Role of histidine residues in EcoP15I DNA methyltransferase activity as probed by chemical modification and site-directed mutagenesis

2008 ◽  
Vol 416 (3) ◽  
pp. 475-475
Author(s):  
P. S. Jois ◽  
N. Madhu ◽  
D. N. Rao
Keyword(s):  
Chemical Modification ◽  
Dna Methyltransferase ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Methyltransferase Activity ◽  
Histidine Residues

Role of histidine residues in EcoP15I DNA methyltransferase activity as probed by chemical modification and site-directed mutagenesis

2008 ◽  
Vol 410 (3) ◽  
pp. 543-553 ◽  
Author(s):  
Prashanth S. Jois ◽  
Nagaraj Madhu ◽  
Desirazu N. Rao
Keyword(s):  
Chemical Modification ◽  
Dna Methyltransferase ◽  
Catalytic Mechanism ◽  
Gel Filtration ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Recognition Sequence ◽  
Native Page ◽  
Histidine Residues

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.

Role of lysines in human angiogenin: chemical modification and site-directed mutagenesis

Biochemistry ◽  
1989 ◽  
Vol 28 (4) ◽  
pp. 1726-1732 ◽  
Author(s):  
Robert Shapiro ◽  
Edward A. Fox ◽  
James F. Riordan
Keyword(s):  
Chemical Modification ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis

Heparinase I from Flavobacterium heparinum: The Role of the Cysteine Residue in Catalysis as Probed by Chemical Modification and Site-Directed Mutagenesis

Biochemistry ◽  
1995 ◽  
Vol 34 (44) ◽  
pp. 14441-14448 ◽  
Author(s):  
Ram Sasisekharan ◽  
Deborah Leckband ◽  
Ranga Godavarti ◽  
Ganesh Venkataraman ◽  
Charles L. Cooney ◽  
...  
Keyword(s):  
Chemical Modification ◽  
Cysteine Residue ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Heparinase I ◽  
Flavobacterium Heparinum
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