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 KmAdoMet (60 nM), KiAdoHye (0.4 nM), and Kcat (0.22 s-1) 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(1) and had unit-cell dimensions of a = 55.3 A, b = 72.7 A, c = 91.0 A, and beta = 102.5 degrees, with two molecules of M.HhaI in each of the two asymmetric units. The crystals diffract beyond 2.5 A and are suitable for structure determination.
Kumar
S
S
Cold Spring Harbor Laboratory, New York 11724.
Cheng
X
X
Pflugrath
J W
JW
Roberts
R J
RJ
D003460:Crystallization; D015257:DNA-Cytosine Methylases / Q000096:biosynthesis / Q000737:chemistry* / Q000302:isolation & purification; D004926:Escherichia coli / Q000235:genetics; D046911:Macromolecular Substances; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D012436:S-Adenosylmethionine / Q000737:chemistry*; D014961:X-Ray Diffraction
D046911:Macromolecular Substances; D011994:Recombinant Proteins; D012436:S-Adenosylmethionine; C037664:DNA modification methylase HhaI; D015257:DNA-Cytosine Methylases