PDBsum entry 2ign

Oxidoreductase

PDB id: 2ign

Contents

Protein chains

(+ 2 more) 577 a.a. *

Ligands

FAD ×8

MES ×8

Waters ×4758

* Residue conservation analysis

PDB id:

2ign

Name:

Oxidoreductase

Title:

Crystal structure of recombinant pyranose 2-oxidase h167a mutant

Structure:

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

Source:

Trametes ochracea. Organism_taxid: 230624. Gene: p2o. Expressed in: escherichia coli bl21(de3). Expression_system_taxid: 469008.

Biol. unit:

Tetramer (from PQS)

Resolution:

1.65Å R-factor: 0.172 R-free: 0.205

Authors:

C.Divne

Key ref:

M.Kujawa et al. (2006). Structural basis for substrate binding and regioselective oxidation of monosaccharides at C3 by pyranose 2-oxidase. J Biol Chem, 281, 35104-35115. PubMed id: 16984920 DOI: 10.1074/jbc.M604718200

Date:

22-Sep-06 Release date: 10-Oct-06

Protein chains

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

Seq: 623 a.a.

Struc: 577 a.a.*

* PDB and UniProt seqs differ at 1 residue position (black cross)

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

DOI no: 10.1074/jbc.M604718200

J Biol Chem 281:35104-35115 (2006)

PubMed id: 16984920

Structural basis for substrate binding and regioselective oxidation of monosaccharides at C3 by pyranose 2-oxidase.

M.Kujawa, H.Ebner, C.Leitner, B.M.Hallberg, M.Prongjit, J.Sucharitakul, R.Ludwig, U.Rudsander, C.Peterbauer, P.Chaiyen, D.Haltrich, C.Divne.

ABSTRACT

Pyranose 2-oxidase (P2Ox) participates in fungal lignin degradation by producing the H2O2 needed for lignin-degrading peroxidases. The enzyme oxidizes cellulose- and hemicellulose-derived aldopyranoses at C2 preferentially, but also on C3, to the corresponding ketoaldoses. To investigate the structural determinants of catalysis, covalent flavinylation, substrate binding, and regioselectivity, wild-type and mutant P2Ox enzymes were produced and characterized biochemically and structurally. Removal of the histidyl-FAD linkage resulted in a catalytically competent enzyme containing tightly, but noncovalently bound FAD. This mutant (H167A) is characterized by a 5-fold lower kcat, and a 35-mV lower redox potential, although no significant structural changes were seen in its crystal structure. In previous structures of P2Ox, the substrate loop (residues 452-457) covering the active site has been either disordered or in a conformation incompatible with carbohydrate binding. We present here the crystal structure of H167A in complex with a slow substrate, 2-fluoro-2-deoxy-D-glucose. Based on the details of 2-fluoro-2-deoxy-D-glucose binding in position for oxidation at C3, we also outline a probable binding mode for D-glucose positioned for regioselective oxidation at C2. The tentative determinant for discriminating between the two binding modes is the position of the O6 hydroxyl group, which in the C2-oxidation mode can make favorable interactions with Asp452 in the substrate loop and, possibly, a nearby arginine residue (Arg472). We also substantiate our hypothesis with steady-state kinetics data for the alanine replacements of Asp452 and Arg472 as well as the double alanine 452/472 mutant.

Selected figure(s)

Figure 3.
FIGURE 3. Details of ligand binding in nsP2Ox[ACT] and His6-H167A[2FG]. Side-by-side view showing the active site in the nsP2Ox[ACT] complex (A) and the His[6]-H167A[2FG] complex (B). In B, the carbohydrate is positioned for oxidation at C3. Firm interactions are noted for the C3 hydroxyl group and the side chains of His^548 and Asn^593. The substrate loop is highlighted in magenta. Atom-coloring scheme: carbon, beige (protein), yellow (FAD), green (ligand); nitrogen, blue; oxygen, red. C, superposition of the active sites in nsP2Ox[ACT] (beige) and His[6]-H167A[2FG] (green).

Figure 4.
FIGURE 4. Productive binding modes of D-glucose in the C2 and C3 orientations. A, observed binding of 2FG to the His[6]-H167A[2FG] active site. Unbiased electron density for the carbohydrate and part of the flavin ring. The electron density was calculated using an early model where the ligand had not yet been included. B, observed binding of 2FG in the C3-oxidation orientation, and in the theoretical C2-oxidation orientation (C). In C, the protein model from the His[6]-H167A[2FG] complex was used for modeling the C2 orientation. The 2FG molecule was rotated 180° about an axis defined roughly by a line running through a point midway between the glucose atoms C5 and O5, and a point midway between atoms C2 and C3. The same coloring scheme is used as in Fig. 3.

The above figures are reprinted by permission from the ASBMB: J Biol Chem (2006, 281, 35104-35115) copyright 2006.

Figures were selected by an automated process.