Role of histidine residues in EcoP15I DNA methyltransferase activity as probed by chemical modification and site-directed mutagenesis.
Towards understanding the catalytic mechanism of M.EcoP15I [EcoP15I MTase (DNA methyltransferase); an adenine methyltransferase], we investigated the role of histidine residues in catalysis. M.EcoP15I, when incubated with DEPC (diethyl pyrocarbonate), a histidine-specific reagent, shows a time- and concentration-dependent inactivation of methylation of DNA containing its recognition sequence of 5'-CAGCAG-3'. The loss of enzyme activity was accompanied by an increase in absorbance at 240 nm. A difference spectrum of modified versus native enzyme shows the formation of N-carbethoxyhistidine that is diminished by hydroxylamine. This, along with other experiments, strongly suggests that the inactivation of the enzyme by DEPC was specific for histidine residues. Substrate protection experiments show that pre-incubating the methylase with DNA was able to protect the enzyme from DEPC inactivation. Site-directed mutagenesis experiments in which the 15 histidine residues in the enzyme were replaced individually with alanine corroborated the chemical modification studies and established the importance of His-335 in the methylase activity. No gross structural differences were detected between the native and H335A mutant MTases, as evident from CD spectra, native PAGE pattern or on gel filtration chromatography. Replacement of histidine with alanine residue at position 335 results in a mutant enzyme that is catalytically inactive and binds to DNA more tightly than the wild-type enzyme. Thus we have shown in the present study, through a combination of chemical modification and site-directed mutagenesis experiments, that His-335 plays an essential role in DNA methylation catalysed by M.EcoP15I.
Jois
Prashanth S
PS
Department of Biochemistry, Indian Institute of Science, Bangalore 560012, India.
Madhu
Nagaraj
N
Rao
Desirazu N
DN
D001483:Base Sequence; D002384:Catalysis; D002942:Circular Dichroism; D019175:DNA Methylation; D015254:DNA Modification Methylases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D017931:DNA Primers; D024202:Electrophoretic Mobility Shift Assay; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D005822:Genetic Vectors; D006639:Histidine / Q000502:physiology*; D015335:Molecular Probes; D016297:Mutagenesis, Site-Directed; D010957:Plasmids; D013050:Spectrometry, Fluorescence; D020349:Surface Plasmon Resonance
D017931:DNA Primers; D015335:Molecular Probes; D006639:Histidine; D015254:DNA Modification Methylases