Purification, crystallization, and preliminary X-ray diffraction analysis of an M. HhaI-AdoMet complex

The type-II DNA-(cytosine-5)-methyltransferase M.HhaI was overexpressed in Escherichia coli and purified to apparent homogeneity. The purification scheme exploits a unique high salt back-extraction step to solubilize M.HhaI selectively, followed by FPLC chromatography. The yield of purified protein was 0.75-1.0 mg per gram of bacterial paste. M.HhaI could be isolated in two forms: bound with its cofactor S-adenosylmethionine (AdoMet) or devoid of the cofactor. The AdoMet-bound form was capable of methylating DNA in vitro in the absence of exogenous AdoMet. From kinetic studies of the purified enzyme, values for K sub(m) super(AdoMet) (60 nM), K sub(i) super(AdoHyc) (0.4 nM), and K sub(cat) (0.22/s) were determined. The purified enzyme bound with its cofactor was crystallized by the hanging drop vapor diffusion technique. Crystals were of monoclinic space group P2 sub(1) and had unit-cell dimensions of a = 55.3 angstrom, b = 72.7 angstrom, c = 91.0 angstrom, and beta = 102.5 degree , with two molecules of M.HhaI in each of the two asymmetric units. The crystals diffract beyond 2.5 angstrom and are suitable for structure determination. (DBO)