EXPRESSION, PURIFICATION, MASS-SPECTROMETRY, CRYSTALLIZATION AND MULTIWAVELENGTH ANOMALOUS DIFFRACTION OF SELENOMETHIONYL PVUII DNA METHYLTRANSFERASE (CYTOSINE-N4-SPECIFIC)

The type II DNA-methyltransferase (cytosine NLC-specific) M.PvuII was overexpressed in Escherichia coli, starting From the internal translat ion initiator at Met14. Selenomethionine was efficiently incorporated into this short form of M.PvuII by a strain prototrophic for methionin e. Both native and selenomethionyl M.PvuII were purified to apparent h omogeneity by a two-column chromatography procedure. The yield of puri fied protein was approximately 1.8 mg/g bacterial paste. Mass spectrom etry analysis of selenomethionyl M.PvuII revealed three major forms th at probably differ in the degree of selenomethionine incorporation and the extent of selenomethionine oxidation. Amino acid sequencing and m ass spectrometry analysis of selenomethionine-containing peptides sugg ests that Met30, Met51, and Met261 were only partially replaced by sel enomethionine. Furthermore, amino acid 261 may be preferentially oxidi zed in both native and selenomethionyl form. Selenomethionyl and nativ e M.PvuII were crystallized separately as binary complexes of the meth yl donor S-adenosyl-L-methionine in the monoclinic space group P2(1). Two complexes were present per asymmetric unit. Six out of nine seleni um positions (per molecule), including the three that were found to be partially substituted, were identified crystallographically.