TY - JOUR
T1 - Hyperthermophilic DNA methyltransferase M.PabI from the archaeon Pyrococcus abyssi
AU - Watanabe, Miki
AU - Yuzawa, Harumi
AU - Handa, Naofumi
AU - Kobayashi, Ichizo
N1 - Copyright:
Copyright 2008 Elsevier B.V., All rights reserved.
PY - 2006/8
Y1 - 2006/8
N2 - Genome sequence comparisons among multiple species of Pyrococcus, a hyperthermophilic archaeon, revealed a linkage between a putative restriction-modification gene complex and several large genome polymorphisms/rearrangements. From a region apparently inserted into the Pyrococcus abyssi genome, a hyperthermoresistant restriction enzyme [PabI; 5′-(GTA/C)] with a novel structure was discovered. In the present work, the neighboring methyltransferase homologue, M.PabI, was characterized. Its N-terminal half showed high similarities to the M subunit of type I systems and a modification enzyme of an atypical type II system, M.AhdI, while its C-terminal half showed high similarity to the S subunit of type I systems. M.PabI expressed within Escherichia coli protected PabI sites from RsaI, a PabI isoschizomer. M.PabI, purified following overexpression, was shown to generate 5′-GTm6AC, which provides protection against PabI digestion. M.PabI was found to be highly thermophilic; it showed methylation at 95°C and retained at least half the activity after 9 min at 95°C. This hyperthermophilicity allowed us to obtain activation energy and other thermodynamic parameters for the first time for any DNA methyltransferases. We also determined the kinetic parameters of kcat, Km, DNA, and Km, AdoMet. The activity of M.PabI was optimal at a slightly acidic pH and at an NaCl concentration of 200 to 500 mM and was inhibited by Zn2+ but not by Mg2+, Ca2+, or Mn2+. These and previous results suggest that this unique methyltransferase and PabI constitute a type II restriction-modification gene complex that inserted into the P. abyssi genome relatively recently. As the most thermophilic of all the characterized DNA methyltransferases, M.PabI may help in the analysis of DNA methylation and its application to DNA engineering.
AB - Genome sequence comparisons among multiple species of Pyrococcus, a hyperthermophilic archaeon, revealed a linkage between a putative restriction-modification gene complex and several large genome polymorphisms/rearrangements. From a region apparently inserted into the Pyrococcus abyssi genome, a hyperthermoresistant restriction enzyme [PabI; 5′-(GTA/C)] with a novel structure was discovered. In the present work, the neighboring methyltransferase homologue, M.PabI, was characterized. Its N-terminal half showed high similarities to the M subunit of type I systems and a modification enzyme of an atypical type II system, M.AhdI, while its C-terminal half showed high similarity to the S subunit of type I systems. M.PabI expressed within Escherichia coli protected PabI sites from RsaI, a PabI isoschizomer. M.PabI, purified following overexpression, was shown to generate 5′-GTm6AC, which provides protection against PabI digestion. M.PabI was found to be highly thermophilic; it showed methylation at 95°C and retained at least half the activity after 9 min at 95°C. This hyperthermophilicity allowed us to obtain activation energy and other thermodynamic parameters for the first time for any DNA methyltransferases. We also determined the kinetic parameters of kcat, Km, DNA, and Km, AdoMet. The activity of M.PabI was optimal at a slightly acidic pH and at an NaCl concentration of 200 to 500 mM and was inhibited by Zn2+ but not by Mg2+, Ca2+, or Mn2+. These and previous results suggest that this unique methyltransferase and PabI constitute a type II restriction-modification gene complex that inserted into the P. abyssi genome relatively recently. As the most thermophilic of all the characterized DNA methyltransferases, M.PabI may help in the analysis of DNA methylation and its application to DNA engineering.
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U2 - 10.1128/AEM.00433-06
DO - 10.1128/AEM.00433-06
M3 - Article
C2 - 16885288
AN - SCOPUS:33747367212
VL - 72
SP - 5367
EP - 5375
JO - Applied and Environmental Microbiology
JF - Applied and Environmental Microbiology
SN - 0099-2240
IS - 8
ER -