Sandbox Reserved 1072

Structure

Mycobacterium Tuberculosis Catalase Peroxidase (mtCP) is a homodimer with each monomer consisting of two domains. The overall structure is stabilized by 703 water molecules.

Monomer Structure

The two domains of each monomer are primarily alpha helical and have similar foldings. The similar foldings suggests that the monomer results from a gene duplication event; however, the C-terminal domain does not contain the heme b prosthetic group, while the does. The active site is therefore located within the N-terminal domain. The two monomers interact through an interlocking hook formed by the N-terminal domains that stabilizes the formation of the dimer.

The N-terminal is formed through hydrophobic interactions between residues Tyr-28 and Tyr-197 and residues Trp-38 and Trp-204. This interlocking loop region is also found in similar conformations of other catalase peroxidase structures such as: hmCP and bpCP.

Active Site

Active Site

There are 6 conserved key active site residues that suround the . These residues are Arg 104, Trp 107, His 108, His 270, Asp 381.

In hmCP, which share 55% and 69% identity with mtCP, the heme is buried inside HmCP-N, and substrate access to the active site is thorugh a narrow channel that prevents access of a large substrate (3). The location of the binding site for isoniazid (INH) is located near the δ meso heme edge, about 3.8 Å away from the heme iron. This binding site is found within what is considered to be the usual substrate access channel of peroxidases. The reaction between INH and the enzyme must occur from interaction in a binding site intended for the natural substrate (A2). Asp 137 plays a key role in the activation and binding of INH. Asp 137 creates energetically favorable interactions due to its ability to make hydrogen-bond interactions between its carboxylic acid side chain and the pyridinyl N1 of INH.

Catalase Peroxidases

Catalase-peroxidases are enzymes that degrade hydrogen peroxide. Catalase converts two equivalents of hydrogen peroxide into water and oxygen via a two-step reaction cycle in which H₂0₂ alternately oxidizes and reduces the heme iron at the active site. Within peroxidases, oxidation of heme iron involves a H202 molecules, similar to that in the catalase-catalyzed reaction. Reduction of the heme iron, however, involves hydrogen donors such as NADH, not a second H₂0₂ molcule (3). Catalase-Peroxidases that have been characterized are either homodimers or homotetramers and contain a single heme b cofactor at the active site. Usually, the primary struture of the subunit can be divided into two halves that have a high level of sequence similarity, most likely due to a gene duplication event.

Clinical Applications

Isoniazid Structure

Isoniazid Role

INH is a prodrug susceptible to oxidative reactions catalyzed by KatG (4). INH action against mycobacteria requires catalase-peroxidase (KatG) function.

Mechanism

Formation of IN-NAD Adduct

Activation or oxidation of INH by KatG has recently been measured in the terms of production of an IN-NAD adduct molecule that serves as a tight binding inhibitor of InhA. InhA is an enzyme involved in the biosynthesis of mycolic acids, which are components of the mycobacterial cell wall. Therefore, inhibition of InhA alone is sufficient enough to inhibit mycolic acid biosynthesis and induce cell lysis after exposure of bacteria to INH (4).

Mutations

Mutation of Ser315 to a Thr in mtCP results in a loss of the activation to the anti-tuberculosis drug (INH) with no loss of either peroxidase or catalase activity (3).

References

1) Bertrand, T.; Eady, N. A. J.; Jones, J. N.; Jesmin, Nagy, J. M.; Jamart-Gregorie, B.; Raven. E. L.; Brown, K. A. Crystal Structure of Mycobacterium Tubercuosis Catalase-Peroxidase. The J. of Biol. Chem. 2004, 279, 38991-38999.

2) Carpena, X.; Loprasert, Suvit,; Mongkolsuk, S.; Switala, J.; Loewen, P. C.; Fita, I. Catalase-peroxidase KatG of Burkholderia pseudomallei at 1.7 Å resolution. J. Mol. Biol. 2003, 327, 475-489.

3) Yamada, Y.; Fujiwara, T.; Sato, T.; Igarashi, N.; Tanaka, N. The 2.0 Å crystal structure of catalase-peroxidase from Haloarcula marismortui Natur Structural Biol. 2002, 9, 691-695.

4) Zhao, X.; Yu, H.; Yu, S.; Wang, F.; Sacchettini, J. C.; Magliozzo, R. S.; Hydrogen Peroxide-Mediated Isoniazid Activation Catalyzed by Mycobacterium tuberculosis Catalase-Peroxidase (KatG) and Its S315T Mutant. Biochemistry. 2006, 45, 4131-4140.