Functional analysis of an acid adaptive DNA adenine methyltransferase from Helicobacter pylori 26695

Abstract

HP0593 DNA-(N(6)-adenine)-methyltransferase (HP0593 MTase) is a member of a Type III restriction-modification system in Helicobacter pylori strain 26695. HP0593 MTase has been cloned, overexpressed and purified heterologously in Escherichia coli. The recognition sequence of the purified MTase was determined as 5'-GCAG-3'and the site of methylation was found to be adenine. The activity of HP0593 MTase was found to be optimal at pH 5.5. This is a unique property in context of natural adaptation of H. pylori in its acidic niche. Dot-blot assay using antibodies that react specifically with DNA containing m6A modification confirmed that HP0593 MTase is an adenine-specific MTase. HP0593 MTase occurred as both monomer and dimer in solution as determined by gel-filtration chromatography and chemical-crosslinking studies. The nonlinear dependence of methylation activity on enzyme concentration indicated that more than one molecule of enzyme was required for its activity. Analysis of initial velocity with AdoMet as a substrate showed that two molecules of AdoMet bind to HP0593 MTase, which is the first example in case of Type III MTases. Interestingly, metal ion cofactors such as Co(2+), Mn(2+), and also Mg(2+) stimulated the HP0593 MTase activity. Preincubation and isotope partitioning analyses clearly indicated that HP0593 MTase-DNA complex is catalytically competent, and suggested that DNA binds to the MTase first followed by AdoMet. HP0593 MTase shows a distributive mechanism of methylation on DNA having more than one recognition site. Considering the occurrence of GCAG sequence in the potential promoter regions of physiologically important genes in H. pylori, our results provide impetus for exploring the role of this DNA MTase in the cellular processes of H. pylori.

Figure 1. Multiple sequence alignment between HP0593, EcoP1I and EcoP15I MTases.

(*) represents sequence identity and (:) represents sequence similarity. The differences exist in the target recognition domain (TRD), which is shown by a box. The amino acid sequences of conserved motifs (motif IV and motif I) are boxed.

Figure 2. Characterization of (His)₆-HP0593 recombinant protein.

A. Two-Dimensional PAGE analysis of (His)₆-HP0593 recombinant protein. The arrow indicates HP0593 protein, which migrates at a pI of ∼5.8. B. MALDI-TOF-MS analysis of tryptic digested pH 5.8 protein band. The matching peptide ion fragments are shown by arrows marked.

Figure 3. Sequence specificity of HP0593 MTase.

A. Restriction digestion pattern of pET14b-hp0593 plasmid DNA. Lane 1, DNA alone, lane 2, DNA + R.PstI; lane 3, DNA + R.PvuII; lane 4, DNA + R.HpyCH4V; lane 5, DNA + R.AluI; lane 6, 1.0 kb DNA ladder. B. Vector DNA methylation. Methylation reaction was carried out in presence of 2.5 µM of site (5’-GCAG-3’) concentration of pET14b DNA or pET14b-hp0593 plasmid DNA or duplex 2 DNA, 150 nM HP0593 MTase and 1.5 µM [methyl-³H] AdoMet in methylation buffer at 37°C for 10 min. 1  =  pET14b DNA, 2  =  pET14b-hp0593 plasmid DNA and 3  =  duplex 2 DNA (positive control). C. Methylation activity of HP0593 MTase in presence of duplex DNAs of varying length (25 bp to 41 bp). Duplexes 1–4 contain 5’-GCAG-3’ sequence, duplexes 5 and 6 contain 5’-ACAG-3’ and 5’-GCTG-3’ sequences, respectively. This experiment was done in duplicates.

Figure 4. Determination of methylation activity.

A. Dot blot assay. Lanes 1 and 2, HP0593 MTase methylated duplex 2 (duplicates); lane 3, unmethylated duplex 2. B. Characterization of HP0593 MTase C54G and Y107G mutants. Increasing concentrations of wild-type or Y107G or C54G mutants of HP0593 MTase (70–500 nM) were incubated with 5.0 µM duplex 2 and 1.5 µM AdoMet in methylation buffer at 37°C for 10 min. The reactions were stopped and analyzed as described in the materials and methods. (•) wild type, (○) C54G, (▴) Y107G.

