Xie, D.; Wang, L.; Xiao, Q.; Wu, X.; Zhang, L.; Yang, Q.; Wang, L. New Insight into the Octamer of TYMS Stabilized by Intermolecular Cys43-Disulfide. Int. J. Mol. Sci.2018, 19, 1393.
Xie, D.; Wang, L.; Xiao, Q.; Wu, X.; Zhang, L.; Yang, Q.; Wang, L. New Insight into the Octamer of TYMS Stabilized by Intermolecular Cys43-Disulfide. Int. J. Mol. Sci. 2018, 19, 1393.
Xie, D.; Wang, L.; Xiao, Q.; Wu, X.; Zhang, L.; Yang, Q.; Wang, L. New Insight into the Octamer of TYMS Stabilized by Intermolecular Cys43-Disulfide. Int. J. Mol. Sci.2018, 19, 1393.
Xie, D.; Wang, L.; Xiao, Q.; Wu, X.; Zhang, L.; Yang, Q.; Wang, L. New Insight into the Octamer of TYMS Stabilized by Intermolecular Cys43-Disulfide. Int. J. Mol. Sci. 2018, 19, 1393.
Abstract
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.
Subject Areas
thymidylate synthase (TYMS); dimer; octamer; biochemical reconstitution; overexpression and purification
Copyright:
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Dan Xie