Structure/function analysis of a critical disulfide bond in the active site of l-xylulose reductase |
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Authors: | H-T Zhao S Endo S Ishikura R Chung P J Hogg A Hara O El-Kabbani |
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Institution: | (1) Medicinal Chemistry and Drug Action, Monash Institute of Pharmaceutical Sciences, 381 Royal Parade, Parkville, Victoria, 3052, Australia;(2) Laboratory of Biochemistry, Gifu Pharmaceutical University, Mitahora-higashi, Gifu 502 – 8585, Japan;(3) UNSW Cancer Research Centre, University of New South Wales, Sydney, New South Wales, 2052, Australia;(4) Children’s Cancer Institute Australia for Medical Research, Randwick, New South Wales, 2031, Australia |
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Abstract: | l-Xylulose reductase (XR) is involved in water re-absorption and cellular osmoregulation. The crystal structure of human XR
complemented with site-directed mutagenesis (Cys138Ala) indicated that the disulfide bond in the active site between Cys138
and Cys150 is unstable and may affect the reactivity of the enzyme. The effects of reducing agents on the activities of the
wild-type and mutant enzymes indicated the reversibility of disulfide-bond formation, which resulted in three-fold decrease
in catalytic efficiency. Furthermore, the addition of cysteine (>2 mM) inactivated human XR and was accompanied by a 10-fold
decrease in catalytic efficiency. TOF-MS analysis of the inactivated enzyme showed the S-cysteinylation of Cys138 in the wild-type
and Cys150 in the mutant enzymes. Thus, the action of human XR may be regulated by cellular redox conditions through reversible
disulfide-bond formation and by S-cysteinylation.
Received 25 January 2009; received after revision 12 February 2009; accepted 16 February 2009
H.-T. Zhao, S. Endo: These two authors contribute equally to this work. |
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Keywords: | " target="_blank"> l-xylulose reductase" target="_blank">l-xylulose reductase short-chain dehydrogenase/reductase X-ray crystallography site-directed mutagenesis disulfide bond protein structure enzyme regulation |
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