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|Title:||A conserved active-site threonine is important for both sugar and flavin oxidations of pyranose 2-oxidase|
Tien Chye Tan
AlbaNova University Center
University of Natural Natural Resources and Applied Life Sciences
|Keywords:||Biochemistry, Genetics and Molecular Biology;Medicine|
|Citation:||Journal of Biological Chemistry. Vol.285, No.13 (2010), 9697-9705|
|Abstract:||Pyranose 2-oxidase (P2O) catalyzes the oxidation by O2 of D-glucose and several aldopyranoses to yield the 2-ketoaldoses and H 2O2. Based on crystal structures, in one rotamer conformation, the threonine hydroxyl of Thr169 forms H-bonds to the flavin-N5/O4 locus, whereas, in a different rotamer, it may interact with either sugar or other parts of the P2O·sugar complex. Transient kinetics of wild-type (WT) and Thr169 → S/N/G/A replacement variants show that D-Glc binds to T169S, T169N, and WT with the same Kd (45-47mM), and the hydride transfer rate constants (kred) are similar (15.3-9.7 s -1 at 4 °C ). kred of T169G with D-glucose (0.7 s -1, 4 °C) is significantly less than that of WT but not as severely affected as in T169A (kred of 0.03 s-1 at 25 °C). Transient kinetics of WT and mutants using D-galactose show that P2O binds D-galactose with a one-step binding process, different from binding of D-glucose. In T169S, T169N, and T169G, the overall turnover with D-Gal is faster than that of WT due to an increase of kred. In the crystal structure of T169S, Ser169 Oγassumes a position identical to that of Oγ1 in Thr169; in T169G, solvent molecules may be able to rescue H-bonding. Our data suggest that a competent reductive half-reaction requires a side chain at position 169 that is able to form an H-bond within the ES complex. During the oxidative half-reaction, all mutants failed to stabilize a C4a-hydroperoxyflavin intermediate, thus suggesting that the precise position and geometry of the Thr169 side chain are required for intermediate stabilization. © 2010 by The American Society for Biochemistry and Molecular Biology, Inc.|
|Appears in Collections:||Scopus 2006-2010|
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