Structural basis for efficient phosphorylation of 3'-azidothymidine monophosphate by Escherichia coli thymidylate kinase.
The crystal structures of Escherichia coli thymidylate kinase (TmpK) in complex with P1-(5'-adenosyl)-P5-(5'-thymidyl)pentaphosphate and P1-(5'-adenosyl)P5-[5'-(3'-azido-3'-deoxythymidine)] pentaphosphate have been solved to 2.0-A and 2.2-A resolution, respectively. The overall structure of the bacterial TmpK is very similar to that of yeast TmpK. In contrast to the human and yeast TmpKs, which phosphorylate 3'-azido-3'-deoxythymidine 5'-monophosphate (AZT-MP) at a 200-fold reduced turnover number (kcat) in comparison to the physiological substrate dTMP, reduction of kcat is only 2-fold for the bacterial enzyme. The different kinetic properties toward AZT-MP between the eukaryotic TmpKs and E. coli TmpK can be rationalized by the different ways in which these enzymes stabilize the presumed transition state and the different manner in which a carboxylic acid side chain in the P loop interacts with the deoxyribose of the monophosphate. Yeast TmpK interacts with the 3'-hydroxyl of dTMP through Asp-14 of the P loop in a bidentate manner: binding of AZT-MP results in a shift of the P loop to accommodate the larger substituent. In E. coli TmpK, the corresponding residue is Glu-12, and it interacts in a side-on fashion with the 3'-hydroxyl of dTMP. This different mode of interaction between the P loop carboxylic acid with the 3' substituent of the monophosphate deoxyribose allows the accommodation of an azido group in the case of the E. coli enzyme without significant P loop movement. In addition, although the yeast enzyme uses Arg-15 (a glycine in E. coli) to stabilize the transition state, E. coli seems to use Arg-153 from a region termed Lid instead. Thus, the binding of AZT-MP to the yeast TmpK results in the shift of a catalytic residue, which is not the case for the bacterial kinase.
Lavie
A
A
Department of Physical Biochemistry, Max Planck Institute for Molecular Physiology, Rheinlanddamm 201, 44139 Dortmund, Germany.
Ostermann
N
N
Brundiers
R
R
Goody
R S
RS
Reinstein
J
J
Konrad
M
M
Schlichting
I
I
D000595:Amino Acid Sequence; D000998:Antiviral Agents / Q000378:metabolism*; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D054306:Dideoxynucleotides; D004926:Escherichia coli / Q000201:enzymology*; D006801:Humans; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009703:Nucleoside-Phosphate Kinase / Q000737:chemistry* / Q000378:metabolism*; D010766:Phosphorylation; D017433:Protein Structure, Secondary; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D016415:Sequence Alignment; D015215:Zidovudine / Q000031:analogs & derivatives* / Q000378:metabolism
D000998:Antiviral Agents; D054306:Dideoxynucleotides; C057288:3'-azido-2',3'-dideoxyuridine 5'-monophosphate; D015215:Zidovudine; D009703:Nucleoside-Phosphate Kinase; C020093:dTMP kinase