Limits...
Kinetics of Methylation by EcoP1I DNA Methyltransferase.

Bheemanaik S, Sistla S, Krishnamurthy V, Arathi S, Desirazu NR - Enzyme Res (2010)

Bottom Line: M.EcoP1I is shown to exist as dimer in solution, and even at high salt concentrations (0.5 M) the dimeric M.EcoP1I does not dissociate into monomers suggesting a strong interaction between the monomer subunits.EcoP1I DNA MTase catalyzes the transfer of methyl groups using a distributive mode of methylation on DNA containing more than one recognition site.A chemical modification of EcoP1I DNA MTase using N-ethylmaleimide resulted in an irreversible inactivation of enzyme activity suggesting the possible role of cysteine residues in catalysis.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Indian Institute of Science, Bangalore 560 012, India.

ABSTRACT
EcoP1I DNA MTase (M.EcoP1I), an N(6)-adenine MTase from bacteriophage P1, is a part of the EcoP1I restriction-modification (R-M) system which belongs to the Type III R-M system. It recognizes the sequence 5'-AGACC-3' and methylates the internal adenine. M.EcoP1I requires Mg(2+) for the transfer of methyl groups to DNA. M.EcoP1I is shown to exist as dimer in solution, and even at high salt concentrations (0.5 M) the dimeric M.EcoP1I does not dissociate into monomers suggesting a strong interaction between the monomer subunits. Preincubation and isotope partitioning studies with M.EcoP1I indicate a kinetic mechanism where the duplex DNA binds first followed by AdoMet. Interestingly, M.EcoP1I methylates DNA substrates in the presence of Mn(2+) and Ca(2+) other than Mg(2+) with varying affinities. Amino acid analysis and methylation assays in the presence of metal ions suggest that M.EcoP1I has indeed two metal ion-binding sites [(358)ID(x)(n) … ExK(401) and (600)DxDxD(604) motif]. EcoP1I DNA MTase catalyzes the transfer of methyl groups using a distributive mode of methylation on DNA containing more than one recognition site. A chemical modification of EcoP1I DNA MTase using N-ethylmaleimide resulted in an irreversible inactivation of enzyme activity suggesting the possible role of cysteine residues in catalysis.

No MeSH data available.


Determination of the molecular mass of M.EcoP1I by size-exclusion chromatography under nondenaturing conditions. (a) The standard curve Ve/Vo versus log molecular weight was derived from the elution profiles of the standard molecular weight markers with Ve corresponding to the peak elution volume of the protein and Vo representing the void volume of the column determined using Blue dextran (2,000,000 kDa). The peak position of M.EcoP1I is indicated by a line: (1) horse myoglobin (17 kDa); (2) chicken ovalbumin (44 kDa); (3) bovine serum albumin (66 kDa); (4) M.EcoP15I (150 kDa); (5) γ-Globulin (158 kDa) and thyroglobulin (670 kDa). Inset: elution profile of M.EcoP1I. (b) Fractions were collected after gel-filtration chromatography and analyzed on SDS-PAGE for presence of M.EcoP1I. (c) Methylation activity of M.EcoP1I present in the fractions was checked using filter-binding assays.
© Copyright Policy - open-access
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2962900&req=5

fig1: Determination of the molecular mass of M.EcoP1I by size-exclusion chromatography under nondenaturing conditions. (a) The standard curve Ve/Vo versus log molecular weight was derived from the elution profiles of the standard molecular weight markers with Ve corresponding to the peak elution volume of the protein and Vo representing the void volume of the column determined using Blue dextran (2,000,000 kDa). The peak position of M.EcoP1I is indicated by a line: (1) horse myoglobin (17 kDa); (2) chicken ovalbumin (44 kDa); (3) bovine serum albumin (66 kDa); (4) M.EcoP15I (150 kDa); (5) γ-Globulin (158 kDa) and thyroglobulin (670 kDa). Inset: elution profile of M.EcoP1I. (b) Fractions were collected after gel-filtration chromatography and analyzed on SDS-PAGE for presence of M.EcoP1I. (c) Methylation activity of M.EcoP1I present in the fractions was checked using filter-binding assays.

Mentions: Gel-filtration analysis was performed to determine the size and subunit structure of M.EcoP1I in solution. A superose 6 HR 10/30 column was calibrated with proteins of known size (17–670 kDa), and different concentrations of M.EcoP1I were loaded (3–15 μM). M.EcoP1I was eluted as a symmetric peak at a position corresponding to a globular protein of 150 kDa (Figure 1), suggesting that the enzyme exists mostly as a dimer under native conditions. The elution profile of M.EcoP1I shows an additional small peak which corresponded to a high molecular weight protein (Figure 1(a), inset). The presence of protein in both peaks was confirmed by SDS-PAGE analysis (Figure 1(b)). The protein present in both of the fractions showed MTase activity (Figure 1(c)). Rechromatography of the eluted dimer peak followed a similar pattern; a small peak of high molecular weight protein and a symmetric peak corresponding to the dimer (data not shown). When the peak corresponding to a high molecular weight protein was reloaded onto the column, it was too eluted as two peaks: a high molecular weight protein peak and a dimer peak (data not shown). The elution pattern did not change when M.EcoP1I was loaded in the presence of 500 mM NaCl suggesting a stronger interaction between the monomeric subunits of the protein (data not shown).


