Regulation of Catalysis by the Smallpox Virus Topoisomerase

Young Hwang,Nana Minkah,Kay Perry,Gregory D Van Duyne,Frederic D Bushman

doi:10.1074/jbc.m608858200

Abstract

The poxvirus type IB topoisomerases catalyze relaxation of supercoiled DNA by cleaving and rejoining DNA strands via a pathway involving a covalent phosphotyrosine intermediate. Recently we determined structures of the smallpox virus topoisomerase bound to DNA in covalent and non-covalent DNA complexes using x-ray crystallography. Here we analyzed the effects of twenty-two amino acid substitutions on the topoisomerase activity in vitro in assays of DNA relaxation, single cycle cleavage, and equilibrium cleavage-religation. Alanine substitutions at 14 positions impaired topoisomerase function, marking a channel of functionally important contacts along the protein-DNA interface. Unexpectedly, alanine substitutions at two positions (D168A and E124A) accelerated the forward rate of cleavage. These findings and further analysis indicate that Asp(168) is a key regulator of the active site that maintains an optimal balance among the DNA cleavage, religation, and product release steps. Finally, we report that high level expression of the D168A topoisomerase in Escherichia coli, but not other alanine-substituted enzymes, prevented cell growth. These findings help elucidate the amino acid side chains involved in DNA binding and catalysis and provide guidance for designing topoisomerase poisons for use as smallpox antivirals.

Highlights

All poxviruses encode topoisomerases of the type IB family (1, 2)
Very extensive mutagenesis of the vaccinia topoisomerase has identified residues involved in catalysis and additional residues important for activation of cleavage after DNA binding (4, 6, 15–21)
Comparison of the vaccinia amino and carboxyl domain structures determined in the absence of DNA (23, 25) to the smallpox-topoisomerase/DNA complex (22) allowed the conformational changes that mediate activation to be visualized

Summary

Ϯ 2a Ͼ1440a

9Ϯ1 Ͼ1440a a Indicates previously tested (Ref. 22). 17 Ϯ 6 17 Ϯ 1 1.8 Ϯ 0.1 1.9 Ϯ 1.0 0.023 Ϯ 0.018 0.029 Ϯ 0.023 0.087 Ϯ 0.062 9.7 Ϯ 1.60 1.05 Ϯ 0.07 0.0041. 0.0025 0.98 active site residues (Arg130, Lys167, Arg223, His265, Tyr274), which make a network of contacts involving residue ϩ1 and the scissile phosphate (22). The reaction is catalyzed by a combination of acid-base catalysis, in which the attacking nucleophile is deprotonated and the leaving group protonated, and electrostatic stabilization, in which the developing negative charge at the scissile phosphate is stabilized by basic side chains at the active site We present a study of amino acid side chains of the smallpox topoisomerase that were suggested by the structural analysis to have a functional role. This work revealed a network of new contacts important for enzyme function, and unexpectedly disclosed amino acid substitutions that increased the apparent rate of cleavage of suicide substrates in vitro

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION