A conserved active-site threonine is important for both sugar and flavin oxidations of pyranose 2-oxidase.
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.
Pitsawong
Warintra
W
Department of Biochemistry and Center of Excellence in Protein Structure and Function, Faculty of Science, Mahidol University, Bangkok 10400, Thailand.
Sucharitakul
Jeerus
J
Department of Biochemistry, Faculty of Dentistry, Chulalongkorn University, Henri-Dunant Road, Patumwan, Bangkok 10300, Thailand.
Prongjit
Methinee
M
Department of Biochemistry and Center of Excellence in Protein Structure and Function, Faculty of Science, Mahidol University, Bangkok 10400, Thailand.
Tan
Tien-Chye
TC
Division of Glycoscience, Kungliga Tekniska Högskolan Biotechnology, Royal Institute of Technology, Albanova University Center, SE-106 91 Stockholm, Sweden.
Spadiut
Oliver
O
Division of Glycoscience, Kungliga Tekniska Högskolan Biotechnology, Royal Institute of Technology, Albanova University Center, SE-106 91 Stockholm, Sweden.
Haltrich
Dietmar
D
Department of Food Science and Technology, Bodenkultur-University of Natural Resource and Applied Life Sciences, A-1190 Vienna, Austria.
Divne
Christina
C
Division of Glycoscience, Kungliga Tekniska Högskolan Biotechnology, Royal Institute of Technology, Albanova University Center, SE-106 91 Stockholm, Sweden.
Chaiyen
Pimchai
P
Department of Biochemistry and Center of Excellence in Protein Structure and Function, Faculty of Science, Mahidol University, Bangkok 10400, Thailand. Electronic address: scpcy@mahidol.ac.th.
D002237:Carbohydrate Dehydrogenases / Q000737:chemistry*; D002241:Carbohydrates / Q000737:chemistry*; D020134:Catalytic Domain; D018360:Crystallography, X-Ray / Q000379:methods; D005415:Flavins / Q000737:chemistry*; D005690:Galactose / Q000737:chemistry; D005947:Glucose / Q000737:chemistry; D006860:Hydrogen Bonding; D006861:Hydrogen Peroxide / Q000737:chemistry; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D010100:Oxygen / Q000737:chemistry*; D013696:Temperature; D013912:Threonine / Q000737:chemistry*; D055454:Trametes / Q000201:enzymology
D002241:Carbohydrates; D005415:Flavins; D013912:Threonine; D006861:Hydrogen Peroxide; D002237:Carbohydrate Dehydrogenases; C022489:pyranose oxidase; D005947:Glucose; D010100:Oxygen; D005690:Galactose