title: Reversible inactivation of the CG specific SssI DNA (cytosine-C5)-methyltransferase with a photocleavable protecting group.
abstract: Caging of proteins by conjugation with a photocleavable group is a powerful approach for reversibly blocking enzymatic activity. Here we describe the covalent modification of the bacterial SssI DNA methyltransferase (M.SssI) with the cysteine-specific reagent 4,5-dimethoxy-2-nitrobenzylbromide (DMNBB). M.SssI contains two cysteine residues; replacement of the active-site Cys141 with Ser resulted in an approximately 100-fold loss of enzymatic activity; this indicates an important role for this residue in catalysis. However, replacement of Cys368 with Ala did not affect methyltransferase activity. Treatment of the Cys368Ala mutant enzyme with DMNBB led to an almost complete loss of activity. Irradiation of the inactivated enzyme with near-ultraviolet light (320-400 nm) restored 60 % of the catalytic activity. This indicates that caging by DMNBB can be used for the reversible inactivation of M.SssI.
authors.affiliation: Biochemistry Laboratory International University Bremen, School of Engineering and Science, Campus Ring 1, 28759 Bremen, Germany.
authors.forename: Philipp
authors.initials: P
authors.lastname: Rathert
authors.forename: Tamás
authors.initials: T
authors.lastname: Raskó
authors.forename: Markus
authors.initials: M
authors.lastname: Roth
authors.forename: Krystyna
authors.initials: K
authors.lastname: Slaska-Kiss
authors.forename: Alfred
authors.initials: A
authors.lastname: Pingoud
authors.forename: Antal
authors.initials: A
authors.lastname: Kiss
authors.forename: Albert
authors.initials: A
authors.lastname: Jeltsch
mesh_terms: D001483:Base Sequence; D002384:Catalysis; D003001:Cloning, Molecular; D003597:Cytosine Nucleotides / Q000378:metabolism*; D004248:DNA (Cytosine-5-)-Methyltransferases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D004789:Enzyme Activation; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D015870:Gene Expression; D006150:Guanine Nucleotides / Q000378:metabolism*; D008745:Methylation; D010777:Photochemistry; D013379:Substrate Specificity
chemical_list: D003597:Cytosine Nucleotides; D006150:Guanine Nucleotides; D004248:DNA (Cytosine-5-)-Methyltransferases