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J. Biol. Chem., Vol. 280, Issue 30, 27990-27997, July 29, 2005

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

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

From the Laboratory of Structural Biology, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709 and the Institut für Biochemie, FB08, Justus-Liebig-Universität, Heinrich-Buff-Ring 58, D-35392 Giessen, Germany

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 (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 metal finger motif and a hydrated Mn²⁺ ion at the active site. Unexpectedly, NucA was found to contain additional metal binding sites 26 Å 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 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.

Received for publication, February 17, 2005 , and in revised form, May 12, 2005.

The atomic coordinates and structure factors (code 1ZM8) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

^* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^¶ To whom correspondence should be addressed: Laboratory of Structural Biology, NIEHS, MR-01, 111 Alexnder Dr., Box 12233, Research Triangle Park, NC. Tel.: 919-541-4879; Fax: 919-541-5707; E-mail: london{at}niehs.nih.gov.

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