Tyr-51 is the proton donor-acceptor for NAD(H)-dependent interconversion of xylose and xylitol by Candida tenuis xylose reductase (AKR2B5).
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 ( approximately 2x10(3)-fold at 300mM) for NAD(+)-dependent xylitol oxidation by Y51A. Activity of the wild-type reductase was dependent on a single ionizable protein group exhibiting a pK 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.
Pival
Simone L
SL
Institute of Biotechnology and Biochemical Engineering, Graz University of Technology, Petersgasse 12, A-8010 Graz, Austria.
Klimacek
Mario
M
Kratzer
Regina
R
Nidetzky
Bernd
B
D000409:Alanine / Q000235:genetics / Q000378:metabolism; D000449:Aldehyde Reductase / Q000235:genetics / Q000378:metabolism*; D019943:Amino Acid Substitution; D002175:Candida / Q000201:enzymology*; D006860:Hydrogen Bonding; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009243:NAD / Q000378:metabolism; D011522:Protons; D014443:Tyrosine / Q000235:genetics / Q000378:metabolism*; D014993:Xylitol / Q000378:metabolism*; D014994:Xylose / Q000378:metabolism*
D011522:Protons; D009243:NAD; D014443:Tyrosine; D014994:Xylose; D000449:Aldehyde Reductase; D000409:Alanine; D014993:Xylitol