1E0Y

Structure of the D170S/T457E double mutant of vanillyl-alcohol oxidase

Summary for 1E0Y

• Entry DOI: 10.2210/pdb1e0y/pdb
• Related: 1AHU 1AHV 1AHZ 1VAO 2VAO
• Descriptor: VANILLYL-ALCOHOL OXIDASE, FLAVIN-ADENINE DINUCLEOTIDE, ALPHA,ALPHA,ALPHA-TRIFLUORO-P-CRESOL, ... (4 entities in total)
• Functional Keywords: flavoenzyme, specificity
• Biological source: PENICILLIUM SIMPLICISSIMUM
• Total number of polymer chains: 2
• Total formula weight: 127747.17

Authors

Van Der heuvel, R.H.H.,Van Berkel, W.J.H.,Mattevi, A. (deposition date: 2000-04-11, release date: 2000-04-12, Last modification date: 2024-11-06)

Primary citation

Van Der Heuvel, R.H.H.,Fraaije, M.W.,Espinosa, M.F.,Mattevi, A.,Van Berkel, W.J.H.
Inversion of Stereospecificity in Vanillyl-Alcohol Oxidase
Proc.Natl.Acad.Sci.USA, 97:9455-, 2000

PubMed Abstract: Vanillyl-alcohol oxidase (VAO) is the prototype of a newly recognized family of structurally related oxidoreductases sharing a conserved FAD-binding domain. The active site of VAO is formed by a cavity where the enzyme is able to catalyze many reactions with phenolic substrates. Among these reactions is the stereospecific hydroxylation of 4-ethylphenol-forming (R)-1-(4'-hydroxyphenyl)ethanol. During this conversion, Asp-170 is probably critical for the hydration of the initially formed p-quinone methide intermediate. By site-directed mutagenesis, the putative active site base has been relocated to the opposite face of the active site cavity. In this way, a change in stereospecificity has been achieved. Like native VAO, the single mutants T457E, D170A, and D170S preferentially converted 4-ethylphenol to the (R)-enantiomer of 1-(4'-hydroxyphenyl)ethanol. The double mutants D170A/T457E and D170S/T457E exhibited an inverted stereospecificity with 4-ethylphenol. Particularly, D170S/T457E was strongly (S)-selective, with an enantiomeric excess of 80%. The crystal structure of D170S/T457E, in complex with trifluoromethylphenol, showed a highly conserved mode of ligand binding and revealed that the distinctive catalytic properties of this mutant are not caused by major structural changes.

PubMed: 10920192
DOI: 10.1073/PNAS.160175897

Experimental method

X-RAY DIFFRACTION (2.75 Å)