PDBsum entry 2wsd

Oxidoreductase

PDB id: 2wsd

Contents

Protein chain

506 a.a. *

Ligands

OXY

EDO ×12

Metals

_CU ×4

Waters ×609

* Residue conservation analysis

PDB id:

2wsd

Name:

Oxidoreductase

Title:

Proximal mutations at the type 1 cu site of cota-laccase: i494a mutant

Structure:

Spore coat protein a. Chain: a. Engineered: yes. Mutation: yes. Other_details: ethylene glycol (edo) as solvent

Source:

Bacillus subtilis. Organism_taxid: 1423. Expressed in: escherichia coli. Expression_system_taxid: 562

Resolution:

1.60Å R-factor: 0.178 R-free: 0.190

Authors:

C.S.Silva,P.Durao,Z.Chen,C.M.Soares,M.M.Pereira,S.Todorovic, P.Hildebrandt,L.O.Martins,P.F.Lindley,I.Bento

Key ref:

P.Durão et al. (2008). Proximal mutations at the type 1 copper site of CotA laccase: spectroscopic, redox, kinetic and structural characterization of I494A and L386A mutants. Biochem J, 412, 339-346. PubMed id: 18307408

Date:

04-Sep-09 Release date: 29-Sep-10

Protein chain

P07788  (COTA_BACSU) -  Laccase from Bacillus subtilis (strain 168)

Seq: 513 a.a.

Struc: 506 a.a.*

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

Enzyme reactions

• Enzyme class 1: E.C.1.10.3.2 - laccase.
• Reaction: 4 hydroquinone + O2 = 4 benzosemiquinone + 2 H2O

4 × hydroquinone + O2 (Bound ligand (Het Group name = OXY) corresponds exactly) = 4 × benzosemiquinone + 2 × H2O

• Cofactor: Cu cation
• Enzyme class 2: E.C.1.3.3.5 - bilirubin oxidase.

Pathway:

• Reaction: 2 (4Z,15Z)-bilirubin IXalpha + O2 = 2 biliverdin IXalpha + 2 H2O

2 × (4Z,15Z)-bilirubin IXalpha + O2 (Bound ligand (Het Group name = OXY) corresponds exactly) = 2 × biliverdin IXalpha + 2 × H2O

Note, where more than one E.C. class is given (as above), each may correspond to a different protein domain or, in the case of polyprotein precursors, to a different mature protein.

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

Added reference

Biochem J 412:339-346 (2008)

PubMed id: 18307408

Proximal mutations at the type 1 copper site of CotA laccase: spectroscopic, redox, kinetic and structural characterization of I494A and L386A mutants.

P.Durão, Z.Chen, C.S.Silva, C.M.Soares, M.M.Pereira, S.Todorovic, P.Hildebrandt, I.Bento, P.F.Lindley, L.O.Martins.

ABSTRACT

In the present study the CotA laccase from Bacillus subtilis has been mutated at two hydrophobic residues in the vicinity of the type 1 copper site. The mutation of Leu(386) to an alanine residue appears to cause only very subtle alterations in the properties of the enzyme indicating minimal changes in the structure of the copper centres. However, the replacement of Ile(494) by an alanine residue leads to significant changes in the enzyme. Thus the major visible absorption band is upshifted by 16 nm to 625 nm and exhibits an increased intensity, whereas the intensity of the shoulder at approx. 330 nm is decreased by a factor of two. Simulation of the EPR spectrum of the I494A mutant reveals differences in the type 1 as well as in the type 2 copper centre reflecting modifications of the geometry of these centres. The intensity weighted frequencies <nu(Cu-S)>, calculated from resonance Raman spectra are 410 cm(-1) for the wild-type enzyme and 396 cm(-1) for the I494A mutant, indicating an increase of the Cu-S bond length in the type 1 copper site of the mutant. Overall the data clearly indicate that the Ile(494) mutation causes a major alteration of the structure near the type 1 copper site and this has been confirmed by X-ray crystallography. The crystal structure shows the presence of a fifth ligand, a solvent molecule, at the type 1 copper site leading to an approximate trigonal bipyramidal geometry. The redox potentials of the L386A and I494A mutants are shifted downwards by approx. 60 and 100 mV respectively. These changes correlate well with decreased catalytic efficiency of both mutants compared with the wild-type.