Crystal Structure of Mouse Thymidylate Synthase in Tertiary Complex with dUMP and Raltitrexed Reveals N-Terminus Architecture and Two Different Active Site Conformations

Dowiercial, A.; Wilk, P.; Rypniewski, W.; Rode, Wojciech; Jarmuła, Adam

doi:10.1155/2014/945803

Cited by 11 publications

(13 citation statements)

References 21 publications

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“…The Ca RMSD values between the liganded and unliganded mTS structures presented here, as well as between these structures and the crystal structures of other ternary complexes with dUMP and Tomudex, including those with hTS [15,16], rTS [17] and mTS [18], are shown in Table 3. The superimposition of the mTS, mTS-dUMP and mTS-FdUMP-meTHF structures is shown in Fig.…”

Section: Resultsmentioning

confidence: 99%

Mouse thymidylate synthase does not show the inactive conformation, observed for the human enzyme

et al. 2016

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Crystal structures of mouse thymidylate synthase (mTS) in complexes with (1) sulfate anion, (2) 2 0 -deoxyuridine 5 0 -monophosphate (dUMP) and (3) 5-fluorodUMP (FdUMP) and N 5,10 -methylenetetrahydrofolate (meTHF) have been determined and deposited in Protein Data Bank under the accession codes 3IHI, 4E5O and 5FCT, respectively. The structures show a strong overall similarity to the corresponding structures of rat and human thymidylate synthases (rTS and hTS, respectively). Unlike with hTS, whose unliganded and liganded forms assume different conformations (''inactive'' and ''active,'' respectively) in the loop 181-197, in each of the three mTS structures, the loop 175-191, homologous to hTS loop 181-197, populates the active conformer, with catalytic Cys 189 buried in the active site and directed toward C(6) of the pyrimidine ring of dUMP/FdUMP, pointing to protein's inability to adopt the inactive conformation. The binary structures of either dUMP-or sulfate-bound mTS, showing the enzyme with open active site and extended C-terminus, differ from the structure of the mTS-5-FdUMP-meTHF ternary complex, with the active site closed and C-terminus folded inward, thus covering the active site cleft. Another difference pertains to the conformation of the Arg44 side chain in the active site-flanking loop 41-47, forming strong hydrogen bonds with the dUMP/FdUMP phosphate moiety in each of the two liganded mTS structures, but turning away from the active site entrance and loosing the possibility of H-bonding with sulfate in the sulfate-bound mTS structure.

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Section: Resultsmentioning

confidence: 99%

Mouse thymidylate synthase does not show the inactive conformation, observed for the human enzyme

et al. 2016

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“…The cavity in the dimer interface could serve as an allosteric site used to regulate the conformational switching between the active and inactive states [ 1 , 30 ]. In addition, TYMS performs at least two different functions with specific interaction regions: the dimer obligates catalytic function, while both the monomer and the dimer are believed to play crucial roles in TYMS–mRNA recognition and regulation [ 31 , 32 , 33 , 34 ]. In this study, we found TYMS not only formed a dimer, but also an octamer by intermolecular Cys43-disulfide ( Figure 3 ).…”

Section: Discussionmentioning

confidence: 99%

New Insight into the Octamer of TYMS Stabilized by Intermolecular Cys43-Disulfide

Xie

Wang

Xiao

et al. 2018

IJMS

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Thymidylate synthase (TYMS) is an essential enzyme for the de novo synthesis of deoxythymidine monophosphate (dTMP) and has been a primary target for cancer chemotherapy. Although the physical structure of TYMS and the molecular mechanisms of TYMS catalyzing the conversion of deoxyuridine monophosphate (dUMP) to dTMP have been the subject of thorough studies, its oligomeric structure remains unclear. Here, we show that human TYMS not only exists in dimer form but also as an octamer by intermolecular Cys43-disulfide formation. We optimized the expression conditions of recombinant human TYMS using the Escherichia coli system. Using high-performance liquid chromatography–tandem mass spectrometry (HPLC–MS/MS), we have shown that purified TYMS has catalytic activity for producing dTMP. In the absence of reductant β-mercaptoethanol, SDS-PAGE and size exclusion chromatography (SEC) showed that the size of the TYMS protein is about 35 kDa, 70 kDa, and 280 kDa. When the Cys43 was mutated to Gly, the band of ~280 kDa and the peak of the octamer disappeared. Therefore, TYMS was determined to form an octamer, depending on the presence of Cys43-disulfide. By measuring steady-state parameters for the monomer, dimer, and octamer, we found the kcat of the octamer was increased slightly more than the monomer. On the basis of these findings, we suggest that the octamer in the active state might have a potential influence on the design of new drug targets.

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“…The cavity in the dimer interface could serve as an allosteric site of to regulate the conformational switching between the active and inactive states [1,30]. In addition, TYMS performs at least two different functions with specific interaction regions: dimer obligates catalytic function, while both the monomer and dimer are believed to play a crucial role in TYMS-mRNA recognition and regulation [31][32][33][34]. In this study, we found TYMS not only formed dimer, but also octamer by intermolecular Cys43-disulfide ( Figure 3).…”

Section: Analysis Of Kinetic Properties Of All Oligomeric Formsmentioning

confidence: 99%

New Insight into the Octamer of Thymidylate Synthetase Stabilized by Intermolecular Cys43-Disulfide

Xie¹,

Wang²,

Xiao³

et al. 2018

Preprint

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Thymidylate synthase (TYMS) is an essential enzyme for the de novo synthesis of dTMP and has been a primary target for cancer chemotherapy. Although the physical structure of TYMS and the molecular mechanisms of TYMS catalyzing the conversion of dUMP to dTMP have been conducted thorough studies, oligomeric structure remains unclear. Here, we show that human TYMS not only exists in dimer but also octamer by intermolecular Cys43-disulfide formation. We optimize the expression condition of recombinant human TYMS using Escherichia coli system. Using HPLC-MS/MS, we show that purified TYMS has catalytic activity for producing dTMP. In the absence of reductant β-mercaptoethanol, SDS-PAGE and size exclusion chromatography (SEC) showed size of TYMS protein is about 35 KDa, 70 KDa, and 280 KDa. While the Cys43 was mutated to Gly, the band of ~280 KDa and the peak of octamer disappeared. Therefore, TYMS was determined to form octamer, dependent on the presence of Cys43-disulfide. By measuring Steady-State Parameters for monomer, dimer and octamer, we found the kcat of octamer is increased slightly than monomer. On the basis of these findings, we suggest that octamer in the active state might have a potential influence on the design of new drug targets.

show abstract

Crystal Structure of Mouse Thymidylate Synthase in Tertiary Complex with dUMP and Raltitrexed Reveals N-Terminus Architecture and Two Different Active Site Conformations

Cited by 11 publications

References 21 publications

Mouse thymidylate synthase does not show the inactive conformation, observed for the human enzyme

Mouse thymidylate synthase does not show the inactive conformation, observed for the human enzyme

New Insight into the Octamer of TYMS Stabilized by Intermolecular Cys43-Disulfide

New Insight into the Octamer of Thymidylate Synthetase Stabilized by Intermolecular Cys43-Disulfide

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