Identification of two conserved aspartic acid residues required for DNA digestion by a novel thermophilic Exonuclease VII in Thermotoga maritima.
Exonuclease VII was first identified in 1974 as a DNA exonuclease that did not require any divalent cations for activity. Indeed, Escherichia coli ExoVII was identified in partially purified extracts in the presence of EDTA. ExoVII is comprised of two subunits (XseA and XseB) that are highly conserved and present in most sequenced prokaryotic genomes, but are not seen in eukaryotes. To better understand this exonuclease family, we have characterized an ExoVII homolog from Thermotoga maritima. Thermotoga maritima XseA/B homologs TM1768 and TM1769 were co-expressed and purified, and show robust nuclease activity at 80 degrees C. This activity is magnesium dependent and is inhibited by phosphate ions, which distinguish it from E. coli ExoVII. Nevertheless, both E. coli and T. maritima ExoVII share a similar putative active site motif with two conserved aspartate residues in the large (XseA/TM1768) subunit. We show that these residues, Asp235 and Asp240, are essential for the nuclease activity of T. maritima ExoVII. We hypothesize that the ExoVII family of nucleases can be sub-divided into two sub-families based on EDTA resistance and that T. maritima ExoVII is the first member of the branch that is characterized by EDTA sensitivity and inhibition by phosphate.
Larrea
Andres A
AA
Department of Biochemistry and Molecular Biology, University of Miami Miller School of Medicine, Miami, FL 33136, USA.
Pedroso
Ilene M
IM
Malhotra
Arun
A
Myers
Richard S
RS
D001224:Aspartic Acid / Q000737:chemistry*; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D020134:Catalytic Domain; D004277:DNA, Single-Stranded / Q000737:chemistry / Q000378:metabolism; D005090:Exodeoxyribonucleases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D023281:Genomics; D007700:Kinetics; D008274:Magnesium / Q000737:chemistry; D009841:Oligonucleotides / Q000032:analysis; D010710:Phosphates / Q000737:chemistry; D021122:Protein Subunits / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013431:Sulfates / Q000737:chemistry; D020124:Thermotoga maritima / Q000201:enzymology* / Q000235:genetics
D001426:Bacterial Proteins; D004277:DNA, Single-Stranded; D009841:Oligonucleotides; D010710:Phosphates; D021122:Protein Subunits; D013431:Sulfates; D001224:Aspartic Acid; D005090:Exodeoxyribonucleases; C026870:exodeoxyribonuclease VII; D008274:Magnesium