Cofactor requirement of HpyAV restriction endonuclease.
BACKGROUND
Helicobacter pylori is the etiologic agent of common gastritis and a risk factor for gastric cancer. It is also one of the richest sources of Type II restriction-modification (R-M) systems in microorganisms.


RESULTS
We have cloned, expressed and purified a new restriction endonuclease HpyAV from H. pylori strain 26695. We determined the HpyAV DNA recognition sequence and cleavage site as CCTTC 6/5. In addition, we found that HpyAV has a unique metal ion requirement: its cleavage activity is higher with transition metal ions than in Mg(++). The special metal ion requirement of HpyAV can be attributed to the presence of a HNH catalytic site similar to ColE9 nuclease instead of the canonical PD-X-D/EXK catalytic site found in many other REases. Site-directed mutagenesis was carried out to verify the catalytic residues of HpyAV. Mutation of the conserved metal-binding Asn311 and His320 to alanine eliminated cleavage activity. HpyAV variant H295A displayed approximately 1% of wt activity.


CONCLUSIONS
Some HNH-type endonucleases have unique metal ion cofactor requirement for optimal activities. Homology modeling and site-directed mutagenesis confirmed that HpyAV is a member of the HNH nuclease family. The identification of catalytic residues in HpyAV paved the way for further engineering of the metal binding site. A survey of sequenced microbial genomes uncovered 10 putative R-M systems that show high sequence similarity to the HpyAV system, suggesting lateral transfer of a prototypic HpyAV-like R-M system among these microorganisms.
Research Department, New England Biolabs, Inc., Ipswich, Massachusetts, United States of America.
Siu-Hong
SH
Chan
Lars
L
Opitz
Lauren
L
Higgins
Diana
D
O'loane
Shuang-Yong
SY
Xu
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D020134:Catalytic Domain / Q000235:genetics; D002413:Cations, Divalent / Q000737:chemistry / Q000378:metabolism; D003035:Cobalt / Q000737:chemistry / Q000378:metabolism; D004247:DNA / Q000235:genetics / Q000378:metabolism; D004262:DNA Restriction Enzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015280:DNA Restriction-Modification Enzymes / Q000235:genetics / Q000378:metabolism; D016480:Helicobacter pylori / Q000201:enzymology* / Q000235:genetics; D008345:Manganese / Q000737:chemistry / Q000378:metabolism; D008670:Metals / Q000737:chemistry / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009532:Nickel / Q000737:chemistry / Q000378:metabolism; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid
D001426:Bacterial Proteins; D002413:Cations, Divalent; D015280:DNA Restriction-Modification Enzymes; D008670:Metals; D003035:Cobalt; D008345:Manganese; D009532:Nickel; D004247:DNA; D004262:DNA Restriction Enzymes