St Maurice Martin, Cremades Nunilo, Croxen Matthew A, Sisson Gary, Sancho Javier, Hoffman Paul S
Department of Medicine, Division of Infectious Diseases, University of Virginia Health Systems, 409 Lane Road, Charlottesville, VA 22908, USA.
J Bacteriol. 2007 Jul;189(13):4764-73. doi: 10.1128/JB.00287-07. Epub 2007 Apr 27.
Pyruvate-dependent reduction of NADP has been demonstrated in cell extracts of the human gastric pathogen Helicobacter pylori. However, NADP is not a substrate of purified pyruvate:ferredoxin oxidoreductase (PFOR), suggesting that other redox active enzymes mediate this reaction. Here we show that fqrB (HP1164), which is essential and highly conserved among the epsilonproteobacteria, exhibits NADPH oxidoreductase activity. FqrB was purified by nickel interaction chromatography following overexpression in Escherichia coli. The protein contained flavin adenine dinucleotide and exhibited NADPH quinone reductase activity with menadione or benzoquinone and weak activity with cytochrome c, molecular oxygen, and 5,5'-dithio-bis-2-nitrobenzoic acid (DTNB). FqrB exhibited a ping-pong catalytic mechanism, a k(cat) of 122 s(-1), and an apparent K(m) of 14 muM for menadione and 26 muM for NADPH. FqrB also reduced flavodoxin (FldA), the electron carrier of PFOR. In coupled enzyme assays with purified PFOR and FldA, FqrB reduced NADP in a pyruvate- and reduced coenzyme A (CoA)-dependent manner. Moreover, in the presence of NADPH, CO(2), and acetyl-CoA, the PFOR:FldA:FqrB complex generated pyruvate via CO(2) fixation. PFOR was the rate-limiting enzyme in the complex, and nitazoxanide, a specific inhibitor of PFOR of H. pylori and Campylobacter jejuni, also inhibited NADP reduction in cell-free lysates. These capnophilic (CO(2)-requiring) organisms contain gaps in pathways of central metabolism that would benefit substantially from pyruvate formation via CO(2) fixation. Thus, FqrB provides a novel function in pyruvate metabolism and, together with production of superoxide anions via quinone reduction under high oxygen tensions, contributes to the unique microaerobic lifestyle that defines the epsilonproteobacterial group.
已在人类胃部病原体幽门螺杆菌的细胞提取物中证实了丙酮酸依赖的NADP还原作用。然而，NADP并非纯化的丙酮酸：铁氧化还原蛋白氧化还原酶（PFOR）的底物，这表明其他氧化还原活性酶介导了此反应。在此，我们表明fqrB（HP1164）在ε-变形菌中是必需且高度保守的，它具有NADPH氧化还原酶活性。在大肠杆菌中过表达后，通过镍亲和色谱法纯化了FqrB。该蛋白质含有黄素腺嘌呤二核苷酸，对甲萘醌或苯醌表现出NADPH醌还原酶活性，而对细胞色素c、分子氧和5,5'-二硫代双-2-硝基苯甲酸（DTNB）表现出较弱活性。FqrB表现出乒乓催化机制，对甲萘醌的k(cat)为122 s(-1)，表观K(m)为14 μM，对NADPH的表观K(m)为26 μM。FqrB还还原了PFOR的电子载体黄素氧还蛋白（FldA）。在与纯化的PFOR和FldA进行的偶联酶测定中，FqrB以丙酮酸和还原型辅酶A（CoA）依赖的方式还原NADP。此外，在存在NADPH、CO₂和乙酰辅酶A的情况下，PFOR:FldA:FqrB复合物通过CO₂固定生成丙酮酸。PFOR是该复合物中的限速酶，硝唑尼特，一种幽门螺杆菌和空肠弯曲菌PFOR的特异性抑制剂，也抑制无细胞裂解物中的NADP还原。这些嗜二氧化碳菌在中心代谢途径中存在缺口，通过CO₂固定形成丙酮酸将使其受益匪浅。因此，FqrB在丙酮酸代谢中提供了一种新功能，并且与在高氧张力下通过醌还原产生超氧阴离子一起，促成了定义ε-变形菌群体的独特微需氧生活方式。
