Escherichia coli DNA Adenine Methyltransferase: THE STRUCTURAL BASIS OF PROCESSIVE CATALYSIS AND INDIRECT READ-OUT

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American Society for Biochemistry and Molecular Biology

RESUMO

We have investigated the structural basis of processive GATC methylation by the Escherichia coli DNA adenine methyltransferase, which is critical in chromosome replication and mismatch repair. We determined the contribution of the orthologically conserved phosphate interactions involving residues Arg95, Asn126, Asn132, Arg116, and Lys139, which directly contact the DNA outside the cognate recognition site (GATC) to processive catalysis, and that of residue Arg137, which is not conserved and contacts the DNA backbone within the GATC sequence. Alanine substitutions at the conserved positions have large impacts on processivity yet do not impact kcat/KmDNA or DNA affinity (KDDNA). However, these mutants cause large preferences for GATC sites varying in flanking sequences when considering the pre-steady state efficiency constant kchem/KDDNA. These changes occur mainly at the level of the methylation rate constant, which results in the observed decreases in processive catalysis. Thus, processivity and catalytic efficiency (kcat/KmDNA) are uncoupled in these mutants. These results reveal that the binding energy involved in DNA recognition contributes to the assembly of the active site rather than tight binding. Furthermore, the conserved residues (Arg95, Asn126, Asn132, and Arg116) repress the modulation of the response of the enzyme to flanking sequence effects. Processivity impacted mutants do not show substrate-induced dimerization as is observed for the wild type enzyme. This study describes the structural means by which an enzyme that does not completely enclose its substrate has evolved to achieve processive catalysis, and how interactions with DNA flanking the recognition site alter this processivity.

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