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

Two Alternative Conformations of S-Adenosyl-L-homocysteine Bound to Escherichia coli DNA Adenine Methyltransferase and the Implication of Conformational Changes in Regulating the Catalytic Cycle

“…Previously we have shown that there is communication between the cofactor-binding site and the active site (4,12). The structure of the binary EcoDam-AdoHcy complex reported here suggests that Lys 14 is essential in this coupling, a conclusion that is supported by biochemical evidence as well.…”

“…The absence of this interaction between the target amino group and the DPPY as well as the presence of the additional amino group at position 2 is expected to make binding of 2AP into the active site pocket less favorable. In this regard, it should be noticed that the time scale of insertion of the flipped 2-aminopurine into the binding pocket is not in agreement with the single turnover rate constant, which is also the case for the wild type enzyme, where the rate constant of binding 2AP to the active site is smaller than 0.1 s Ϫ1 (12), whereas the single turnover rate constant is in the order of 0.15 s Ϫ1 (4). These observations point toward a limitation in using 2AP for base flipping studies as far as analyzing binding of the modified base into the active site pocket is concerned.…”

“…The largest difference in protein conformation between the binary and the ternary structures lies in the hexapeptide ( 9 KWAGGK 14 ) ending with Lys 14 whose side chain is disordered in the binary structure (Fig. 1D), with the C-␣ atoms of Gly 12 and Gly 13 moved more than 2.0 Å. This hexapeptide sequence motif is highly conserved with two invariant residues of GXK among the GATC-related DNA-(adenine-N ) forms the only sequence-specific contact of EcoDam to the Gua of the GATC sequence (4).…”

“…We have previously shown that in EcoDam cofactor binding, DNA binding and binding of the flipped target base into the active site pocket are coupled (4,12). Here, on the basis of a new binary structure of EcoDam in complex with AdoHcy and bio- chemical studies, we describe some of the structural and mechanistic details of this process.…”

“…The mixture was then subjected to UV crosslinking and AdoMet binding to the enzyme analyzed by SDS-PAGE. To avoid DNA methylation in the experiments, a catalytically inactive EcoDam variant (D181A) was used, which was purified as described (12).…”