Emptage Caroline D, Knox Richard J, Danson Michael J, Hough David W
Centre for Extremophile Research, Department of Biology & Biochemistry, University of Bath, Bath BA2 7AY, UK.
Biochem Pharmacol. 2009 Jan 1;77(1):21-9. doi: 10.1016/j.bcp.2008.09.010. Epub 2008 Sep 17.
5-aziridinyl-2,4-dinitrobenzamide (CB1954) has potential applications in enzyme/prodrug targeted anti-cancer therapies since it can be activated by nitroreductases to form a cytotoxic, bifunctional hydroxylamine derivative. A nitroreductase that can activate CB1954 has been previously isolated from Escherichia coli, but its usefulness is limited by its poor stability and low catalytic efficiency for CB1954. We now report the identification and characterization of a nitroreductase enzyme from the thermophilic bacterium Bacillus licheniformis. Although there is only 28% amino acid sequence identity between this enzyme and the previously isolated E. coli nitroreductase, the two enzymes have a number of characteristics in common. Both enzymes have been shown to reduce both CB1954 and menadione in the presence of NADH and NADPH. However, whereas E. coli nitroreductase produces equimolar amounts of the 2- and 4- hydroxylamine derivative of CB1954, the B. licheniformis enzyme produces only the desired 4-hydroxylamine derivative. It has a preference for NADPH as cosubstrate, and is also active with a range of CB1954 derivatives as substrate and reduced pyridinium cofactor analogues. Moreover, the enzyme is much more thermostable than the E. coli nitroreductase and shows maximum activity at 30 degrees C. These characteristics suggest that the B. licheniformis nitroreductase may be a possible candidate enzyme for enzyme/prodrug therapies due to its bacterial origin, the high activity observed with CB1954 and its enhanced stability.
5-氮杂环丙基-2,4-二硝基苯甲酰胺（CB1954）在酶/前药靶向抗癌治疗中具有潜在应用，因为它可被硝基还原酶激活，形成具有细胞毒性的双功能羟胺衍生物。此前已从大肠杆菌中分离出一种能激活CB1954的硝基还原酶，但其稳定性差且对CB1954的催化效率低，限制了其用途。我们现在报告从嗜热细菌地衣芽孢杆菌中鉴定和表征一种硝基还原酶。尽管该酶与先前分离的大肠杆菌硝基还原酶之间只有28%的氨基酸序列同一性，但这两种酶有许多共同特征。两种酶在NADH和NADPH存在下均能还原CB1954和甲萘醌。然而，大肠杆菌硝基还原酶产生等摩尔量的CB1954的2-和4-羟胺衍生物，而地衣芽孢杆菌的酶只产生所需的4-羟胺衍生物。它更倾向于以NADPH作为辅酶，并且对一系列CB1954衍生物作为底物以及还原型吡啶辅酶类似物也有活性。此外，该酶比大肠杆菌硝基还原酶热稳定性高得多，在30℃时显示出最大活性。这些特性表明，由于其细菌来源、对CB1954观察到的高活性以及增强的稳定性，地衣芽孢杆菌硝基还原酶可能是酶/前药疗法的一种可能的候选酶。
