2z6a
From Proteopedia
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S-Adenosyl-L-methionine-Dependent Methyl Transfer: Observable Precatalytic Intermediates during DNA Cytosine Methylation
Overview
S-Adenosyl-l-methionine- (AdoMet-) dependent methyltransferases are, widespread, play critical roles in diverse biological pathways, and are, antibiotic and cancer drug targets. Presently missing from our, understanding of any AdoMet-dependent methyl-transfer reaction is a, high-resolution structure of a precatalytic enzyme/AdoMet/DNA complex. The, catalytic mechanism of DNA cytosine methylation was studied by, structurally and functionally characterizing several active site mutants, of the bacterial enzyme M.HhaI. The 2.64 A resolution protein/DNA/AdoMet, structure of the inactive C81A M.HhaI mutant suggests that active site, water, a approximately 13 degrees tilt of the target base toward the, active site nucleophile, and the presence or absence of the cofactor, methylsulfonium are coupled via a hydrogen-bonding network involving, Tyr167. The active site in the mutant complex is assembled to optimally, align the pyrimidine for nucleophilic attack and subsequent methyl, transfer, consistent with previous molecular dynamics ab initio and, quantum mechanics/molecular mechanics calculations. The mutant/DNA/AdoHcy, structure (2.88 A resolution) provides a direct comparison to the, postcatalytic complex. A third C81A ternary structure (2.22 A resolution), reveals hydrolysis of AdoMet to adenosine in the active site, further, validating the coupling between the methionine portion of AdoMet and, ultimately validating the structural observation of a, prechemistry/postchemistry water network. Disruption of this, hydrogen-bonding network by a Tyr167 to Phe167 mutation does not alter the, kinetics of nucleophilic attack or methyl transfer. However, the Y167F, mutant shows detectable changes in kcat, caused by the perturbed kinetics, of AdoHcy release. These results provide a basis for including an, extensive hydrogen-bonding network in controlling the rate-limiting, product release steps during cytosine methylation.
About this Structure
2Z6A is a Single protein structure of sequence from Haemophilus parahaemolyticus with as ligand. Active as DNA (cytosine-5-)-methyltransferase, with EC number 2.1.1.37 Full crystallographic information is available from OCA.
Reference
S-Adenosyl-l-methionine-Dependent Methyl Transfer: Observable Precatalytic Intermediates during DNA Cytosine Methylation., Youngblood B, Shieh FK, Buller F, Bullock T, Reich NO, Biochemistry. 2007 Jul 31;46(30):8766-75. Epub 2007 Jul 7. PMID:17616174
Page seeded by OCA on Wed Jan 23 14:05:32 2008
