Abstract
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 Arg(95), Asn(126), Asn(132), Arg(116), and Lys(139), which directly contact the DNA outside the cognate recognition site (GATC) to processive catalysis, and that of residue Arg(137), 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 k(cat)/K(m)(DNA) or DNA affinity (K(D)(DNA)). However, these mutants cause large preferences for GATC sites varying in flanking sequences when considering the pre-steady state efficiency constant k(chem)/K(D)(DNA). 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 (k(cat)/K(m)(DNA)) 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 (Arg(95), Asn(126), Asn(132), and Arg(116)) 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.
Highlights
Processive enzyme catalysis, whereby a single substrate binding event is coupled to multiple rounds of catalytic turnovers, occurs frequently with enzymes that act on polymeric substrates such as DNA and in many cases, is essential for the viability of the organism [1,2,3,4,5]
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
The differential and regulated methylation of a small number of GATC sites found within the highly methylated E. coli genome [31, 32] in addition to preferential methylation observed in vitro [14, 25, 33] suggests that Escherichia coli DNA adenine methyltransferase (EcoDam) specificity is regulated by higher-order specificity involving the DNA flanking the target GATC sequences
Summary
Six EcoDam mutants were produced (Arg137 Ala, Arg116 Ala, Arg Ala, Asn126 Ala, Asn132 Ala, and Lys139 Ala) using the QuikChange PCR mutagenesis kit (Stratagene) with vector pDal572 as a template and six sets of primers (Operon). WT and mutant enzymes were diluted into protein dilution buffer so that each reaction contained 10 nM enzyme, 600 nM DNA, and 30 M AdoMet. Reactions were quenched by removing aliquots from the reaction mixture into pre-heated TE at 75 °C, and incubated for at least 15 min to ensure heat inactivation of EcoDam. After cooling to room temperature, samples were digested with DpnII for at least 12 h at 37 °C. Initial Velocity Studies—The initial velocity of WT and mutant EcoDams were monitored at various enzyme concentrations at time points that were within the early phase of product formation (less than 35% conversion). At specific time points that varied with enzyme concentration, 10 l of the reaction was quenched by submerging the mixture into 1% SDS.
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