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

Mattevi, A.; Fraaije, Marco W.; Mozzarelli, Andrea; Olivi, Luca; Coda, Alessandro; Berkel, Willem J.H. van

doi:10.1016/s0969-2126(97)00245-1

Cited by 164 publications

(228 citation statements)

References 37 publications

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“…In fact, the neighboring residues Tyr 503 and Arg 504 stabilize the phenolate form of the substrate by forming hydrogen bonds (23). The two mutations E502G and T505S did not induce any major conformational changes in this loop or in the side chains of Tyr 503 and Arg 504 (Fig.…”

Section: Directed Evolution Of Vanillyl-alcohol Oxidasementioning

confidence: 88%

“…Moreover, p-cresol makes an angle of 34°with the flavin, whereas the noncovalently bound inhibitors isoeugenol and 2-nitro-p-cresol are less tilted with respect to the FAD. From this it was argued that steric restrictions imposed by the shape of the active-site cavity are a key factor in preventing enzyme inactivation through covalent adduct stabilization (23). The x-ray models of I238T, F454Y, E502G, and T505S did not reveal any conformational perturbations in the active-site cavities, and the orientation of the flavin and isoeugenol was highly conserved.…”

Section: Discussionmentioning

confidence: 99%

“…X-ray crystallography has shown that VAO His 422 is covalently linked to the FAD prosthetic group via a FAD C-8␣- His 422 N-⑀3 bond (23). Fluorescence analysis of unstained SDSpolyacrylamide gels in 5% (v/v) acetic acid showed that the four mutants were as fluorescent as the wild-type enzyme, strongly indicating that the FAD is covalently bound to His 422 in all four mutants.…”

Section: Directed Evolution Of Vao and Characterization Of Mutantmentioning

confidence: 99%

“…The enzyme is the prototype of a large family of structurally related oxidoreductases sharing a conserved FAD-binding domain (22,23). VAO catalyzes the oxidation of a wide range of phenolic compounds, including 4-alkylphenols (24, 25).…”

mentioning

confidence: 99%

“…The flavoprotein subunit of PCMH shares 32% sequence identity with VAO, and their active sites are remarkably conserved. Superposition of the crystallographic models of VAO (Protein Data Bank code 1VAO) (23) and PCMH (code 1DIQ) (29) has revealed a root mean square deviation of 1.0 Å for 470 C-␣ atoms. The two enzymes catalyze similar reactions, but p-cresol (4-methylphenol), the physiological substrate of PCMH (30), is an extremely poor substrate for VAO (27).…”

mentioning

confidence: 99%

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Laboratory-evolved Vanillyl-alcohol Oxidase Produces Natural Vanillin

Heuvel

Berg

Rovida

et al. 2004

Journal of Biological Chemistry

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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.The increased use of enzymes and other proteins in the pharmaceutical, chemical, and agricultural industry has generated considerable interest in the design of proteins with new or improved properties. Two different but complementary technologies have been applied to this goal: (i) rational design, which relies on the availability of the three-dimensional structure and knowledge about the relationship between sequence, structure, and mechanism, and (ii) directed evolution methods, which use random mutagenesis of the gene encoding the protein or recombination of gene fragments to create diversity and then experimental screening of the libraries generated for the desired properties. Rational design has been used to elucidate and change enzyme mechanism, substrate and product specificity, enantioselectivity, cofactor specificity, and protein stability (1-3). Directed evolution has been applied to increase catalytic activity; to invert or improve enantioselectivity; and to alter substrate and product specificity, protein stability, pH optimum, and tolerance against organic solvents (1, 4 -7). An obvious advantage of directed evolution methods over sitedirected mutagenesis is that enzymes can be tailored for the production of (intermediate) products without detailed knowledge of protein structure and structure-function relationships.In this study, we engineered the flavoenzyme vanillyl-alcohol oxidase (VAO) 1 by random mutagenesis such that the evolved mutants are capable of producing natural vanillin (4-hydroxy-3-methoxybenzaldehyde) from the precursor creosol (2-methoxy...

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Section: Directed Evolution Of Vanillyl-alcohol Oxidasementioning

confidence: 88%

Section: Discussionmentioning

confidence: 99%

Section: Directed Evolution Of Vao and Characterization Of Mutantmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

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Laboratory-evolved Vanillyl-alcohol Oxidase Produces Natural Vanillin

Heuvel

Berg

Rovida

et al. 2004

Journal of Biological Chemistry

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First partial three-dimensional model of human monoamine oxidase A

Wouters

Baudoux

1998

Proteins

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A survey of the major known structural aspects of monoamine oxidase (MAO) is given and a first partial model of human MAO A is presented. This 3D model has been established using secondary structure predictions and fold recognition methods. It shows two alpha/beta domains (the FAD-binding N-terminal and central domains) and an alpha+beta domain. The C-terminal region is predicted to be responsible for anchoring the protein into the mitochondrial membrane and was not modeled. The covalent binding of the flavin cofactor to a cysteine residue is well predicted. The model is validated with experimental data from the literature and should be useful in designing new experimental studies (site-directed mutagenesis, chemical modification, specific antibodies). This first step towards the 3D structure of monoamine oxidase should contribute to a better understanding of the mechanisms of action and inhibition of this drug target in the treatment of clinical depression.

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Enantioselective hydroxylation of 4-alkylphenols by vanillyl alcohol oxidase

Drijfhout¹,

Fraaije²,

Jongejan³

et al. 1998

Biotechnol. Bioeng.

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Vanillyl alcohol oxidase (VAO) from Penicillium simplicissimum catalyzes the enantioselective hydroxylation of 4‐ethylphenol, 4‐propylphenol, and 2‐methoxy‐4‐propylphenol into 1‐(4′‐hydroxyphenyl)ethanol, 1‐(4′‐hydroxyphenyl)propanol, and 1‐(4′‐hydroxy‐3′‐methoxyphenyl)propanol, respectively, with an ee of 94% for the R enantiomer. The stereochemical outcome of the reactions was established by comparing the chiral GC retention times of the products to those of chiral alcohols obtained by the action of the lipases from Candida antarctica and Pseudomonas cepacia. Isotope labeling experiments revealed that the oxygen atom incorporated into the alcoholic products is derived from water. During the VAO‐mediated conversion of 4‐ethylphenol/4‐propylphenol, 4‐vinylphenol/4‐propenylphenol are formed as side products. With 2‐methoxy‐4‐propylphenol as a substrate, this competing side reaction is nearly abolished, resulting in less than 1% of the vinylic product, isoeugenol. The VAO‐mediated conversion of 4‐alkylphenols also results in small amounts of phenolic ketones indicative for a consecutive oxidation step. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 59:171–177, 1998.

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

Cited by 164 publications

References 37 publications

Laboratory-evolved Vanillyl-alcohol Oxidase Produces Natural Vanillin

Laboratory-evolved Vanillyl-alcohol Oxidase Produces Natural Vanillin

First partial three-dimensional model of human monoamine oxidase A

Enantioselective hydroxylation of 4-alkylphenols by vanillyl alcohol oxidase

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