Cloning, over-expression and the catalytic properties of the EcoP15 modification methylase from Escherichia coli.
The EcoP15 modification methylase gene from the p15B plasmid of Escherichia coli 15T-has been cloned and expressed at high levels in a plasmid vector system. We have purified the enzyme to near homogeneity in large amounts and have studied some of its enzymatic properties. Initial rates of methyl transfer are first order in methylase concentration and, with pUC19 DNA as substrate, the reaction proceeds by a random mechanism in which either DNA or S-adenosylmethionine can bind to the free enzyme. After methyltransfer to DNA, the methylated DNA and S-adenosylhomocysteine appear to dissociate in random order. As expected in such a mechanism, S-adenosylhomocysteine is a non-competitive inhibitor by S-adenosylmethionine at concentrations not much above its KM suggests that release of methylated DNA may be the rate-limiting step. This suggestion is strengthened by the fact that a mutant of the closely related EcoP1 does not show such substrate inhibition.
Rao
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Department of Microbiology, University of Basel, Switzerland.
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Bickle
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D001667:Binding, Competitive; D004247:DNA / Q000378:metabolism*; D015263:Deoxyribonucleases, Type III Site-Specific / Q000037:antagonists & inhibitors / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D005796:Genes; D005822:Genetic Vectors; D007700:Kinetics; D008745:Methylation; D010957:Plasmids / Q000235:genetics
D004247:DNA; C023451:endodeoxyribonuclease EcoP15I; D015263:Deoxyribonucleases, Type III Site-Specific