Mutational study of the role of tyrosine-49 in the Saccharomyces cerevisiae xylose reductase.
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.
Jeong
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University of Guelph, Department of Environmental Biology, Guelph, Ontario, Canada N1G 2W1.
Sopher
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Kim
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Lee
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D000449:Aldehyde Reductase / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D009249:NADP / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics; D016415:Sequence Alignment; D014443:Tyrosine / Q000737:chemistry / Q000502:physiology*; D014994:Xylose / Q000378:metabolism
D011994:Recombinant Proteins; D014443:Tyrosine; D009249:NADP; D014994:Xylose; D000449:Aldehyde Reductase