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A Conserved Active-site Threonine Is Important for Both Sugar and Flavin Oxidations of Pyranose 2-Oxidase
KTH, School of Biotechnology (BIO), Glycoscience.
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2010 (English)In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 285, no 13, 9697-9705 p.Article in journal (Refereed) Published
Abstract [en]

Pyranose 2-oxidase (P2O) catalyzes the oxidation by O-2 of D-glucose and several aldopyranoses to yield the 2-ketoaldoses and H2O2. Based on crystal structures, in one rotamer conformation, the threonine hydroxyl of Thr(169) 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 center dot sugar complex. Transient kinetics of wild-type (WT) and Thr(169)-> S/N/G/A replacement variants show that D-Glc binds to T169S, T169N, and WT with the same K-d (45-47 mM), and the hydride transfer rate constants (k(red)) are similar (15.3-9.7 s(-1) at 4 degrees C). k(red) of T169G with D-glucose (0.7 s(-1), 4 degrees C) is significantly less than that of WT but not as severely affected as in T169A (k(red) of 0.03 s(-1) at 25 degrees 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, Ser(169) O gamma assumes a position identical to that of O gamma 1 in Thr(169); 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 Thr(169) side chain are required for intermediate stabilization.

Place, publisher, year, edition, pages
2010. Vol. 285, no 13, 9697-9705 p.
National Category
Biochemistry and Molecular Biology Biochemistry and Molecular Biology
URN: urn:nbn:se:kth:diva-28373DOI: 10.1074/jbc.M109.073247ISI: 000276165900042ScopusID: 2-s2.0-77951245033OAI: diva2:389361
FormasSwedish Research Council
QC 20110119Available from: 2011-01-19 Created: 2011-01-14 Last updated: 2011-01-19Bibliographically approved

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