Kaczmarek F S, Zaniewski R P, Gootz T D, Danley D E, Mansour M N, Griffor M, Kamath A V, Cronan M, Mueller J, Sun D, Martin P K, Benton B, McDowell L, Biek D, Schmid M B
Department of Infectious Diseases, Pfizer Central Research, Groton, CT 06340, USA.
J Bacteriol. 2001 May;183(10):3016-24. doi: 10.1128/JB.183.10.3016-3024.2001.
A Staphylococcus aureus mutant conditionally defective in DNA ligase was identified by isolation of complementing plasmid clones that encode the S. aureus ligA gene. Orthologues of the putative S. aureus NAD(+)-dependent DNA ligase could be identified in the genomes of Bacillus stearothermophilus and other gram-positive bacteria and confirmed the presence of four conserved amino acid motifs, including motif I, KXDG with lysine 112, which is believed to be the proposed site of adenylation. DNA sequence comparison of the ligA genes from wild type and temperature-sensitive S. aureus strain NT64 identified a single base alteration that is predicted to result in the amino acid substitution E46G. The S. aureus ligA gene was cloned and overexpressed in Escherichia coli, and the enzyme was purified to near homogeneity. NAD(+)-dependent DNA ligase activity was demonstrated with the purified enzyme by measuring ligation of (32)P-labeled 30-mer and 29-mer oligonucleotides annealed to a complementary strand of DNA. Limited proteolysis of purified S. aureus DNA ligase by thermolysin produced products with apparent molecular masses of 40, 22, and 21 kDa. The fragments were purified and characterized by N-terminal sequencing and mass analysis. The N-terminal fragment (40 kDa) was found to be fully adenylated. A fragment from residues 1 to 315 was expressed as a His-tagged fusion in E. coli and purified for functional analysis. Following deadenylation with nicotinamide mononucleotide, the purified fragment could self-adenylate but lacked detectable DNA binding activity. The 21- and 22-kDa C-terminal fragments, which lacked the last 76 amino acids of the DNA ligase, had no adenylation activity or DNA binding activity. The intact 30-kDa C terminus of the S. aureus LigA protein expressed in E. coli did demonstrate DNA binding activity. These observations suggest that, as in the case with the NAD(+)-dependent DNA ligase from B. stearothermophilus, two independent functional domains exist in S. aureus DNA ligase, consisting of separate adenylation and DNA binding activities. They also demonstrate a role for the extreme C terminus of the ligase in DNA binding. As there is much evidence to suggest that DNA ligase is essential for bacterial survival, its discovery in the important human pathogen S. aureus indicates its potential as a broad-spectrum antibacterial target for the identification of novel antibiotics.
通过分离编码金黄色葡萄球菌连接酶A基因的互补质粒克隆，鉴定出一种DNA连接酶存在条件性缺陷的金黄色葡萄球菌突变体。在嗜热脂肪芽孢杆菌和其他革兰氏阳性菌的基因组中可以鉴定出假定的金黄色葡萄球菌NAD(+)依赖性DNA连接酶的直系同源物，并证实存在四个保守的氨基酸基序，包括基序I，即含有赖氨酸112的KXDG，据信它是腺苷化的位点。对野生型和温度敏感型金黄色葡萄球菌菌株NT64的连接酶A基因进行DNA序列比较，发现一个单碱基改变，预计会导致氨基酸替换E46G。将金黄色葡萄球菌连接酶A基因克隆并在大肠杆菌中过量表达，然后将该酶纯化至接近均一状态。通过测量连接到DNA互补链上的(32)P标记的30聚体和29聚体寡核苷酸的连接情况，用纯化后的酶证实了NAD(+)依赖性DNA连接酶活性。嗜热菌蛋白酶对纯化的金黄色葡萄球菌DNA连接酶进行有限的蛋白水解，产生了表观分子量分别为40 kDa、22 kDa和21 kDa的产物。对这些片段进行纯化，并通过N端测序和质谱分析进行表征。发现N端片段（40 kDa）已完全腺苷化。将1至315位残基的片段在大肠杆菌中表达为His标签融合蛋白，并进行纯化以进行功能分析。用烟酰胺单核苷酸进行去腺苷化后，纯化的片段可以自我腺苷化，但缺乏可检测到的DNA结合活性。缺少DNA连接酶最后76个氨基酸的21 kDa和22 kDa C端片段没有腺苷化活性或DNA结合活性。在大肠杆菌中表达的金黄色葡萄球菌连接酶A蛋白完整的30 kDa C端确实表现出DNA结合活性。这些观察结果表明，与嗜热脂肪芽孢杆菌的NAD(+)依赖性DNA连接酶一样，金黄色葡萄球菌DNA连接酶中存在两个独立的功能域，分别由腺苷化和DNA结合活性组成。它们还证明了连接酶极端C端在DNA结合中的作用。由于有许多证据表明DNA连接酶对细菌生存至关重要, 因此在重要的人类病原体金黄色葡萄球菌中发现它表明其作为鉴定新型抗生素的广谱抗菌靶点的潜力。