Kinetics of Methylation by EcoP1I DNA Methyltransferase.

Bheemanaik S, Sistla S, Krishnamurthy V, Arathi S, Desirazu NR - Enzyme Res (2010)

Determination of the molecular mass of M.EcoP1I by size-exclusion chromatography under nondenaturing conditions. (a) The standard curve Ve/Vo versus log molecular weight was derived from the elution profiles of the standard molecular weight markers with Ve corresponding to the peak elution volume of the protein and Vo representing the void volume of the column determined using Blue dextran (2,000,000 kDa). The peak position of M.EcoP1I is indicated by a line: (1) horse myoglobin (17 kDa); (2) chicken ovalbumin (44 kDa); (3) bovine serum albumin (66 kDa); (4) M.EcoP15I (150 kDa); (5) γ-Globulin (158 kDa) and thyroglobulin (670 kDa). Inset: elution profile of M.EcoP1I. (b) Fractions were collected after gel-filtration chromatography and analyzed on SDS-PAGE for presence of M.EcoP1I. (c) Methylation activity of M.EcoP1I present in the fractions was checked using filter-binding assays.
© Copyright Policy - open-access
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2962900&req=5

fig1: Determination of the molecular mass of M.EcoP1I by size-exclusion chromatography under nondenaturing conditions. (a) The standard curve Ve/Vo versus log molecular weight was derived from the elution profiles of the standard molecular weight markers with Ve corresponding to the peak elution volume of the protein and Vo representing the void volume of the column determined using Blue dextran (2,000,000 kDa). The peak position of M.EcoP1I is indicated by a line: (1) horse myoglobin (17 kDa); (2) chicken ovalbumin (44 kDa); (3) bovine serum albumin (66 kDa); (4) M.EcoP15I (150 kDa); (5) γ-Globulin (158 kDa) and thyroglobulin (670 kDa). Inset: elution profile of M.EcoP1I. (b) Fractions were collected after gel-filtration chromatography and analyzed on SDS-PAGE for presence of M.EcoP1I. (c) Methylation activity of M.EcoP1I present in the fractions was checked using filter-binding assays.
Mentions: Gel-filtration analysis was performed to determine the size and subunit structure of M.EcoP1I in solution. A superose 6 HR 10/30 column was calibrated with proteins of known size (17–670 kDa), and different concentrations of M.EcoP1I were loaded (3–15 μM). M.EcoP1I was eluted as a symmetric peak at a position corresponding to a globular protein of 150 kDa (Figure 1), suggesting that the enzyme exists mostly as a dimer under native conditions. The elution profile of M.EcoP1I shows an additional small peak which corresponded to a high molecular weight protein (Figure 1(a), inset). The presence of protein in both peaks was confirmed by SDS-PAGE analysis (Figure 1(b)). The protein present in both of the fractions showed MTase activity (Figure 1(c)). Rechromatography of the eluted dimer peak followed a similar pattern; a small peak of high molecular weight protein and a symmetric peak corresponding to the dimer (data not shown). When the peak corresponding to a high molecular weight protein was reloaded onto the column, it was too eluted as two peaks: a high molecular weight protein peak and a dimer peak (data not shown). The elution pattern did not change when M.EcoP1I was loaded in the presence of 500 mM NaCl suggesting a stronger interaction between the monomeric subunits of the protein (data not shown).

Bottom Line: M.EcoP1I is shown to exist as dimer in solution, and even at high salt concentrations (0.5 M) the dimeric M.EcoP1I does not dissociate into monomers suggesting a strong interaction between the monomer subunits.EcoP1I DNA MTase catalyzes the transfer of methyl groups using a distributive mode of methylation on DNA containing more than one recognition site.A chemical modification of EcoP1I DNA MTase using N-ethylmaleimide resulted in an irreversible inactivation of enzyme activity suggesting the possible role of cysteine residues in catalysis.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Indian Institute of Science, Bangalore 560 012, India.

ABSTRACT
EcoP1I DNA MTase (M.EcoP1I), an N(6)-adenine MTase from bacteriophage P1, is a part of the EcoP1I restriction-modification (R-M) system which belongs to the Type III R-M system. It recognizes the sequence 5'-AGACC-3' and methylates the internal adenine. M.EcoP1I requires Mg(2+) for the transfer of methyl groups to DNA. M.EcoP1I is shown to exist as dimer in solution, and even at high salt concentrations (0.5 M) the dimeric M.EcoP1I does not dissociate into monomers suggesting a strong interaction between the monomer subunits. Preincubation and isotope partitioning studies with M.EcoP1I indicate a kinetic mechanism where the duplex DNA binds first followed by AdoMet. Interestingly, M.EcoP1I methylates DNA substrates in the presence of Mn(2+) and Ca(2+) other than Mg(2+) with varying affinities. Amino acid analysis and methylation assays in the presence of metal ions suggest that M.EcoP1I has indeed two metal ion-binding sites [(358)ID(x)(n) … ExK(401) and (600)DxDxD(604) motif]. EcoP1I DNA MTase catalyzes the transfer of methyl groups using a distributive mode of methylation on DNA containing more than one recognition site. A chemical modification of EcoP1I DNA MTase using N-ethylmaleimide resulted in an irreversible inactivation of enzyme activity suggesting the possible role of cysteine residues in catalysis.

No MeSH data available.