Binding of the anti-tubercular drug isoniazid to the arylamine N-acetyltransferase protein from Mycobacterium smegmatis

Abstract

Isoniazid is a frontline drug used in the treatment of tuberculosis (TB). Isoniazid is a prodrug, requiring activation in the mycobacterial cell by the catalase/peroxidase activity of the katG gene product. TB kills two million people every year and the situation is getting worse due to the increase in prevalence of HIV/AIDS and emergence of multidrug-resistant strains of TB. Arylamine N-acetyltransferase (NAT) is a drug-metabolizing enzyme (E.C. 2.1.3.5). NAT can acetylate isoniazid, transferring an acetyl group from acetyl coenzyme A onto the terminal nitrogen of the drug, which in its N-acetylated form is therapeutically inactive. The bacterium responsible for TB, Mycobacterium tuberculosis, contains and expresses the gene encoding the NAT protein. Isoniazid binds to the NAT protein from Salmonella typhimurium and we report here the mode of binding of isoniazid in the NAT enzyme from Mycobacterium smegmatis, closely related to the M. tuberculosis and S. typhimurium NAT enzymes. The mode of binding of isoniazid to M. smegmatis NAT has been determined using data collected from two distinct crystal forms. We can say with confidence that the observed mode of binding of isoniazid is not an artifact of the crystallization conditions used. The NAT enzyme is active in mycobacterial cells and we propose that isoniazid binds to the NAT enzyme in these cells. NAT activity in M. tuberculosis is likely therefore to modulate the degree of activation of isoniazid by other enzymes within the mycobacterial cell. The structure of NAT with isoniazid bound will facilitate rational drug design for anti-tubercular therapy.

Figure 1.

Schematic to show activation and inactivation of INH. INH is a prodrug and requires activation by the catalase peroxidase protein (the KatG gene product). The NAT enzymes can N-acetylate INH, rendering the drug therapeutically inactive. The asterisk next to the terminal nitrogen in the active form of INH denotes a range of oxidized species (Bodiguel et al. 2001).

Figure 2.

Crystals of arylamine N-acetyltransferase from M. smegmatis grown in the presence of INH. NAT protein was incubated for 30 min with 20 mM INH at 37°C prior to setting crystal trials. Crystal trials were incubated at 20°C and crystals appeared within 3 d. Two typical examples of the crystals produced are shown here. Conditions are (A) 0.1 M MES at pH 6.5, 12% PEG 20.00; (B) 0.2 M ammonium sulphate, 0.1 M MES at pH 6.5, 30% PEG MME 5000.

Figure 3.

INH bound in the active site of the NAT enzyme. The INH moiety can be seen at the bottom of the active site cleft. (A) The backbone is shown in ribbon format, color ramped from red through white to blue over the length of the amino acid chain. The catalytic triad of residues (Cys⁷⁰-His¹¹⁰-Asp¹²⁷) and well-conserved Tyr¹⁷⁷ are also shown, with carbons colored magenta. (B) Electrostatic surface of NAT in complex with INH. The location of the active site at the bottom of a long deep cleft is apparent, as is the generally negative electrostatic potential of the catalytic site. The figure was generated using the programs ElectroSurface and AESOP (M. Noble and J. Gruber, unpubl.).

Figure 4.

Orientation of INH in the active site of the NAT enzyme. Electron density can be clearly seen for INH in this omit map contoured at 1.5σ. The active site triad residues (Cys⁷⁰-His¹¹⁰-Asp¹²⁷) are shown in ball-and-stick format and the associated electron density is shaded in blue. The INH molecule is also shown in ball-and-stick format with the associated electron density shown colored in pink. The backbone is shown in ribbon format. This figure was produced using AESOP.

Figure 5.

Ligplot analysis of the mode of binding of INH. Ligplot (Wallace et al. 1995) was used to analyze the mode of binding of INH to the NAT protein from M. smegmatis. Ligplot was run using the ‘A’ chain from the P2₁2₁2₁ structure. The output suggests hydrogen bonding to the amide nitrogen from Cys⁷⁰ to the carbonyl group from INH and also from the carbonyl group of the backbone from Thr¹⁰⁹ to both of the nitrogens in the hydrazyl group of INH. Hydrophobic interactions with the aromatic ring of INH are proposed from the residues Phe³⁸, Val⁹⁵, Phe¹³⁰, and Phe²⁰⁴.

Figure 6.

Modeling of substrates into the active site of NAT from M. smegmatis. Substrates were overlaid based upon the structure of INH using the terminal nitrogen as a reference point for alignment. Section A shows INH as seen in the crystal structure. Sections B–F represent the modeled mode of binding of substrates hydralazine, 5-aminosalicylate, 4-aminosalicylate, anisidine, and p-aminobenzoic acid, respectively. The secondary structure is colored blue with the molecular surface colored grey. Potential hydrogen bonds between the catalytic triad of residues and the Tyr¹⁷⁷ residue are indicated with dotted lines. In all frames the protein structure is exactly superimposable; the Cys⁷⁰ label has been placed to avoid occlusion of model substrates in frames B–F.