, suggesting deprotonation is impaired in these variants. Binding studies disclosed that the competitive inhibitor isoeugenol is predominantly in its deprotonated form when bound to wild-type VAO, but predominantly in its protonated form when bound to the variants. These results indicate that Tyr-108 and Tyr-503 are responsible for the activation of substrates in VAO, providing new insights into the catalytic mechanism of VAO and related enzymes that oxidize para-substituted phenols. 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. 2017 Sep 1;292(35):14668-14679. doi: 10.1074/jbc.M117.778449. Epub, 2017 Jul 17. PMID:28717004[1] From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. See AlsoReferences
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