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Please use this identifier to cite or link to this item: http://repository.li.mahidol.ac.th/dspace/handle/123456789/28754
Title: A conserved active-site threonine is important for both sugar and flavin oxidations of pyranose 2-oxidase
Authors: Warintra Pitsawong
Jeerus Sucharitakul
Methinee Prongjit
Tien Chye Tan
Oliver Spadiut
Dietmar Haltrich
Christina Divne
Pimchai Chaiyen
Mahidol University
Chulalongkorn University
AlbaNova University Center
University of Natural Natural Resources and Applied Life Sciences
Keywords: Biochemistry, Genetics and Molecular Biology;Medicine
Issue Date: 26-Mar-2010
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.
URI: https://www.scopus.com/inward/record.uri?partnerID=HzOxMe3b&scp=77951245033&origin=inward
http://repository.li.mahidol.ac.th/dspace/handle/123456789/28754
ISSN: 1083351X
00219258
Appears in Collections:Scopus 2006-2010

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