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Biochemical Characterizations of Human TMPK Mutations Identified in Patients with Severe Microcephaly: Single Amino Acid Substitutions Impair Dimerization and Abolish Their Catalytic Activity

Frisk, Jun Mei Hu and Vanoevelen, J. M. and Bierau, Jörgen and Pejler, Gunnar and Eriksson, Staffan and Wang, Liya (2021). Biochemical Characterizations of Human TMPK Mutations Identified in Patients with Severe Microcephaly: Single Amino Acid Substitutions Impair Dimerization and Abolish Their Catalytic Activity. ACS Omega. 6 :49 , 33943–33952
[Research article]

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Abstract

Deoxythymidylate kinase (TMPK) is a key enzyme in the synthesis of deoxythymidine triphosphate (dTTP). Four TMPK variants (P81L, A99T, D128N, and a frameshift) have been identified in human patients who suffered from severe neurodegenerative diseases. However, the impact of these mutations on TMPK function has not been clarified. Here we show that in fibroblasts derived from a patient, the P81L and D128N mutations led to a complete loss of TMPK activity in mitochondria and extremely low and unstable TMPK activity in cytosol. Despite the lack of TMPK activity, the patient-derived fibroblasts apparently grew normal. To investigate the impact of the mutations on the enzyme function, the mutant TMPKs were expressed, purified, and characterized. The wild-type TMPK mainly exists as a dimer with high substrate binding affinity, that is, low KM value and high catalytic efficiency, that is, kcat/KM. In contrast, all mutants were present as monomers with dramatically reduced substrate binding affinity and catalytic efficiencies. Based on the human TMPK structure, none of the mutated amino acids interacted directly with the substrates. By structural analysis, we could explain why the respective amino acid substitutions could drastically alter the enzyme structure and catalytic function. In conclusion, TMPK mutations identified in patients represent loss of function mutations but surprisingly the proliferation rate of the patient-derived fibroblasts was normal, suggesting the existence of an alternative and hitherto unknown compensatory TMPK-like enzyme for dTTP synthesis. Further studies of the TMPK enzymes will help to elucidate the role of TMPK in neuropathology.

Authors/Creators:Frisk, Jun Mei Hu and Vanoevelen, J. M. and Bierau, Jörgen and Pejler, Gunnar and Eriksson, Staffan and Wang, Liya
Title:Biochemical Characterizations of Human TMPK Mutations Identified in Patients with Severe Microcephaly: Single Amino Acid Substitutions Impair Dimerization and Abolish Their Catalytic Activity
Series Name/Journal:ACS Omega
Year of publishing :2021
Volume:6
Number:49
Page range:33943–33952
Number of Pages:10
ISSN:2470-1343
Language:English
Publication Type:Research article
Article category:Scientific peer reviewed
Version:Published version
Copyright:Creative Commons: Attribution 4.0
Full Text Status:Public
Subjects:(A) Swedish standard research categories 2011 > 1 Natural sciences > 106 Biological Sciences (Medical to be 3 and Agricultural to be 4) > Biochemistry and Molecular Biology
URN:NBN:urn:nbn:se:slu:epsilon-p-114570
Permanent URL:
http://urn.kb.se/resolve?urn=urn:nbn:se:slu:epsilon-p-114570
Additional ID:
Type of IDID
DOI10.1021/acsomega.1c05288
ID Code:26944
Faculty:VH - Faculty of Veterinary Medicine and Animal Science
Department:(VH) > Dept. of Anatomy, Physiology and Biochemistry
Deposited By: SLUpub Connector
Deposited On:08 Feb 2022 16:25
Metadata Last Modified:12 Sep 2022 17:30

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