A Conserved Active-site Threonine Is Important for Both Sugar and Flavin Oxidations of Pyranose 2-Oxidase

• Published in: The Journal of biological chemistry Vol. 285; no. 13; pp. 9697 - 9705
• Main Authors: Pitsawong, Warintra, Sucharitakul, Jeerus, Prongjit, Methinee, Tan, Tien-Chye, Spadiut, Oliver, Haltrich, Dietmar, Divne, Christina, Chaiyen, Pimchai
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 26-03-2010; American Society for Biochemistry and Molecular Biology
• Subjects: Biochemistry; Biokemi; Carbohydrate Dehydrogenases - chemistry; Carbohydrates - chemistry; Catalytic Domain; Chemistry; Crystallography, X-Ray - methods; Electron Transfer; Enzymes/Catalysis; Enzymes/Flavin; Enzymes/Kinetics; Enzymes/Oxidase; Enzymes/Oxidation-Reduction; Enzymology; Flavins - chemistry; Galactose - chemistry; Glucose - chemistry; Hydrogen Bonding; Hydrogen Peroxide - chemistry; Kemi; Kinetics; Medicin och hälsovetenskap; Molecular biology; Molekylärbiologi; Mutagenesis, Site-Directed; NATURAL SCIENCES; NATURVETENSKAP; Oxygen - chemistry; Temperature; Threonine - chemistry; Trametes - enzymology; Enzymes/Catalysis; Enzymes/Kinetics; Enzymes/Oxidase; Electron Transfer; Enzymes/Flavin; Enzymes/Oxidation-Reduction
• ISSN: 0021-9258; 1083-351X; 1083-351X
• DOI: 10.1074/jbc.M109.073247

Pyranose 2-oxidase (P2O)5 catalyzes the oxidation by O2 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 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 (45–47 mm), 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 Oγ assumes 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.