The Tuberculosis Prodrug Isoniazid Bound to Activating Peroxidases

Abstract

Isoniazid (INH, isonicotinic acid hydrazine) is one of only two therapeutic agents effective in treating tuberculosis. This prodrug is activated by the heme enzyme catalase peroxidase (KatG) endogenous to Mycobacterium tuberculosis but the mechanism of activation is poorly understood, in part because the binding interaction has not been properly established. The class I peroxidases ascorbate peroxidase (APX) and cytochrome c peroxidase (CcP) have active site structures very similar to KatG and are also capable of activating isoniazid. We report here the first crystal structures of complexes of isoniazid bound to APX and CcP. These are the first structures of isoniazid bound to any activating enzymes. The structures show that isoniazid binds close to the ␦-heme edge in both APX and CcP, although the precise binding orientation varies slightly in the two cases. A second binding site for INH is found in APX at the ␥-heme edge close to the established ascorbate binding site, indicating that the ␥-heme edge can also support the binding of aromatic substrates. We also show that in an active site mutant of soybean APX (W41A) INH can bind directly to the heme iron to become an inhibitor and in a different mode when the distal histidine is replaced by alanine (H42A). These structures provide the first unambiguous evidence for the location of the isoniazid binding site in the class I peroxidases and provide rationalization of isoniazid resistance in naturally occurring KatG mutant strains of M. tuberculosis.

Isonicotinic Acid Hydrazide Conversion to Isonicotinyl-NAD by Catalase-peroxidases

“…For reasons which are not clear, the occupancy of INH, based on the electron density and B-factors of the INH atoms relative to nearby protein segments and overlapping waters, was invariably higher in the B subunit compared with the A subunit. These binding sites are not in the heme cavity as predicted by work with eukaryotic peroxidases (43,44), but rather they are found at the end of a funnel shaped channel (27) on the opposite side of the subunit from the heme entrance channel. A change in the protein side chain location of Glu 198 , relative to the native structure, creates a cavity that is occupied by a chloride ion adjacent to which INH binds (Fig.…”

Neutron cryo-crystallography captures the protonation state of ferryl heme in a peroxidase

“…The original MKT variant of CcP (35) was a gift from Professor Grant Mauk (University of British Columbia) and was cloned into the expression plasmid pLEICS-03 carrying kanamycin resistance and a TEV-cleavable His-Tag sequence and expressed in BL21 DE3 Gold. The protein was purified and crystallised at pH 6.0 according to published methods (36,37). We have used deuterated CcP crystals in which labile hydrogen atoms are exchanged for deuterium atoms to enhance their visibility in nuclear scattering density maps.…”

Oxidised dextrans influence on reactive oxygen species generation by murine peritoneal exudate phagocytic cells

“…На это указывают данные об индукции окислительного стресса изониазидом в условиях in vitro [24] и in vivo [25], а также обнаружение изопропильного радикала при перфузии изониазидом печени мышей [26]. Ферменты, принимающие участие в метаболизме этого лекарственного средства в клетках млекопитающих, до настоящего времени не идентифицированы, однако известно о способности активных центров некоторых пероксидаз (отличных от KatG) связывать изониазид [27]. Можно предположить, что при некоторых условиях может происходить активация изониазида и в макрофагах, необходимым условием которой является усиление внутриклеточной продукции АФК, наблюдаемой, в частности, при стимуляции фагоцитов окисленными декстранами.…”