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Updated: Oct 7, 2025
Characterization at the Molecular Level using Robust Biochemical Approaches of a New Kinase Protein
Published on: June 30, 2019
Structural and functional analysis of human thymidylate kinase isoforms
Junmei Hu Frisk1, Gunnar Pejler1,2, Staffan Eriksson1
1Department of Anatomy, Physiology and Biochemistry, Swedish University of Agricultural Sciences, Uppsala, Sweden.
Abstract:
Thymidylate kinase (TMPK) phosphorylates deoxythymidine monophosphate (dTMP) and plays an important role in genome stability. Deficiency in TMPK activity due to genetic alterations of DTYMK, i.e., the gene coding for TMPK, causes severe microcephaly in humans. However, no defects were observed in other tissues, suggesting the existence of a compensatory enzyme for dTTP synthesis. In search for this compensatory enzyme we analyzed 6 isoforms of TMPK mRNA deposited in the GenBank. Of these, only isoform 1 has been characterized and represents the known human TMPK. Our results reveal that isoform 2, 3, 4 and 5 lack essential structural elements for substrate binding and, thus, they are considered as nonfunctional isoforms. Isoform 6, however, has intact catalytic centers, i.e., dTMP-binding, DRX motif, ATP-binding p-loop and lid region, which are the key structural elements of an active TMPK, suggesting that isoform 6 may function as TMPK. When isoform 6 was expressed and purified, it showed only minimal activity (<0.1%) as compared with isoform 1. A putative isoform 6 was detected in a cancer cell line, in addition to the dominant isoform 1. However, because of its low activity, isoform 6 is unlikely be able to compensate for the loss of TMPK activity caused by deletions and/or point mutations of the DTYMK gene. Thereby, future studies to identify and characterize the compensatory TMPK enzyme found in patients with DTYMK mutations may contribute to the understanding of dTTP synthesis and of the pathophysiological role of DTYMK mutations in neurodegenerative disorders.
Insights
Researchers investigated thymidylate kinase (TMPK) and its gene (DTYMK). They found a potential compensatory enzyme isoform 6, but its low activity suggests it cannot fully replace TMPK function in patients with DTYMK mutations.
Area of Science:
- Biochemistry
- Molecular Biology
- Genetics
Background:
- Thymidylate kinase (TMPK) is crucial for genome stability by phosphorylating deoxythymidine monophosphate (dTMP).
- Genetic defects in the DTYMK gene, encoding TMPK, lead to severe microcephaly in humans.
- The absence of defects in other tissues suggests a compensatory enzyme for dTTP synthesis exists.
Purpose of the Study:
- To identify potential compensatory enzymes for TMPK activity.
- To characterize different isoforms of TMPK mRNA.
- To investigate the functional significance of TMPK isoforms in dTTP synthesis.
Main Methods:
- Analysis of 6 TMPK mRNA isoforms from GenBank.
- Structural analysis of isoforms for substrate-binding elements.
- Expression and purification of Isoform 6.
- Enzymatic activity assays comparing Isoform 6 to Isoform 1.
- Detection of isoforms in cancer cell lines.
Main Results:
- Isoforms 2-5 lack essential structural elements and are nonfunctional.
- Isoform 6 possesses intact catalytic centers but exhibits minimal activity (<0.1% of Isoform 1).
- Isoform 6 was detected alongside the dominant Isoform 1 in a cancer cell line.
Conclusions:
- Isoform 6 is unlikely to compensate for the loss of TMPK activity due to DTYMK gene mutations.
- Further research is needed to identify and characterize the compensatory enzyme responsible for dTTP synthesis in patients with DTYMK mutations.
- Understanding this compensatory enzyme could elucidate dTTP synthesis pathways and the pathophysiology of DTYMK-related neurodevelopmental disorders.
