Structural Insights into the Mechanism of Nuclease A, a ββα Metal Nuclease from Anabaena

Alfred Pingoud,Mahua Ghosh,Lars C Pedersen,Gregor Meiss,Robert E London

doi:10.1074/jbc.m501798200

Abstract

Nuclease A (NucA) is a nonspecific endonuclease from Anabaena sp. capable of degrading single- and double-stranded DNA and RNA in the presence of divalent metal ions. We have determined the structure of the Δ(2-24),D121A mutant of NucA in the presence of Zn2+ and Mn2+ (PDB code 1ZM8). The mutations were introduced to remove the N-terminal signal peptide and to reduce the activity of the nonspecific nuclease, thereby reducing its toxicity to the Escherichia coli expression system. NucA contains a ββα metal finger motif and a hydrated Mn2+ ion at the active site. Unexpectedly, NucA was found to contain additional metal binding sites ∼26 A apart from the catalytic metal binding site. A structural comparison between NucA and the closest analog for which structural data exist, the Serratia nuclease, indicates several interesting differences. First, NucA is a monomer rather than a dimer. Second, there is an unexpected structural homology between the N-terminal segments despite a poorly conserved sequence, which in Serratia includes a cysteine bridge thought to play a regulatory role. In addition, although a sequence alignment had suggested that NucA lacks a proposed catalytic residue corresponding to Arg57 in Serratia, the structure determined here indicates that Arg93 in NucA is positioned to fulfill this role. Based on comparison with DNA-bound nuclease structures of the ββα metal finger nuclease family and available mutational data on NucA, we propose that His124 acts as a catalytic base, and Arg93 participates in the catalysis possibly through stabilization of the transition state.

Highlights

Nuclease A (NucA) is a nonspecific endonuclease from Anabaena sp. capable of degrading single- and doublestranded DNA and RNA in the presence of divalent metal ions
Based on comparison with DNA-bound nuclease structures of the ␤␤␣ metal finger nuclease family and available mutational data on NucA, we propose that His124 acts as a catalytic base, and Arg93 participates in the catalysis possibly through stabilization of the transition state
Structure of the Active Site in the Manganese-substituted Enzyme—To obtain a structure of NucA with Mn2ϩ in the active site, crystals grown in the presence of Zn2ϩ were transferred into solution containing no Zn2ϩ and only Mn2ϩ as a divalent cation

Summary

Introduction

Nuclease A (NucA) is a nonspecific endonuclease from Anabaena sp. capable of degrading single- and doublestranded DNA and RNA in the presence of divalent metal ions. Based on comparison with DNA-bound nuclease structures of the ␤␤␣ metal finger nuclease family and available mutational data on NucA, we propose that His124 acts as a catalytic base, and Arg participates in the catalysis possibly through stabilization of the transition state. The best characterized of these is the Serratia nuclease [7], a homodimer (8 –11) that contains two disulfide bonds per subunit [12] and is characterized by an unusual Mg2ϩ binding site optimized for binding a hydrated metal ion [13, 14]. If the cysteine residues in the N-terminal signal peptide are not included, there is only one additional cysteine in NucA, and it is not anticipated to contain disulfide bonds, which could prevent the nuclease from being active in the cell. The three-dimensional structure of NuiA has recently been determined [20]

Methods

Results

Discussion

Conclusion