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Tyr‐51 is the proton donor–acceptor for NAD(H)‐dependent interconversion of xylose and xylitol by Candida tenuis xylose reductase (AKR2B5)
Author(s) -
Pival Simone L.,
Klimacek Mario,
Kratzer Regina,
Nidetzky Bernd
Publication year - 2008
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/j.febslet.2008.11.003
Subject(s) - xylitol , nad+ kinase , chemistry , xylose , protonation , bromide , stereochemistry , aldo keto reductase , reductase , biochemistry , enzyme , medicinal chemistry , organic chemistry , fermentation , ion
Substitution of active‐site Tyr‐51 by Ala (Y51A) disrupted the activity of Candida tenuis xylose reductase by six orders of magnitude. External bromide brought about unidirectional rate enhancement (≈2 × 10 3 ‐fold at 300 mM) for NAD + ‐dependent xylitol oxidation by Y51A. Activity of the wild‐type reductase was dependent on a single ionizable protein group exhibiting a p K of 9.2 ± 0.1 and 7.3 ± 0.3 in the holo‐enzyme bound with NADH and NAD + , respectively. This group which had to be protonated for xylose reduction and unprotonated for xylitol oxidation was eliminated in Y51A, consistent with a catalytic acid–base function of Tyr‐51. Bromide may complement the xylitol dehydrogenase activity of Y51A by partly restoring the original hydrogen bond between the reactive alcohol and the phenolate of Tyr‐51.