Abstract
The need for new antibiotics has become pressing in light of the emergence of antibiotic-resistant strains of human pathogens. Yersinia pestis, the causative agent of plague, is a public health threat and also an agent of concern in biodefence. It is a recently emerged clonal derivative of the enteric pathogen Yersinia pseudotuberculosis. Previously, we developed a bioinformatic approach to identify proteins that may be suitable targets for antimicrobial therapy and in particular for the treatment of plague. One such target was cytidine monophosphate (CMP) kinase, which is an essential gene in some organisms. Previously, we had thought CMP kinase was essential for Y. pseudotuberculosis, but by modification of the mutagenesis approach, we report here the production and characterization of a Δcmk mutant. The isogenic mutant had a growth defect relative to the parental strain, and was highly attenuated in mice. We have also elucidated the structure of the CMP kinase to 2.32 Å, and identified three key residues in the active site that are essential for activity of the enzyme. These findings will have implications for the development of novel CMP kinase inhibitors for therapeutic use.
Highlights
Cytidine monophosphate (CMP) kinase is a member of the nucleoside monophosphate (NMP) kinase family, and plays a crucial role in biosynthesis of nucleoside precursors
This differs from the activity of eukaryotic CMP kinases, which catalyse the conversion of UMP and CMP to the respective diphosphate form [1], and, structurally, bacterial CMP kinases are different from other members of the NMP kinase family [2]
The Y. pseudotuberculosis cmk gene was inactivated by replacement of the gene with a kanamycin-resistance cassette driven by the cmk promoter
Summary
Cytidine monophosphate (CMP) kinase is a member of the nucleoside monophosphate (NMP) kinase family, and plays a crucial role in biosynthesis of nucleoside precursors. CMP kinase catalyses the transfer of a phosphoryl group from ATP to CMP or dCMP. This differs from the activity of eukaryotic CMP kinases, which catalyse the conversion of UMP and CMP to the respective diphosphate form [1], and, structurally, bacterial CMP kinases are different from other members of the NMP kinase family [2]. CMP kinase over-expression can suppress the lethal effects of inactivation of the UMP. License http://creativecommons.org/licenses/by/3.0/, which permits unrestricted use, provided the original author and source are credited. Inactivation of cmk is reportedly not lethal in Escherichia coli [8] nor in Salmonella enterica [9]. We had attempted to create a cmk mutant in Yersinia pseudotuberculosis, without success, which indicated that cmk is an essential gene in this member of the Enterobacteriaceae [13]
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