title: Isonicotinic acid hydrazide conversion to Isonicotinyl-NAD by catalase-peroxidases.
abstract: 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.
authors.affiliation: Department of Microbiology, University of Manitoba,Winnipeg, Manitoba, Canada.
authors.forename: Ben
authors.initials: B
authors.lastname: Wiseman
authors.forename: Xavi
authors.initials: X
authors.lastname: Carpena
authors.forename: Miguel
authors.initials: M
authors.lastname: Feliz
authors.forename: Lynda J
authors.initials: LJ
authors.lastname: Donald
authors.forename: Miquel
authors.initials: M
authors.lastname: Pons
authors.forename: Ignacio
authors.initials: I
authors.lastname: Fita
authors.forename: Peter C
authors.initials: PC
authors.lastname: Loewen
mesh_terms: D000995:Antitubercular Agents / Q000378:metabolism; D001426:Bacterial Proteins / Q000378:metabolism*; D001665:Binding Sites; D016957:Burkholderia pseudomallei / Q000201:enzymology; D002374:Catalase / Q000378:metabolism*; D018360:Crystallography, X-Ray; D007538:Isoniazid / Q000031:analogs & derivatives* / Q000378:metabolism*; D007700:Kinetics; D009243:NAD / Q000031:analogs & derivatives* / Q000096:biosynthesis / Q000378:metabolism; D010544:Peroxidases; D011355:Prodrugs
chemical_list: D000995:Antitubercular Agents; D001426:Bacterial Proteins; D011355:Prodrugs; C525017:isonicotinyl-NAD; D009243:NAD; D010544:Peroxidases; D002374:Catalase; C480353:katG protein, Mycobacterium tuberculosis; D007538:Isoniazid