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J. Biol. Chem., Vol. 275, Issue 48, 38111-38119, December 1, 2000
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From the Department Of Medical Microbiology, University of
Manitoba, Winnipeg, Manitoba R3E 0W3, Canada
In all organisms the deoxyribonucleotide
precursors required for DNA synthesis are synthesized from
ribonucleotides, a reaction catalyzed by ribonucleotide reductase. In a
previous study we showed that Chlamydia trachomatis growth
was inhibited by hydroxyurea, an inhibitor of ribonucleotide reductase,
and a mutant resistant to the cytotoxic effects of the drug was
isolated. Here we report the cloning, expression, and purification of
the R1 and R2 subunits of the C. trachomatis ribonucleotide
reductase. In comparison with other ribonucleotide reductases, the
primary sequence of protein R1 has an extended amino terminus, and the
R2 protein has a phenylalanine where the essential tyrosine is normally
located. Despite its unusual primary structure, the recombinant enzyme catalyzes the reduction of CDP to dCDP. Results from deletion mutagenesis experiments indicate that while the extended amino terminus
of the R1 protein is not required for enzyme activity, it is needed for
allosteric inhibition mediated by dATP. Results with site-directed
mutants of protein R2 suggest that the essential tyrosine is situated
two amino acids downstream of its normal location. Finally, Western
blot analysis show that the hydroxyurea-resistant mutant C. trachomatis isolate overexpresses both subunits of ribonucleotide reductase. At the genetic level, compared with wild type C. trachomatis, the resistant isolate has a single base mutation
just upstream of the ATG start codon of the R2 protein. The possibility
that this mutation affects translational efficiency is discussed.
Cloning and Characterization of Ribonucleotide Reductase from
Chlamydia trachomatis*
*
This work was supported by Medical Research Council of
Canada Grant GR-13301 (to G. M.).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: Dept. of Medical
Microbiology, University of Manitoba, 730 William Ave., Winnipeg, Manitoba R3E 0W3, Canada. Tel.: 204-789-3307; Fax: 204-789-3926; E-mail: mcclart@cc.umanitoba.ca.
Copyright © 2000 by The American Society for Biochemistry and Molecular Biology, Inc.
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