Functional analysis of BamHI DNA cytosine-N4 methyltransferase.
We show that the kinetic mechanism of the DNA (cytosine-N(4)-)-methyltransferase M.BamHI, which modifies the underlined cytosine (GGATCC), differs from cytosine C(5) methyltransferases, and is similar to that observed with adenine N(6) methyltransferases. This suggests that the obligate order of ternary complex assembly and disassembly depends on the type of methylation reaction. In contrast, the single-turnover rate of catalysis for M.BamHI (0.10s(-1)) is closer to the DNA (cytosine-C(5)-)-methyltransferases (0.14s(-1)) than the DNA (adenine-N(6)-)-methyltransferases (>200s(-1)). The nucleotide flipping transition dominates the single-turnover constant for adenine N(6) methyltransferases, and, since the disruption of the guanine-cytosine base-pair is essential for both types of cytosine DNA methyltransferases, this transition may be a common, rate-limiting step for methylation for these two enzyme subclasses. The similar overall rate of catalysis by M.BamHI and other DNA methyltransferases is consistent with a common rate-limiting catalytic step of product dissociation. Our analyses of M.BamHI provide functional insights into the relationship between the three different classes of DNA methyltransferases that complement both prior structural and evolutionary insights.
Lindstrom
William M
WM
Department of Chemistry and Biochemistry, University of California-Santa Barbara, Santa Barbara, CA 93106-9510, USA.
Malygin
Ernst G
EG
Ovechkina
Lidiya G
LG
Zinoviev
Victor V
VV
Reich
Norbert O
NO
D020134:Catalytic Domain; D003596:Cytosine / Q000737:chemistry / Q000378:metabolism; D015257:DNA-Cytosine Methylases / Q000737:chemistry / Q000378:metabolism*; D007700:Kinetics; D046911:Macromolecular Substances; D008745:Methylation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012436:S-Adenosylmethionine / Q000737:chemistry / Q000378:metabolism; D013379:Substrate Specificity
D046911:Macromolecular Substances; D011994:Recombinant Proteins; D012436:S-Adenosylmethionine; D003596:Cytosine; C042525:DNA modification methylase BamHI; D015257:DNA-Cytosine Methylases