PDBsum entry 2vcs

Oxidoreductase

PDB id: 2vcs

Contents

Protein chain

249 a.a. *

Ligands

HEM

ISZ ×2

SO4

Waters ×299

* Residue conservation analysis

PDB id:

2vcs

Name:

Oxidoreductase

Title:

Structure of isoniazid (inh) bound to cytosolic soybean ascorbate peroxidase mutant h42a

Structure:

Ascorbate peroxidase. Chain: a. Fragment: residues 2-250. Synonym: cytosolic ascorbate peroxidase 1. Engineered: yes. Mutation: yes

Source:

Glycine max. Soybean. Organism_taxid: 3847. Expressed in: escherichia coli. Expression_system_taxid: 562.

Resolution:

1.68Å R-factor: 0.168 R-free: 0.210

Authors:

C.L.Metcalfe,I.K.Macdonald,K.A.Brown,E.L.Raven,P.C.E.Moody

Key ref:

C.L.Metcalfe et al. (2007). The tuberculosis prodrug isoniazid bound to activating peroxidases. J Biol Chem, 283, 6193. PubMed id: 18056997 DOI: 10.1074/jbc.M707412200

Date:

26-Sep-07 Release date: 04-Dec-07

Protein chain

Q43758  (Q43758_SOYBN) -  L-ascorbate peroxidase from Glycine max

Seq: 250 a.a.

Struc: 249 a.a.*

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

Enzyme reactions

• Enzyme class: E.C.1.11.1.11 - L-ascorbate peroxidase.
• Reaction: L-ascorbate + H2O2 = L-dehydroascorbate + 2 H2O
• Cofactor: Heme

Heme (Bound ligand (Het Group name = HEM) matches with 95.45% similarity)

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

reference

DOI no: 10.1074/jbc.M707412200

J Biol Chem 283:6193 (2007)

PubMed id: 18056997

The tuberculosis prodrug isoniazid bound to activating peroxidases.

C.L.Metcalfe, I.K.Macdonald, E.J.Murphy, K.A.Brown, E.L.Raven, P.C.Moody.

ABSTRACT

Isoniazid (INH, isonicotinic acid hydrazine) is one of only two therapeutic agents effective in treating tuberculosis. This prodrug is activated by the heme enzyme catalase-peroxidase (KatG) endogenous to Mycobacterium tuberculosis, but the mechanism of activation is poorly understood, in part because the binding interaction has not been properly established. The class I peroxidases ascorbate peroxidase (APX) and cytochrome c peroxidase (CcP) have very similar active site structures to KatG, and are also capable of activating isoniazid. We report here the first crystal structures of complexes of isoniazid bound to APX and CcP. These are the first structures of isoniazid bound to any activating enzymes. The structures show that isoniazid binds close to the d-heme edge in both APX and CcP, although the precise binding orientation varies slightly in the two cases. A second binding site for INH is found in APX at the g-heme edge close to the established ascorbate binding site, indicating that the g-heme edge can also support the binding of aromatic substrates. We also show that in an active site mutant of sAPX (W41A) INH can bind directly to the heme iron to become an inhibitor, and in a different mode when the distal histidine is replaced by alanine(H42A). These structures provide the first unambiguous evidence for the location of the isoniazid binding site in the class I peroxidases, and provide rationalisation of isoniazid resistance in naturally occurring KatG mutant strains of M. tuberculosis.

Selected figure(s)

Figure 4.
FIGURE 4. Stereo diagrams showing INH bound in the active site mutants of sAPX W41A and H42A. A, in sAPX·(W41A), two molecules of INH (brown) are bound in the distal cavity, one in the same position as sAPX and a second coordinated directly to the heme iron. The figure shows observed F[o] - F[c] difference density (in green, contoured at 3 ). B, in sAPX (H42A) the orientation of INH (brown) is rotated relative to the wild type and is held in position by a hydrogen bond to Trp-41. Observed F[o] - F[c] difference density is shown in green. In both cases the occupancy of the INH is partial and shared with water molecules that are represented as red spheres.

Figure 6.
FIGURE 6. Stereo diagram showing INH bound in the ascorbate binding pocket of sAPX. Hydrogen bonding interactions are observed between INH and Arg-172 and Lys-31 and via a water to the propionate group of the heme. There are INH molecules bound in identical positions in the sAPX (W41A) and sAPX (H42A) structures. The first INH molecule bound in the distal cavity is also shown to aid orientation. The protein is shown in green, the heme group in blue, and INH in pink. Waters are represented as red spheres. The observed F[o] - F[c] difference density (contoured at 3 ) for the INH molecules is shown in green. Figs. 2, 3, 4, 5, 6 were prepared with PyMOL (22).

The above figures are reprinted by permission from the ASBMB: J Biol Chem (2007, 283, 6193) copyright 2007.

Figures were selected by an automated process.