DNA (Cytosine-N 4-)- and -(Adenine-N 6-)-methyltransferases Have Different Kinetic Mechanisms but the Same Reaction Route

Malygin, E. G.; Zinoviev, Victor V.; Evdokimov, Alexey A.; Lindstrom, William M.; Reich, Norbert O.; Hattman, Stanley

doi:10.1074/jbc.m213213200

Cited by 12 publications

(5 citation statements)

References 29 publications

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“…Similar mechanisms to avoid binary deadend complexes seem to be operative also in other DNA MTases. 50 Therefore, DNA recognition, cofactor binding and target base recognition of DNA MTases are coupled processes involving interactions of the enzyme to the target site prior to and after base flipping as well as contacts to the flipped base itself.…”

Section: Discussionmentioning

confidence: 99%

Stopped-flow and Mutational Analysis of Base Flipping by the Escherichia coli Dam DNA-(adenine-N6)-methyltransferase

Liebert¹,

Hermann²,

Schlickenrieder³

et al. 2004

Journal of Molecular Biology

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Section: Discussionmentioning

confidence: 99%

Stopped-flow and Mutational Analysis of Base Flipping by the Escherichia coli Dam DNA-(adenine-N6)-methyltransferase

Liebert¹,

Hermann²,

Schlickenrieder³

et al. 2004

Journal of Molecular Biology

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“…The methylation products methylated DNA and S-adenosylhomocysteine are known to inhibit the methyltransferase activity in some cases (28,51). Therefore, initial reaction velocities were determined by keeping the molarity of these products at less than 20% of the added oligoduplex substrate at the start of the reaction.…”

Section: Effect Of Cationsmentioning

confidence: 99%

Hyperthermophilic DNA Methyltransferase M.PabI from the Archaeon Pyrococcus abyssi

Watanabe

Yuzawa

Handa

et al. 2006

Appl Environ Microbiol

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Genome sequence comparisons among multiple species of Pyrococcus, a hyperthermophilic archaeon, revealed a linkage between a putative restriction-modification gene complex and several large genome polymorphisms/rearrangements. From a region apparently inserted into the Pyrococcus abyssi genome, a hyperthermoresistant restriction enzyme [PabI; 5-(GTA/C)] with a novel structure was discovered. In the present work, the neighboring methyltransferase homologue, M.PabI, was characterized. Its N-terminal half showed high similarities to the M subunit of type I systems and a modification enzyme of an atypical type II system, M.AhdI, while its C-terminal half showed high similarity to the S subunit of type I systems. M.PabI expressed within Escherichia coli protected PabI sites from RsaI, a PabI isoschizomer. M.PabI, purified following overexpression, was shown to generate 5-GTm6AC, which provides protection against PabI digestion. M.PabI was found to be highly thermophilic; it showed methylation at 95°C and retained at least half the activity after 9 min at 95°C. This hyperthermophilicity allowed us to obtain activation energy and other thermodynamic parameters for the first time for any DNA methyltransferases. We also determined the kinetic parameters of k cat , K m, DNA , and K m, AdoMet . The activity of M.PabI was optimal at a slightly acidic pH and at an NaCl concentration of 200 to 500 mM and was inhibited by Zn 2؉ but not by Mg 2؉ , Ca 2؉ , or Mn 2؉ . These and previous results suggest that this unique methyltransferase and PabI constitute a type II restriction-modification gene complex that inserted into the P. abyssi genome relatively recently. As the most thermophilic of all the characterized DNA methyltransferases, M.PabI may help in the analysis of DNA methylation and its application to DNA engineering.DNA methylation plays a crucial role in diverse biological processes, for example, replication and repair of DNA, expression and silencing of genes, and distinction between self and nonself DNAs. DNA methyltransferases require S-adenosyl-Lmethionine (AdoMet) as the donor of a methyl group (7). The common core of the AdoMet-dependent methyltransferases is formed by seven-stranded ␤-sheets (53), and the target base within a double-stranded DNA molecule is installed into its catalytic pocket by a "base-flipping" mechanism (20). The AdoMet-dependent DNA methyltransferases are classified into three groups according to their products: those yielding 5-methylcytosine (m5C), those yielding N 4 -methylcytosine (m4C), and those yielding N 6 -methyladenine (m6A). The latter two groups of amino-nitrogen methyltransferases are further subdivided into ␣, ␤, ␥, and other families, according to the order of motifs (of the catalytic center and AdoMet-binding region) and the target recognition domain (TRD) (4, 27).Most of the prokaryotic DNA methyltransferases constitute restriction-modification (RM) systems, which can be classified into types I, II, and III (45,46). A classic type II RM system is composed of two distinct prote...

