**4. Discussion**

Both prodrugs INH and PZA bind to LPO in the substrate-binding site on the distal heme side. In the unliganded structure of LPO, the space of the substrate-binding site is filled with six water molecules, W1, W2, W3, W4¸ W5 and W6. Upon binding to INH, four water molecules W2, W4¸ W5 and W6 are expelled from the site. In the structure of the complex of LPO with PZA, three water molecules W4, W5 and W6 are displaced. The conserved water molecule W1 occupies a position on distal heme side in the centre between the positions of heme-iron and Nδ2 atom of His109. His109 is linked to a chain of six other conserved water molecules, W1, W2, W3, W4¸ W5 and W6 together with His266 and Asp253 residues interlinked between them. The pyridine ring nitrogen atom is hydrogen bonded to W1 which in turn is hydrogen bonded to Nδ2 of His109 and heme iron. The position and orientation of INH are fixed in the substrate-binding site because of a hydrogen bonded interaction between amino nitrogen atom of INH and N2 of Gln423. The position of PZA also fixed in the substrate-binding site on the distal heme side where amino nitrogen atom of PZA forms a hydrogen bond with water molecule W2 which in turn is hydrogen bonded to the conserved water molecule W1. As in the complex of LPO with INH, W1 is hydrogen bonded to heme iron and His109 Nδ2. On the other side carbonyl oxygen atom of PZA is hydrogen bonded to Gln423 N2. However, unlike INH where pyridine nitrogen atom is hydrogen bonded to W1, in this case carboxamide nitrogen atom is hydrogen bonded to W1 via another water molecule W2. The main difference between the two complexes is that the INH molecule interacts directly with heme water molecule W1 while PZA binds to heme water molecule W1 via another water molecule W2.

As far as the ligand binding sites are concerned all the three enzymes, LPO, *Mt*CP and PncA show considerable similarities with strong preferences for the binding of small aromatic compounds. There is a clear evidence that INH is converted in a similar manner into active form by both LPO and *Mt*CP indicating a direct role of LPO in the treatment of TB. Similarly, PZA also makes a good substrate for LPO which can be converted into active form as an antibacterial agent. However, the final active forms produced by LPO and PncA may not be same and the mechanism of action may be different. Nevertheless, LPO seems to have a role in the treatment of tuberculosis through its interactions with INH and PZA and it should be exploited.

#### **5. Acknowledgements**

The authors acknowledge Department of Science and Technology (DST) of the Central Government, New Delhi for the financial support. NP and MS thank the Indian Council of Medical Research, New Delhi and Council of Scientific and Industrial Research, New Delhi for the award of fellowships. TPS thanks Department of Biotechnology (DBT) for the award of Distinguished Biotechnology Research Professorship.
