Structural basis for efficient phosphorylation of 3′-azidothymidine monophosphate by Escherichia coli thymidylate kinase

Abstract

The crystal structures of Escherichia coli thymidylate kinase (TmpK) in complex with P 1 -(5-adenosyl)-P 5 -(5-thymidyl)pentaphosphate and P 1 -(5-adenosyl)P 5 -[5-(3-azido-3-deoxythymidine)] pentaphosphate have been solved to 2.0-Å and 2.2-Å 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 (k cat ) in comparison to the physiological substrate dTMP, reduction of k cat 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.Phosphorylation of AZT-MP by thymidylate kinase (TmpK; EC 2.7.4.9, ATP:dTMP phosphotransferase) has been implicated as the rate-limiting step in the activation pathway of the anti-HIV prodrug 3Ј-azido-3Ј-deoxythymidine (AZT). AZT must be phosphorylated by cellular enzymes to azidothymidine triphosphate before it can exert its antiviral effect by inhibiting HIV reverse transcriptase and acting as a chain terminator of nascent DNA strands. The bottleneck of AZT activation lies in the second phosphorylation step, the step that adds a phosphate group to azidothymidine monophosphate (AZT-MP) to yield azidothymidine diphosphate and is catalyzed by TmpK. Cells treated with AZT accumulate the toxic AZT-MP (1-3) in millimolar concentration (4, 5), and thus the active AZT triphosphorylated metabolite is at very low concentration. This situation is only slightly improved upon coinfection with herpes simplex virus I thymidine kinase-carrying vectors that efficiently phosphorylate the antiviral drugs acyclovir or ganciclovir but not AZT or AZT-MP (6, 7).Therefore, we set out to understand the structural basis for the slow activation of AZT-MP. The structures of the yeast TmpK (TmpK yeast ) complexed with either the physiological substrate d...

Polyphosphate-dependent synthesis of ATP and ADP by the family-2 polyphosphate kinases in bacteria

“…Structural similarity between the PPK2 domains and thymidylate kinases and the conservation of the key catalytic residues of thymidylate kinases in PPK2 enzymes suggest that these enzymes have a common evolutionary origin and catalytic mechanism. Our model of the PPK2 catalytic mechanism is based on the mechanisms proposed for thymidylate kinases from E. coli (Tmk) and Mycobacterium tuberculosis (TMPK Mtub ) (19,21) and is supported by the results of our mutagenic studies (Fig. 4 and Table 1).…”

“…4). Like in E. coli Tmk (19), the side chain of the Walker A motif Asp-93 (Asp-307 in PA3455) probably interacts with the ribose 3Ј-hydroxyl, whereas its 2Ј-hydroxyl might form a hydrogen bond to Tyr-233 (Tyr-447 in PA3455), which is consistent with the reduced affinity of SMc02148 Y233A to ADP and low activity of D93A (and PA3455 D307A and Y447A) ( Table 1 and Fig. 4).…”

“…SMc02148 has a very similar lid module (␣8, ␣9, and 6), which is covered by the small N-terminal extension domain (␤1, ␤2, and ␣1) on the top. The lid module of the N-terminal PPK2 domain of PA3455 was only partially modeled because of missing density, suggesting significant conformational flexibility of this module like in thymidylate kinases (19). In contrast, the lid modules of the PA3455 C-terminal PPK2 domain and SMc02148 were well resolved.…”

Structural Requirements for Efficient Phosphorylation of Nucleotide Analogs by Human Thymidylate Kinase

“…In support of this was the observation that in E. coli TMPK, which does not have this arginine in the P-loop but rather possesses a number of arginine residues in the Lid region, AZTMP phosphorylation was only 2-fold slower than the TMP phosphorylation rate [32] (this factor is 200 for the yeast TMPK, 60 for the human enzyme). Structural studies on the bacterial TMPK buttress this model.…”

Structure of thymidylate kinase fromEhrlichia chaffeensis

“…Specifically, the P-loop amino-acid sequences of the human and Ehrlichia proteins are GVDRAGKS and GIDGSGKT, respectively. These motifs both contain an acidic Asp residue that is uniquely found in TMPKs compared with other nucleoside monophosphate kinases (Lavie et al, 1998). The human enzyme is a type I TMPK, in which the Asp15 residue is immediately followed by a catalytically important arginine residue.…”