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 synthesis for all living organisms. Although eukaryotic and viral TMP kinases have been studied extensively, little is known about their bacterial counterparts. To characterize the TMP kinase of Yersinia pestis, a chromosomal region encompassing its gene (tmk) was cloned and sequenced; a high degree of conservation with the corresponding region of Escherichia coli was found. The Y. pestis tmk gene was overexpressed in E. coli, where the enzyme represented 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 two enzymes was related to their ability to phosphorylate 3'-deoxy-3'-azidothymidine monophosphate (AZTMP). Although the enzymes of both species had comparable Km values for this analogue, they differed significantly in their Vmax for AZTMP. Whereas E. coli used AZTMP as a relatively good substrate, the Y. pestis enzyme had a Vmax 100 times lower with AZTMP than with TMP. This fact explains why AZT, a potent bactericidal agent against E. coli, is only moderately active on Y. enterocolitica. Sequence comparisons between E. coli and Y. pestis TMP kinases along with the three-dimensional structure of the E. coli enzyme suggest that segments lying outside the main regions involved in nucleotide binding and catalysis are responsible for the different rates of AZTMP phosphorylation.
Chenal-Francisque
V
V
Laboratoire des Yersinia, Institut Pasteur, Paris, France.
Tourneux
L
L
Carniel
E
E
Christova
P
P
Li de la Sierra
I
I
Bârzu
O
O
Gilles
A M
AM
D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D054306:Dideoxynucleotides; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D008969:Molecular Sequence Data; D009703:Nucleoside-Phosphate Kinase / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013045:Species Specificity; D013379:Substrate Specificity; D013942:Thymine Nucleotides / Q000378:metabolism*; D015007:Yersinia / Q000201:enzymology; D015010:Yersinia pestis / Q000201:enzymology* / Q000235:genetics; D015215:Zidovudine / Q000031:analogs & derivatives* / Q000378:metabolism
D054306:Dideoxynucleotides; D011994:Recombinant Proteins; D013942:Thymine Nucleotides; C057287:3'-azido-3'-deoxythymidine 5'phosphate; D015215:Zidovudine; D009703:Nucleoside-Phosphate Kinase; C020093:dTMP kinase