Cofactor requirement of HpyAV restriction endonuclease

• Published in: PloS one Vol. 5; no. 2; p. e9071
• Main Authors: Chan, Siu-Hong, Opitz, Lars, Higgins, Lauren, O'loane, Diana, Xu, Shuang-Yong
• Format: Journal Article
• Language: English
• Published: United States Public Library of Science 05.02.2010; Public Library of Science (PLoS)
• Subjects: Alanine; Amino Acid Sequence; Amino acids; Bacterial Proteins - chemistry; Bacterial Proteins - genetics; Bacterial Proteins - metabolism; Base Sequence; Binding sites; Binding Sites - genetics; Biochemistry/Biocatalysis; Bioinformatics; Biotechnology/Applied Microbiology; Biotechnology/Protein Chemistry and Proteomics; Catalysis; Catalytic Domain - genetics; Cations, Divalent - chemistry; Cations, Divalent - metabolism; Cleavage; Cloning; Cobalt - chemistry; Cobalt - metabolism; Deoxyribonucleic acid; DNA; DNA - genetics; DNA - metabolism; DNA methylation; DNA Restriction Enzymes - chemistry; DNA Restriction Enzymes - genetics; DNA Restriction Enzymes - metabolism; DNA Restriction-Modification Enzymes - genetics; DNA Restriction-Modification Enzymes - metabolism; Ductile-brittle transition; E coli; Endonuclease; Enzymes; Escherichia coli; Etiology; Fracture mechanics; Gastric cancer; Gastritis; Genes; Genomes; Genomics; Haemophilus influenzae; Health aspects; Health risks; Helicobacter pylori; Helicobacter pylori - enzymology; Helicobacter pylori - genetics; Homology; Mammals; Manganese - chemistry; Manganese - metabolism; Metal ions; Metals - chemistry; Metals - metabolism; Microbiology/Applied Microbiology; Microbiology/Microbial Evolution and Genomics; Microorganisms; Models, Molecular; Molecular Biology/DNA Methylation; Molecular Sequence Data; Mutagenesis; Mutagenesis, Site-Directed; Mutation; Next-generation sequencing; Nickel - chemistry; Nickel - metabolism; Nuclease; Nucleases; Nucleotide sequence; Protein Structure, Tertiary; Proteins; Residues; Restriction-modification; Risk factors; Sequence Homology, Amino Acid; Site-directed mutagenesis; Stomach cancer; United States > US; Massachusetts
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0009071

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. 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. 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.