3k4b | pdb_00003k4b
Pyranose 2-oxidase T169S mutant
Structural highlights
Evolutionary Conservation Check, as determined by ConSurfDB. You may read the explanation of the method and the full data available from ConSurf. Publication Abstract from PubMedPyranose 2-oxidase (P2O) catalyzes the oxidation by O(2) of d-glucose and several aldopyranoses to yield the 2-ketoaldoses and H(2)O(2). 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.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 k(red). 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. A conserved active-site threonine is important for both sugar and flavin oxidations of pyranose 2-oxidase.,Pitsawong W, Sucharitakul J, Prongjit M, Tan TC, Spadiut O, Haltrich D, Divne C, Chaiyen P J Biol Chem. 2010 Mar 26;285(13):9697-705. Epub 2010 Jan 20. PMID:20089849[1] From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. See AlsoReferences
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