Structural insights into the mechanism of nuclease A, a betabeta alpha metal nuclease from Anabaena.
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 delta(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 betabeta alpha metal finger motif and a hydrated Mn2+ ion at the active site. Unexpectedly, NucA was found to contain additional metal binding sites approximately 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 betabeta alpha 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.
Ghosh
Mahua
M
Laboratory of Structural Biology, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709, USA.
Meiss
Gregor
G
Pingoud
Alfred
A
London
Robert E
RE
Pedersen
Lars C
LC
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D017033:Anabaena / Q000201:enzymology*; D001120:Arginine / Q000737:chemistry; D001665:Binding Sites; D002384:Catalysis; D020134:Catalytic Domain; D002412:Cations; D018360:Crystallography, X-Ray; D003545:Cysteine / Q000737:chemistry; D004247:DNA / Q000737:chemistry; D004252:DNA Mutational Analysis; D019281:Dimerization; D004220:Disulfides; D004720:Endonucleases / Q000737:chemistry* / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism; D006639:Histidine / Q000737:chemistry; D006868:Hydrolysis; D007477:Ions; D008345:Manganese / Q000737:chemistry; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010100:Oxygen / Q000737:chemistry; D017510:Protein Folding; D021382:Protein Sorting Signals; D017434:Protein Structure, Tertiary; D012313:RNA / Q000737:chemistry; D011994:Recombinant Proteins / Q000737:chemistry; D017386:Sequence Homology, Amino Acid; D012705:Serratia / Q000378:metabolism; D055672:Static Electricity; D015032:Zinc / Q000737:chemistry
D002412:Cations; D004220:Disulfides; D007477:Ions; D021382:Protein Sorting Signals; D011994:Recombinant Proteins; D008345:Manganese; D006639:Histidine; D012313:RNA; D004247:DNA; D001120:Arginine; D004720:Endonucleases; C085858:sugar-nonspecific nuclease; D015032:Zinc; D003545:Cysteine; D010100:Oxygen