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J. Biol. Chem., Vol. 275, Issue 49, 38722-38730, December 8, 2000
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From the DNA cytosine-5-methyltransferase HhaI
recognizes the GCGC sequence and flips the inner cytosine out of DNA
helix and into the catalytic site for methylation. The 5'-phosphate of
the flipped out cytosine is in contact with the conserved Thr-250 from
the target recognition domain. We have produced 12 mutants of Thr-250 and examined their methylation potential in vivo. Six
active mutants were subjected to detailed biochemical and structural
studies. Mutants with similar or smaller side chains (Ser, Cys, and
Gly) are very similar to wild-type enzyme in terms of steady-state kinetic parameters kcat,
KmDNA,
KmAdoMet. In contrast, the
mutants with bulkier side chains (Asn, Asp, and His) show increased
Km values for both substrates. Fluorescence titrations and stopped-flow kinetic analysis of interactions with duplex oligonucleotides containing 2-aminopurine at the target base
position indicate that the T250G mutation leads to a more polar but
less solvent-accessible position of the flipped out target base. The
x-ray structure of the ternary
M.HhaI(T250G)·DNA·AdoHcy complex shows that the
target cytosine is locked in the catalytic center of enzyme. The space
created by the mutation is filled by water molecules and the adjacent
DNA backbone atoms dislocate slightly toward the missing side chain. In
aggregate, our results suggest that the side chain of Thr-250 is
involved in constraining the conformation the DNA backbone and the
target base during its rotation into the catalytic site of enzyme.
Functional Roles of the Conserved Threonine 250 in the Target
Recognition Domain of HhaI DNA Methyltransferase*
,
auskas
Institute of Biotechnology, Laboratory of
Biological DNA Modification, LT-2028 Vilnius, Lithuania, the
§ Department of Biochemistry, Emory University School of
Medicine, Atlanta, Georgia 30322, and ¶ Max-Planck-Institut
für molekulare Physiologie, Abteilung Physikalische Biochemie,
D-44227 Dortmund, Germany
*
This work was supported in part by a Howard Hughes Medical
Institute International Research Scholarship grant (to S. K.), a
Volkswagen-Stiftung collaborative research grant (to S. K. and E. W.), and National Institutes of Health Grant GM49245 and the Georgia Research Alliance (to X. C.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom correspondence should be addressed. Fax: 370-2-602116;
E-mail: klimasau@ibt.lt.
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