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 i ncludes enzymes involved in phospholipid metabolism, a bacterial toxin, pox virus envelope proteins, and bacterial nucleases. Based on sequence compari sons, we show here that the tyrosyl-DNA phosphodiesterase (Tdp1) that has b een implicated in the repair of topoisomerases I covalent complexes with DN A contains two unusual HKD signature motifs that place the enzyme in a dist inct class within the PLD superfamily. Mutagenesis studies with the human e nzyme in which the invariant histidines and lysines of the HKD motifs are c hanged confirm that these highly conserved residues are essential for Tdp1 activity. Furthermore, we show that, like other members of the family for w hich it has been examined, the reaction involves the formation of an interm ediate in which the cleaved substrate is covalently linked to the enzyme. T hese results reveal that the hydrolytic reaction catalyzed by Tdp1 occurs b y the phosphoryl transfer chemistry that is common to all members of the PL D superfamily.