PDBsum entry 3k4c

Oxidoreductase

PDB id: 3k4c

Contents

Protein chains

576 a.a. *

Ligands

FAD ×4

1PE ×4

MES ×2

Waters ×1900

* Residue conservation analysis

PDB id:

3k4c

Name:

Oxidoreductase

Title:

Pyranose 2-oxidase h167a/t169g mutant

Structure:

Pyranose 2-oxidase. Chain: a, b, c, d. Synonym: pyranose oxidase. Engineered: yes. Mutation: yes

Source:

Trametes ochracea. White-rot fungus. Organism_taxid: 230624. Gene: p2o. Expressed in: escherichia coli. Expression_system_taxid: 562.

Resolution:

1.70Å R-factor: 0.181 R-free: 0.216

Authors:

C.Divne,T.C.Tan

Key ref:

W.Pitsawong et al. (2010). A conserved active-site threonine is important for both sugar and flavin oxidations of pyranose 2-oxidase. J Biol Chem, 285, 9697-9705. PubMed id: 20089849

Date:

05-Oct-09 Release date: 02-Feb-10

Protein chains

Q7ZA32  (Q7ZA32_TRAOC) -  Pyranose 2-oxidase from Trametes ochracea

Seq: 623 a.a.

Struc: 576 a.a.*

* PDB and UniProt seqs differ at 2 residue positions (black crosses)

Enzyme reactions

• Enzyme class: E.C.1.1.3.10 - pyranose oxidase.
• Reaction: D-glucose + O2 = 2-dehydro-D-glucose + H2O2
• Cofactor: FAD

FAD (Bound ligand (Het Group name = FAD) corresponds exactly)

Molecule diagrams generated from .mol files obtained from the KEGG ftp site

reference

J Biol Chem 285:9697-9705 (2010)

PubMed id: 20089849

A conserved active-site threonine is important for both sugar and flavin oxidations of pyranose 2-oxidase.

W.Pitsawong, J.Sucharitakul, M.Prongjit, T.C.Tan, O.Spadiut, D.Haltrich, C.Divne, P.Chaiyen.

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.