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“…Pre-steady-state studies have allowed rate constants for only the methyl transfer step to be extracted in a number of systems. For example, HhaI DNA-(cytosine-C 5 )-MTase has a k methylation of ∼15.6 min -1 (43), whereas BamHI DNA-(cytosine-N 4 )-MTase from Bacillus amyloliquefaciens and bacteriophage T4Dam DNA-(adenine-N 6 )-MTase have k methylations of 5.1 and 33.6 min -1 , respectively(44). It must be mentioned that the EcoRI DNA-(adenine-N 6 )-MTase has a k methylation of 2460 min -1 (45); however, this rate constant is generally not representative of those of most methyltransferases.…”

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confidence: 99%

Isotope and Elemental Effects Indicate a Rate-Limiting Methyl Transfer as the Initial Step in the Reaction Catalyzed by Escherichia coli Cyclopropane Fatty Acid Synthase

et al. 2004

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Cyclopropane fatty acid (CFA) synthases catalyze the formation of cyclopropane rings on unsaturated fatty acids (UFAs) that are natural components of membrane phospholipids. The methylene carbon of the cyclopropane ring derives from the activated methyl group of S-adenosyl-L-methionine (AdoMet), affording S-adenosyl-L-homocysteine (AdoHcys) and a proton as the remaining products. This reaction is unique among AdoMet-dependent enzymes, because the olefin of the UFA substrate is isolated and unactivated toward nucleophilic or electrophilic addition, raising the question as to the timing and mechanism of proton loss from the activated methyl group of AdoMet. Two distinct reaction schemes have been proposed for this transformation; however, neither was based on detailed in vitro mechanistic analysis of the enzyme. In the preceding paper [Iwig, D. F. and Booker, S. J. (2004) Biochemistry 43, http://dx.doi.org/10.1021/bi048693+], we described the synthesis of two analogues of AdoMet, Se-adenosyl-L-selenomethionine (SeAdoMet) and Te-adenosyl-L-telluromethionine (TeAdoMet), and their intrinsic reactivity toward polar chemistry in which AdoMet is known to be involved. We found that the electrophilicity of AdoMet and its onium congeners followed the series SeAdoMet > AdoMet > TeAdoMet, while the acidity of the carbons adjacent to the relevant heteroatom followed the series AdoMet > SeAdoMet > TeAdoMet. When each of these compounds was used as the methylene donor in the CFA synthase reaction, the kinetic parameters of the reaction, k(cat) and k(cat) K(M)(-1), followed the series SeAdoMet > AdoMet > TeAdoMet, suggesting that the reaction takes place via methyl transfer followed by proton loss, rather than by processes that are initiated by proton abstraction from AdoMet. Use of S-adenosyl-L-[methyl-d(3)]methionine as the methylene donor resulted in an inverse isotope effect of 0.87 +/- 0.083, supporting this conclusion and also indicating that the methyl transfer takes place via a tight s(N)2 transition state.

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DNA (Cytosine-N 4-)- and -(Adenine-N 6-)-methyltransferases Have Different Kinetic Mechanisms but the Same Reaction Route

Cited by 12 publications

References 29 publications

Stopped-flow and Mutational Analysis of Base Flipping by the Escherichia coli Dam DNA-(adenine-N6)-methyltransferase

Stopped-flow and Mutational Analysis of Base Flipping by the Escherichia coli Dam DNA-(adenine-N6)-methyltransferase

Hyperthermophilic DNA Methyltransferase M.PabI from the Archaeon Pyrococcus abyssi

Isotope and Elemental Effects Indicate a Rate-Limiting Methyl Transfer as the Initial Step in the Reaction Catalyzed by Escherichia coli Cyclopropane Fatty Acid Synthase

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