Abstract

Mycobacterium tuberculosis cells contain two apurinic/apyrimidinic (AP) endonucleases, endonuclease IV (MtbEnd) and exonuclease III (MtbXthA), the former playing a dominant role in protecting mycobacterial DNA from oxidative stress. Mycobacterial endonuclease IV substantially differs from its homologs found in Escherichia coli and other proteobacteria in a number of conserved positions important for DNA binding and AP site recognition. The M. tuberculosis end gene was cloned, and recombinant MtbEnd purified and characterized. The protein efficiently hydrolyzed DNA at the natural AP site and its 1′-deoxy analog in the presence of divalent cations, of which Ca²⁺, Mn²⁺, and Co²⁺ supported the highest activity. Exonuclease activity was not detected in MtbEnt preparations. The pH optimum was estimated at 7.0–8.0; the ionic strength optimum, at ~50 mM NaCl. Enzymatic activity of MtbEnd was suppressed in the presence of methoxyamine, a chemotherapeutic agent that modifies AP sites. Based on the results, MtbEnd was assumed to provide a possible target for new anti-tuberculosis drugs.

中文翻译：

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结核分枝杆菌重组核酸内切酶IV的表征

摘要

结核分枝杆菌细胞包含两种嘌呤/双嘧啶（AP）核酸内切酶，核酸内切酶IV（MtbEnd）和核酸外切酶III（MtbXthA），前者在保护分枝杆菌DNA免受氧化应激中起主要作用。分枝杆菌内切核酸酶IV与大肠杆菌和其他变形杆菌中发现的同源物在许多对DNA结合和AP位点识别很重要的保守位置上有很大不同。在结核分枝杆菌端基因克隆和重组MtbEnd纯化和表征。该蛋白质在存在二价阳离子的情况下有效水解天然AP位点的DNA及其1'-脱氧类似物，其中Ca 2 ⁺，Mn ²⁺和Co ²⁺支持最高的活动。在MtbEnt制剂中未检测到核酸外切酶活性。最适pH值估计为7.0-8.0。最佳离子强度，约为〜50 mM NaCl。在甲氧基胺（一种修饰AP位点的化学治疗剂）的存在下，MtbEnd的酶活性被抑制。根据结果​​，假定MtbEnd为新的抗结核药物提供了可能的靶标。