Singh Rahul, Wiseman Ben, Deemagarn Taweewat, Donald Lynda J, Duckworth Harry W, Carpena Xavi, Fita Ignacio, Loewen Peter C
Department of Microbiology, University of Manitoba, Winnipeg, Manitoba R3T 2N2, Canada.
J Biol Chem. 2004 Oct 8;279(41):43098-106. doi: 10.1074/jbc.M406374200. Epub 2004 Jul 26.
Catalase-peroxidases (KatG) produced by Burkholderia pseudomallei, Escherichia coli, and Mycobacterium tuberculosis catalyze the oxidation of NADH to form NAD+ and either H2O2 or superoxide radical depending on pH. The NADH oxidase reaction requires molecular oxygen, does not require hydrogen peroxide, is not inhibited by superoxide dismutase or catalase, and has a pH optimum of 8.75, clearly differentiating it from the peroxidase and catalase reactions with pH optima of 5.5 and 6.5, respectively, and from the NADH peroxidase-oxidase reaction of horseradish peroxidase. B. pseudomallei KatG has a relatively high affinity for NADH (Km=12 microm), but the oxidase reaction is slow (kcat=0.54 min(-1)) compared with the peroxidase and catalase reactions. The catalase-peroxidases also catalyze the hydrazinolysis of isonicotinic acid hydrazide (INH) in an oxygen- and H2O2-independent reaction, and KatG-dependent radical generation from a mixture of NADH and INH is two to three times faster than the combined rates of separate reactions with NADH and INH alone. The major products from the coupled reaction, identified by high pressure liquid chromatography fractionation and mass spectrometry, are NAD+ and isonicotinoyl-NAD, the activated form of isoniazid that inhibits mycolic acid synthesis in M. tuberculosis. Isonicotinoyl-NAD synthesis from a mixture of NAD+ and INH is KatG-dependent and is activated by manganese ion. M. tuberculosis KatG catalyzes isonicotinoyl-NAD formation from NAD+ and INH more efficiently than B. pseudomallei KatG.
由类鼻疽伯克霍尔德菌、大肠杆菌和结核分枝杆菌产生的过氧化氢酶-过氧化物酶（KatG）催化NADH氧化形成NAD⁺，并根据pH值生成H₂O₂或超氧自由基。NADH氧化酶反应需要分子氧，不需要过氧化氢，不受超氧化物歧化酶或过氧化氢酶抑制，最适pH值为8.75，这使其与过氧化物酶和过氧化氢酶反应（最适pH值分别为5.5和6.5）以及辣根过氧化物酶的NADH过氧化物酶-氧化酶反应明显区分开来。类鼻疽伯克霍尔德菌KatG对NADH具有相对较高的亲和力（Km = 12微摩尔），但与过氧化物酶和过氧化氢酶反应相比，氧化酶反应较慢（kcat = 0.54分钟⁻¹）。过氧化氢酶-过氧化物酶还在不依赖氧气和H₂O₂的反应中催化异烟肼（INH）的肼解反应，并且由NADH和INH混合物产生的依赖KatG的自由基生成速度比单独与NADH和INH的反应速率之和快两到三倍。通过高压液相色谱分离和质谱鉴定，偶联反应的主要产物是NAD⁺和异烟酰-NAD，异烟肼的活化形式，可抑制结核分枝杆菌中分枝菌酸的合成。由NAD⁺和INH混合物合成异烟酰-NAD依赖于KatG，并由锰离子激活。结核分枝杆菌KatG比类鼻疽伯克霍尔德菌KatG更有效地催化由NAD⁺和INH形成异烟酰-NAD。
