The tyrosyl-DNA phosphodiesterase Tdp1 is a member of the phospholipase D superfamily

The phospholipase D (PLD) superfamily is a diverse group of proteins that includes enzymes involved in phospholipid metabolism, a bacterial toxin, poxvirus envelope proteins, and bacterial nucleases. Based on sequence comparisons, we show here that the tyrosyl-DNA phosphodiesterase (Tdp1) that has been implicated in the repair of topoisomerase I covalent complexes with DNA contains two unusual HKD signature motifs that place the enzyme in a distinct class within the PLD superfamily. Mutagenesis studies with the human enzyme in which the invariant histidines and lysines of the HKD motifs are changed confirm that these highly conserved residues are essential for Tdp1 activity. Furthermore, we show that, like other members of the family for which it has been examined, the reaction involves the formation of an intermediate in which the cleaved substrate is covalently linked to the enzyme. These results reveal that the hydrolytic reaction catalyzed by Tdp1 occurs by the phosphoryl transfer chemistry that is common to all members of the PLD superfamily.

磷脂酶D（phospholipase D, PLD）超家族是一类多样化的蛋白质家族，涵盖参与磷脂代谢的酶、一种细菌毒素、痘病毒包膜蛋白以及细菌核酸酶。基于序列比对分析，本研究证实，此前被认为参与DNA拓扑异构酶I与DNA形成的共价复合物修复的酪氨酰-DNA磷酸二酯酶（tyrosyl-DNA phosphodiesterase, Tdp1），包含两个特殊的HKD特征基序，这使其归属于PLD超家族中一个独特的分支。针对人类Tdp1开展的诱变实验中，研究人员对HKD基序中保守的组氨酸与赖氨酸残基进行突变，结果证实这些高度保守的残基对Tdp1的酶活性至关重要。此外，正如该超家族其他已被研究的成员一样，本研究证实其催化反应会形成一类中间产物：裂解后的底物与酶以共价键相连。上述研究结果表明，Tdp1所催化的水解反应，是通过PLD超家族所有成员共有的磷酸转移化学机制完成的。