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Mutational study of the role of tyrosine‐49 in the Saccharomyces cerevisiae xylose reductase
Author(s) -
Ye Jeong Eun,
Sopher Coralie,
Seon Kim In,
Lee Hung
Publication year - 2001
Publication title -
yeast
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.923
H-Index - 102
eISSN - 1097-0061
pISSN - 0749-503X
DOI - 10.1002/yea.758
Subject(s) - saccharomyces cerevisiae , xylose , biochemistry , recombinant dna , enzyme , biology , xylose metabolism , turnover number , reductase , tyrosine , escherichia coli , yeast , gene , fermentation
The xyl1 gene encoding xylose reductase was cloned from Saccharomyces cerevisiae and expressed in Escherichia coli . The purified enzyme readily carried out xylose reduction in vitro . It prefers NADPH as the co‐enzyme by about 80‐fold over NADH. Compared to the native enzyme purified from S. cerevisiae (Kuhn et al ., 1995), the recombinant xylose reductase displayed slightly higher (about two‐fold) affinities ( K m ) for the substrate (xylose) and co‐factor (NADPH), as well as a 3.9‐fold faster turnover number ( K cat ) and 7.4‐fold greater catalytic efficiency ( K cat / K m ). The reason for the apparent discrepancies in kinetic constants between the recombinant and native S. cerevisiae xylose reductases is not known. Replacement of Tyr49 by Phe in the recombinant enzyme led to greater than 98% loss of activity, suggesting that this residue plays a critical role in catalysis. Intrinsic enzyme fluorescence spectroscopic analysis showed that the wild‐type and the Y49F variant both bound the co‐enzyme NADPH with similar affinity. This supports the view that Tyr49 is involved in interaction with the substrate and not the co‐factor during catalysis. Copyright © 2001 John Wiley & Sons, Ltd.