2. Academic Validation
  3. New Insight into the Octamer of TYMS Stabilized by Intermolecular Cys43-Disulfide

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

  • Int J Mol Sci. 2018 May 7;19(5):1393. doi: 10.3390/ijms19051393.
Dan Xie 1,  Lulu Wang 2,  Qi Xiao 3,  Xiaoyan Wu 4,  Lin Zhang 5,  Qingkai Yang 6,  Lina Wang 7
Affiliations

Affiliations

  • 1 Institute of Cancer Stem Cell, Dalian Medical University, 9 Western Lvshun South Road, Dalian 116044, China. danxie2016@hotmail.com.
  • 2 School of Life Science and Biotechnology, Dalian University of Technology, No. 2 Linggong Road, Dalian 116024, China. wanglulu0813@126.com.
  • 3 Institute of Cancer Stem Cell, Dalian Medical University, 9 Western Lvshun South Road, Dalian 116044, China. xiaoqi1993a@hotmail.com.
  • 4 Institute of Cancer Stem Cell, Dalian Medical University, 9 Western Lvshun South Road, Dalian 116044, China. wuxiaoyan1234@hotmail.com.
  • 5 Institute of Cancer Stem Cell, Dalian Medical University, 9 Western Lvshun South Road, Dalian 116044, China. zhanglindlmed@163.com.
  • 6 Institute of Cancer Stem Cell, Dalian Medical University, 9 Western Lvshun South Road, Dalian 116044, China. Qingkaiyang@hotmail.com.
  • 7 Institute of Cancer Stem Cell, Dalian Medical University, 9 Western Lvshun South Road, Dalian 116044, China. Linawang1109@hotmail.com.
PMID: 29735940 DOI: 10.3390/ijms19051393
Abstract

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.

Keywords

dimer; monomer; octamer; overexpression and purification; thymidylate synthase (TYMS).

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