Intrinsic endonucleolytic activity of snake venom phosphodiesterase

Abstract

Snake venoms contain enzymes whose biochemical activities cannot be inferred reliably from sequence or proteomic detection alone. Snake venom phosphodiesterase (svPDE), an extracellular ENPP-family glycoprotein, is generally regarded as a 3′-to-5′ exonuclease, although endonucleolytic activity was reported in early preparations. Whether this activity is intrinsic to a molecularly defined svPDE and how DNA structure influences cleavage remained unresolved. Here, nuclease activity in Protobothrops flavoviridis venom was tracked by sequential chromatography, and LC–MS/MS identified svPDE in active fractions. Comparable activity was detected in a commercial Crotalus atrox PDE I preparation. Purified, sequence- and structure-defined Naja atra svPDE converted covalently closed circular plasmid DNA to open-circular and then linear forms in a concentration-dependent manner; higher concentrations caused extensive degradation. Moreover, N. atra svPDE converted a plasmid containing a single nick at a defined position into linear DNA, and restriction mapping was consistent with complementary-strand cleavage at or near the nick. These results establish endonucleolytic activity as an intrinsic property of svPDE and suggest that its nuclease action may be influenced by structural features associated with DNA topology and strand discontinuities. Thus, svPDE may facilitate double-strand breakage at or near sites of single-strand discontinuity, expanding the biochemical repertoire of this conserved venom enzyme.