The highly similar TMP kinases of Yersinia pestis and Escherichia coli differ markedly in their AZTMP phosphorylating activity

Thymidine monophosphate (TMP) kinases are key enzymes in nucleotide synthes is for all living organisms. Although eukaryotic and viral TMP kinases have been studied extensively, little is known about their bacterial counterpar ts. To characterize the TMP kinase of Yersinia pestis, a chromosomal region encompassing its gene (tmk) was cloned and sequenced; a high degree of con servation with the corresponding region of Escherichia coli was found. The Y. pestis tmk gene was overexpressed in E. coli, where the enzyme represent ed over 20% of total soluble proteins. The CD spectrum of the purified TMP kinase from Y. pestis was characteristic for proteins rich in alpha-helical structures. Its thermodynamic stability was significantly lower than that of E. coli TMP kinase. However, the most striking difference between the tw o enzymes was related to their ability to phosphorylate 3'-deoxy-3'-azidoth ymidine monophosphate (AZTMP). Although the enzymes of both species had com parable K-m values for this analogue, they differed significantly in their V-max for AZTMP. Whereas E. coli used AZTMP as a relatively good substrate, the Y. pestis enzyme had a V-max 100 times lower with AZTMP than with TMP. This fact explains why AZT, a potent bactericidal agent against E. coli, i s only moderately active on Y. enterocolitica. Sequence comparisons between E. coli and Y. pestis TMP kinases along with the three-dimensional structu re of the E. coli enzyme suggest that segments lying outside the main regio ns involved in nucleotide binding and catalysis are responsible for the dif ferent rates of AZTMP phosphorylation.