Abstract
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.
Highlights
Since the chlamydial R1 has a duplication of approximately 110 amino acids at the N terminus, it contains three reasonable homologues of the signature sequence VXKRDG (Fig. 1A), which occurs at the N terminus of R1s that contain an allosteric activity site [11, 32]
Expression and Purification of Wild Type and Mutant C. trachomatis R1 and R2 Proteins—In agreement with previous findings with cloned overexpressed mouse [22], viral [38, 39], and Arabidopsis thalania [40] R1 proteins using bacterial expression systems, we found that insoluble inclusion body formation decreased and yield of soluble R1 protein increased if lower growth temperature and reduced IPTG concentration were used
The data presented here indicate that the C. trachomatis L2 nrdAB operon is expressed, R1 and R2 proteins are produced, and recombinant enzyme is active at reducing CDP to dCDP
Summary
To initiate more detailed studies on the chlamydial RNR and to define the molecular mechanism of hydroxyurea resistance in our mutant chlamydiae we cloned, expressed, purified, and raised antibody against both subunits of the enzyme. Results from site-directed mutagenesis studies with the chlamydial R2 protein suggest that tyrosine 129 (chlamydiae numbering) is probably the location of the essential tyrosyl free radical.
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
