Cofactor requirement of HpyAV restriction endonuclease

Abstract

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.

Principal findings: 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/significance: 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.

Figure 1. Gene organization of the HpyAV/Hin4II R-M systems and the structural model of the HpyAV catalytic site.

A. Organization of the HpyAV and Hin4II R-M systems. The Hin4II R-M system consists of separate M1 and M2 genes for C5 cytosine (dark grey) and N6 adenine (light grey) methylation, respectively, preceding the ORF of Hin4II . M.HpyAV is a fusion of C5 and N6A MTase domains with high sequence similarity to M1.Hin4II and M2.Hin4II, respectively. B. The structural model of HpyAV catalytic site and structural alignment to HNH endonucleases. Modeling of amino acid residues 281 to 360 of HpyAV to the ββα-Me motif of ColE9 and the structural alignment to ColE9, I-HmuI, Hpy99I and KpnI were done as described in Materials and Methods. The blue dots underneath the alignment indicate metal-binding residues; the red dots indicate the general base His and the green dot indicates the Asn implied to decrease the pKa of the general base His in ColE9 and I-HmuI. Amino acid residues that are assigned to β-strands and the helix of the ββα-Me motif are indicated by red arrows and blue rods, respectively. The conserved residues implicated in catalysis are colored in black or grey.

Figure 2. HpyAV endonuclease activity in buffers with various divalent cations.

A. Cleavage activity of HpyAV in MgSO₄ and NiSO₄. Decreasing concentrations of HpyAV were added to reactions containing 1 µg of λ DNA, 20 mM Tris-HCl, pH 7.9, 200 mM NaCl supplemented with 2 mM of NiSO₄, 4 mM of MgSO₄, 2 mM of NiSO₄ and 4 mM of MgSO₄ or no divalent metal ions. The reactions were carried out as described in Materials and Methods. The reactions that exhibit 1 U of HpyAV activity (complete cleavage) are marked with a dot. B. DNA cleavage activity of HpyAV in buffers supplemented with the indicated concentration of metal ion solutions. Eight units of HpyAV were incubated with 1 µg of λ DNA in 20 mM Tris-HCl, 200 mM NaCl as described in Materials and Methods.

Figure 3. DNA cleavage activity of the catalytic residue mutants.

Purified protein of WT, H294D, H295A and H320A were assayed as described in Materials and Methods in the presence of 2 mM NiSO₄. Five μl of undiluted, three-fold and nine-fold dilutions of 1.5 mg/ml of enzyme solution were assayed on 1 µg of λ DNA.

Figure 4. DNA cleavage activity of KpnI and EcoRI in the presence of different metal ions.

Ten units of KpnI or EcoRI were incubated with 1 µg of pXba or λ DNA, respectively, as described in Materials and Methods.