Figure 5. Binding of substrates to HP0593 MTase.

A. AdoMet binding to HP0593 MTase: Modified Stern-Volmer plot for determining the K _SV AdoMet. B. Electrophoretic mobility shift assay: Binding of HP0593 MTase to duplex 2 DNA in presence of AdoHcy. Lane 1; radiolabeled duplex 2, lanes 2–9, increasing concentrations of HP0593 MTase (0.5 µM–5.0 µM) were incubated with 5’ [γ-³²P] end-labeled duplex 2 (approximately 100 nM) in methylation buffer on ice for 10 min in presence of 5 µM AdoHcy and analyzed as described in materials and methods; lanes 10–13, chased with excess (5, 10, 15, and 20-fold, respectively) of unlabeled DNA (duplex 2).

Figure 6. Kinetics of methylation.

A. Determination of pH optima. Methylation activity of purified HP0593 MTase (50 nM) was measured in buffers with a pH range of 3.5 to 8.5 containing 2.5 µM of duplex 2, 1.5 µM [methyl-³H] AdoMet at 37°C for 10 min. Methylation activities were quantified by a filter-binding assay as described in materials and methods. B. Initial velocity versus square of HP0593 MTase concentration. Methylation assays were carried out in a reaction mixture containing 5 µM of duplex 2 DNA, 1.5 µM [methyl-³H] AdoMet and increasing concentrations of HP0593 MTase (20–300 nM) in methylation buffer at 37°C for 10 min. The samples were analyzed as described in materials and methods. Initial velocity data were plotted against the square of HP0593 MTase concentrations. C. Initial velocity vs. AdoMet concentration. Methylation assays were carried out in reactions containing 5 µM of duplex 2 DNA and increasing concentrations of [³H] AdoMet (0.25–4.0 µM) in methylation buffer at 37°C for 10 min. HP0593 MTase (150 nM) was added to start the reaction. Incorporation of methyl groups were estimated as described in materials and methods.

Figure 7. Preincubation and isotope partitioning analysis.

A. Time course of methylation reaction under different preincubation conditions. HP0593 MTase was preincubated with either DNA (○) or [methyl-³H] AdoMet (▪) or without preincubation (Δ) as described in materials and methods. The reaction was then started with the addition of [methyl-³H] AdoMet or DNA, respectively. Aliquots were removed at the defined time points, and the incorporation of methyl groups measured by the filter binding assay as described in materials and methods. B. Isotope partitioning analysis of HP0593 MTase. Methylation assays were carried out in reaction mixture containing 1.0 µM HP0593 MTase, 1.0 µM DNA and 1.5 µM AdoMet. (-•-), Product formation after enzyme was preincubated at 25°C with high specific activity [methyl-³H] AdoMet (67.3 Ci/mmol) and reaction started with DNA and labeled [methyl-³H] AdoMet. (-▪-), Product formation after enzyme was preincubated with high specific activity [methyl-³H] AdoMet and reaction started with DNA and unlabeled AdoMet. The final concentrations of HP0593, DNA and AdoMet were 0.050 µM, 1.0 µM and 1.5 µM, respectively.

Figure 8. Effect of divalent metal ions on methylation activity of HP0593 MTase.

A. Histogram showing methylation activity of 250 nM HP0593 MTase in the absence of any metal ions (-Me²⁺) and in the presence of cobalt chloride (Co²⁺), magnesium chloride (Mg²⁺), manganese chloride (Mn²⁺), calcium chloride (Ca²⁺), zinc chloride (Zn²⁺), and nickel chloride (Ni²⁺). 1 = 0.4 mM, 2 = 0.5 mM, 3 = 1.0 mM, and 4 = 5.0 mM. Kinetics of DNA binding. HP0593 MTase was injected for 120 sec over streptavidin chip containing immobilized duplex 18 DNA at a flow rate of 20 µl/min followed by dissociation phase of 120 sec. The global fit of the data was used to calculate the binding constants. B. SPR sensorgram displaying the response of increasing HP0593 MTase concentrations (25–100 nM) in presence of 1.0 mM MnCl₂. (Inset) kinetic constants. C. SPR sensorgram displaying the response of increasing HP0593 MTase concentrations (100–200 nM) in absence of metal. (Inset) kinetic constants.