PDBsum entry 1e8h

Oxidoreductase

PDB id: 1e8h

Contents

Protein chains

545 a.a. *

Ligands

ADP ×2

Waters ×89

* Residue conservation analysis

PDB id:

1e8h

Name:

Oxidoreductase

Title:

Structure of the h61t mutant of the flavoenzyme vanillyl-alcohol oxidase in the apo form complexed by adp

Structure:

Vanillyl-alcohol oxidase. Chain: a, b. Synonym: aryl-alcohol oxidase, 4-allylphenol oxidase. Engineered: yes. Mutation: yes

Source:

Penicillium simplicissimum. Organism_taxid: 69488. Atcc: 90172. Organelle: peroxisomes. Cellular_location: intracellular. Expressed in: escherichia coli. Expression_system_taxid: 562. Other_details: fungus

Biol. unit:

Dimer (from PDB file)

Resolution:

2.60Å R-factor: 0.237 R-free: 0.303

Authors:

A.Mattevi,M.W.Fraaije

Key ref:

M.W.Fraaije et al. (2000). Structural analysis of flavinylation in vanillyl-alcohol oxidase. J Biol Chem, 275, 38654-38658. PubMed id: 10984479 DOI: 10.1074/jbc.M004753200

Date:

20-Sep-00 Release date: 21-Sep-00

Protein chains

P56216  (VAOX_PENSI) -  Vanillyl-alcohol oxidase from Penicillium simplicissimum

Seq: 560 a.a.

Struc: 545 a.a.*

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

Enzyme reactions

• Enzyme class: E.C.1.1.3.38 - vanillyl-alcohol oxidase.
• Reaction: 4-hydroxy-3-methoxy-benzenemethanol + O2 = vanillin + H2O2
• Cofactor: FAD

FAD (Bound ligand (Het Group name = ADP) matches with 50.94% similarity)

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

reference

DOI no: 10.1074/jbc.M004753200

J Biol Chem 275:38654-38658 (2000)

PubMed id: 10984479

Structural analysis of flavinylation in vanillyl-alcohol oxidase.

M.W.Fraaije, R.H.van Den Heuvel, W.J.van Berkel, A.Mattevi.

ABSTRACT

Vanillyl-alcohol oxidase (VAO) is member of a newly recognized flavoprotein family of structurally related oxidoreductases. The enzyme contains a covalently linked FAD cofactor. To study the mechanism of flavinylation we have created a design point mutation (His-61 --> Thr). In the mutant enzyme the covalent His-C8alpha-flavin linkage is not formed, while the enzyme is still able to bind FAD and perform catalysis. The H61T mutant displays a similar affinity for FAD and ADP (K(d) = 1.8 and 2.1 microm, respectively) but does not interact with FMN. H61T is about 10-fold less active with 4-(methoxymethyl)phenol) (k(cat) = 0.24 s(-)(1), K(m) = 40 microm) than the wild-type enzyme. The crystal structures of both the holo and apo form of H61T are highly similar to the structure of wild-type VAO, indicating that binding of FAD to the apoprotein does not require major structural rearrangements. These results show that covalent flavinylation is an autocatalytical process in which His-61 plays a crucial role by activating His-422. Furthermore, our studies clearly demonstrate that in VAO, the FAD binds via a typical lock-and-key approach to a preorganized binding site.

Selected figure(s)

Figure 2.
Fig. 2. The active site of the H61T ternary complex structure showing all relevant residues, the isoalloxazine ring of the FAD cofactor (yellow) and the bound 4-(trifluoromethyl)phenol (blue) contoured by the 2F[o] F[c] electron density calculated with the refined model. The picture was generated with DINO (27).

Figure 3.
Fig. 3. A, a section through the C trace of the apoenzyme subunit showing the large internal solvent-filled cavity. For clarity, the FAD cofactor and 4-(trifluoromethyl)phenol as bound in the holo H61T ternary complex were superimposed and shown in yellow and blue, respectively. The surface was calculated using MSMS (28) and the picture was generated using DINO (27). B, superposition of active site residues in the apo (green) and the holo form complexed with 4-(trifluoromethyl)phenol (red) structures of the VAO H61T mutant. The figure was prepared with MOLSCRIPT ( 26).

The above figures are reprinted by permission from the ASBMB: J Biol Chem (2000, 275, 38654-38658) copyright 2000.

Figures were selected by the author.