Uracil as an Alternative to 5-Fluorocytosine
in Addressable Protein Targeting
J.A. Wendel and S.S. Smith*
This is an abstract
for a talk to be given at the
Fifth
Foresight Conference on Molecular Nanotechnology.
The full paper is now available.
Bacterial DNA methyltransferases offer an approach to
addressable protein targeting in macromolecular assembly that may
permit the construction of a variety of usefully ordered protein
arrays (Smith 1995, Smith et al. 1997, Smith et al. 1997a). In
this system for macromolecular assembly, the natural recognition
specificity of the bacterial DNA cytosine methyltransferases is
used to target fusion proteins to preselected sites on a DNA
scaffold through the formation of a stable covalent attachment
with the mechanism-based inhibitor, 5-Fluorocytosine. Current
evidence on the mechanism of action of bacterial DNA
methyltransferases suggests that proton circulation during
catalysis can facilitate substrate binding, nucleophilic attack
and product release. Ab initio modeling of the reaction has been
used to analyze the mechanism of inhibition for commonly studied
inhibitors of the reaction in order to determine whether or not
they can substitute for 5-Fluorocytosine in DNA as covalent
targets for the enzymes. The ab initio data suggest that in
addition to the well known thermodynamic barrier to b-elimination
that blocks the release of the methyltransferase from the
5-fluoro, 5-methyl-dihydrocytosine formed with 5-fluorocytosine,
each of the these inhibitors creates a significant kinetic
barrier to methyl transfer by lowering the energy of the HOMO
(Highest Occupied Molecular Orbital) of the activated pyrimidine
relative to that of activated cytosine. This kinetic barrier
causes methyltransferases to stall after covalent attachment to
C6 of any of the cytosine analogs studied. Subsequent
methyltransfer to produce the 5-methyldihydropyrimidine complex
is slow for each of these compounds with the relative reaction
rates consistent with the calculated values of the HOMO for five
inhibitors studied. Of these inhibitors, dUra is readily attacked
and stable complexes are formed in the absence of significant
methyltransfer. The data suggest that dUra is a valuable and cost
effective substitute for 5FdCyt in the production of ordered
macromolecular assemblies.
*Supported by grant N00014-94-1-1116 from the Office of Naval
Research
References
[1] Smith, S. S., (1995) Nucleoprotein-Based
Nanoscale Fabrication. In: Biological and Biomedical Science and
Technology Division 1995 Programs, ed. E. Eisenstadt, (U.S. Navy
Publication ONR 34196-3), pp. 161-162.
[2] Smith, S.S., Niu, L., Baker, D.J., Wendel,
J.A., Kane, S. E. and Joy, D.S. (1997) Nanoscale Addressing in
Macromolecular Assembly. Miami BiotechnologyShort Reports, Vol.
8. F.Ahmad et al., eds. IRL at Oxford University Press, p.13.
[3] Smith, S.S., Niu, L. Baker, D.J., Wendel, J.A.
Kane, S.E.,and Joy, D.S (1997a) Nucleoprotein Based Nanoscale Assembly. Proc. Natl. Acad. Sci. U.S.A. 94:2162-2167.
*Corresponding Address:
Steven Smith, City of Hope National Medical Center, 1500 E.
Duarte Rd, Duarte, CA 91010-3000, ph: 818-301-8316, fax:
818-301-8972, email: [email protected]
or [email protected]
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