3K4C
Pyranose 2-oxidase H167A/T169G mutant
Summary for 3K4C
| Entry DOI | 10.2210/pdb3k4c/pdb |
| Related | 1TT0 2IGK 2IGM 2IGN 2IGO 3BG6 3K4B |
| Descriptor | Pyranose 2-oxidase, FLAVIN-ADENINE DINUCLEOTIDE, PENTAETHYLENE GLYCOL, ... (5 entities in total) |
| Functional Keywords | oxidoreductase, gmc oxidoreductase, h167a/t169g mutant, rossmann fold, phbh fold, homotetramer, 8-alpha-(n3) histidyl flavinylation |
| Biological source | Trametes ochracea (White-rot fungus) |
| Total number of polymer chains | 4 |
| Total formula weight | 281696.44 |
| Authors | |
| Primary citation | Pitsawong, W.,Sucharitakul, J.,Prongjit, M.,Tan, T.C.,Spadiut, O.,Haltrich, D.,Divne, C.,Chaiyen, P. A conserved active-site threonine is important for both sugar and flavin oxidations of pyranose 2-oxidase. J.Biol.Chem., 285:9697-9705, 2010 Cited by PubMed Abstract: Pyranose 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. PubMed: 20089849DOI: 10.1074/jbc.M109.073247 PDB entries with the same primary citation |
| Experimental method | X-RAY DIFFRACTION (1.7 Å) |
Structure validation
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