Isonicotinic acid hydrazide conversion to Isonicotinyl-NAD by catalase-peroxidases

J Biol Chem. 2010 Aug 20;285(34):26662-73. doi: 10.1074/jbc.M110.139428. Epub 2010 Jun 15.


Activation of the pro-drug isoniazid (INH) as an anti-tubercular drug in Mycobacterium tuberculosis involves its conversion to isonicotinyl-NAD, a reaction that requires the catalase-peroxidase KatG. This report shows that the reaction proceeds in the absence of KatG at a slow rate in a mixture of INH, NAD(+), Mn(2+), and O(2), and that the inclusion of KatG increases the rate by >7 times. Superoxide, generated by either Mn(2+)- or KatG-catalyzed reduction of O(2), is an essential intermediate in the reaction. Elimination of the peroxidatic process by mutation slows the rate of reaction by 60% revealing that the peroxidatic process enhances, but is not essential for isonicotinyl-NAD formation. The isonicotinyl-NAD(*+) radical is identified as a reaction intermediate, and its reduction by superoxide is proposed. Binding sites for INH and its co-substrate, NAD(+), are identified for the first time in crystal complexes of Burkholderia pseudomallei catalase-peroxidase with INH and NAD(+) grown by co-crystallization. The best defined INH binding sites were identified, one in each subunit, on the opposite side of the protein from the entrance to the heme cavity in a funnel-shaped channel. The NAD(+) binding site is approximately 20 A from the entrance to the heme cavity and involves interactions primarily with the AMP portion of the molecule in agreement with the NMR saturation transfer difference results.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Antitubercular Agents / metabolism
  • Bacterial Proteins / metabolism*
  • Binding Sites
  • Burkholderia pseudomallei / enzymology
  • Catalase / metabolism*
  • Crystallography, X-Ray
  • Isoniazid / analogs & derivatives*
  • Isoniazid / metabolism*
  • Kinetics
  • NAD / analogs & derivatives*
  • NAD / biosynthesis
  • NAD / metabolism
  • Peroxidases
  • Prodrugs


  • Antitubercular Agents
  • Bacterial Proteins
  • Prodrugs
  • isonicotinyl-NAD
  • NAD
  • Peroxidases
  • Catalase
  • katG protein, Mycobacterium tuberculosis
  • Isoniazid