Probing the role of cysteine residues in the EcoP15I DNA methyltransferase.
Chemical modification using thiol-directed agents and site-directed mutagenesis has been used to investigate the role of cysteine residues of EcoP15I DNA methyltransferase. Irreversible inhibition of enzymatic activity was provoked by chemical modification of the enzyme by N-ethylmaleimide and iodoacetamide. 5, 5'-Dithiobis(2-nitrobenzoic acid) titration of the enzyme under nondenaturing and denaturing conditions confirmed the presence of six cysteine residues without any disulfides in the protein. Aware that relatively bulky reagents inactivate the methyltransferase by directly occluding the substrate-binding site or by locking the methyltransferase in an inactive conformation, we used site-directed mutagenesis to sequentially replace each of the six cysteines in the protein at positions 30, 213, 344, 434, 553, and 577. All the resultant mutant methylases except for the C344S and C344A enzymes retained significant activity as assessed by in vivo and in vitro assays. The effects of the substitutions on the function of EcoP15I DNA methyltransferase were investigated by substrate binding assays, activity measurements, and steady-state kinetic analysis of catalysis. Our results clearly indicate that the cysteines at positions other than 344 are not essential for activity. In contrast, the C344A enzyme showed a marked loss of enzymatic activity. More importantly, whereas the inactive C344A mutant enzyme bound S-adenosyl-L-methionine, it failed to bind to DNA. Furthermore, in double and triple mutants where two or three cysteine residues were replaced by serine, all such mutants in which the cysteine at position 344 was changed, were inactive. Taken together, these results convincingly demonstrate that the Cys-344 is necessary for enzyme activity and indicate an essential role for it in DNA binding.
Reddy
Y V
YV
Department of Biochemistry, Indian Institute of Science, Bangalore 560 012, India.
Rao
D N
DN
D019943:Amino Acid Substitution; D003001:Cloning, Molecular; D003545:Cysteine; D019175:DNA Methylation; D004228:Dithionitrobenzoic Acid / Q000494:pharmacology; D004926:Escherichia coli / Q000201:enzymology; D005033:Ethylmaleimide / Q000494:pharmacology; D007460:Iodoacetamide / Q000494:pharmacology; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D011489:Protein Denaturation; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D015265:Site-Specific DNA-Methyltransferase (Adenine-Specific) / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
D011994:Recombinant Proteins; D004228:Dithionitrobenzoic Acid; C089712:DNA modification methylase EcoP15I; D015265:Site-Specific DNA-Methyltransferase (Adenine-Specific); D003545:Cysteine; D005033:Ethylmaleimide; D007460:Iodoacetamide