PDBsum entry 1b80

Oxidoreductase

PDB id: 1b80

Contents

Protein chains

349 a.a. *

Ligands

HEM ×2

Metals

_CA ×4

Waters ×573

* Residue conservation analysis

References listed in PDB file

Key reference

Title

Crystal structures of pristine and oxidatively processed lignin peroxidase expressed in escherichia coli and of the w171f variant that eliminates the redox active tryptophan 171. Implications for the reaction mechanism.

Authors

W.Blodig, A.T.Smith, W.A.Doyle, K.Piontek.

Ref.

J Mol Biol, 2001, 305, 851-861. [DOI no: 10.1006/jmbi.2000.4346]

PubMed id

11162097

Abstract

The heme enzyme lignin peroxidase (LiP) from the white rot fungus Phanerochaete chrysosporium contains a solvent exposed redox active tryptophan residue (Trp171) that carries a unique hydroxy group stereo-specifically attached to its C(beta) atom. A Trp171Phe mutant has no activity at all towards the substrate veratryl alcohol. The mechanism of veratryl alcohol oxidation involving beta-hydroxy-Trp171 is largely unknown. Here, we present the first crystal structures of LiP isozyme H8 at high resolution in its pristine non-hydroxylated form, of the C(beta)-hydroxylated form, and of the Trp171Phe mutant using recombinantly expressed and refolded protein produced from Escherichia coli. As a consequence, all structures are unglycosylated. Structural changes in response to the mutation are marginal and allow us to attribute the complete lack of activity exclusively to the absence of the redox active indole side-chain. The origin of the stereospecificity of the Trp171 hydroxylation can be explained on structural grounds. A reaction mechanism involving Trp171 is proposed and the possible function of the modification is discussed. Another important result regarding the ongoing debate on the co-ordination state of the heme iron in the resting state is that the iron is six co-ordinate in all cases the data being collected at room temperature. The mean distance from the iron to the distal water ligand is 2.18(+/-0.08) A. The radical scavenger orcinol was found to decrease radiation damage to the crystals, during data collection at room temperature.

Figure 3.
Figure 3. Difference omit maps of (a) the LiPH8-H[2]O[2]structure and (b) the pristine LiPH8 structure for residue Trp171 at 1.73 and 1.8 Å resolution, respectively. The maps in cyan are (2F[o] -F[c])exp(ia[c]) electron densities contoured at 2 s where all atoms of Trp171 were omitted for phase calculation. In red a (F[o] -F[c])exp(ia[c]) electron density map is shown, contoured at 8 s where only the hydroxy group was omitted. The picture was produced with O [Jones et al 1991].

Figure 5.
Figure 5. Difference omit map ((F[o] -F[c])exp(ia[c])) at 1.85 Å resolution of the W171F mutant structure of recombinant LiPH8 for residue Phe171 contoured at 7s. The water molecule (Wat417) which hydrogen bonds to the hydroxy group at the C^b atom of Trp171 in the LiP-H[2]O[2] structure is shown as a red asterisk (see also Figure 4). The picture was produced with O [Jones et al 1991].

The above figures are reprinted by permission from Elsevier: J Mol Biol (2001, 305, 851-861) copyright 2001.

Secondary reference #1

Title

The crystal structure of lignin peroxidase at 1.70 a resolution reveals a hydroxy group on the cbeta of tryptophan 171: a novel radical site formed during the redox cycle.

Authors

T.Choinowski, W.Blodig, K.H.Winterhalter, K.Piontek.

Ref.

J Mol Biol, 1999, 286, 809-827. [DOI no: 10.1006/jmbi.1998.2507]

PubMed id

10024453

Figure 6.
Figure 6. Stereoscopic view of the (a) proximal and (b) distal calcium-binding sites in LiP415. Both difference omit maps are contoured at 15σ. The bonds are shown as broken lines and the liganding residues and bond lengths are labelled.

Figure 15.
Figure 15. The molecular surface charge distribution of LiP415 showing the “back side” of the protein. The negative potentials on the surface are shaded in red and the positive potentials in blue. The heme, and several hydrophobic surface residues at the vicinity of Trp171 are depicted by bonds. The picture was generated using the program GRASP [Nicholls 1993].

The above figures are reproduced from the cited reference with permission from Elsevier

Secondary reference #2

Title

Two substrate interaction sites in lignin peroxidase revealed by site-Directed mutagenesis.

Authors

W.A.Doyle, W.Blodig, N.C.Veitch, K.Piontek, A.T.Smith.

Ref.

Biochemistry, 1998, 37, 15097-15105. [DOI no: 10.1021/bi981633h]

PubMed id

9790672

Secondary reference #3

Title

Autocatalytic formation of a hydroxy group at c beta of trp171 in lignin peroxidase.

Authors

W.Blodig, W.A.Doyle, A.T.Smith, K.Winterhalter, T.Choinowski, K.Piontek.

Ref.

Biochemistry, 1998, 37, 8832-8838. [DOI no: 10.1021/bi9727186]

PubMed id

9636023