Kinetic and structural characterization of an alternatively spliced variant of human mitochondrial 5′(3′)-deoxyribonucleotidase

Abstract

Human mitochondrial 5′(3′)-deoxyribonucleotidase (mdN) catalyzes dephosphorylation of nucleoside monophosphates, and thus helps maintain homeostasis of deoxynucleosides required for mitochondrial DNA synthesis. Mature mdN is a 23-kDa dimeric protein with highest expression levels in the heart, brain and skeletal muscle. We have identified an alternative splice variant of the mdN gene containing an 18-nucleotide insertion encoding 6 amino acids (GKWPAT) at the 3′-end of the penultimate exon 4. We recombinantly expressed this enzyme variant and characterized its biochemical and kinetic properties as well as its three-dimensional structure. Our high-resolution (1.27 Å) crystal structure revealed that the insertion forms a loop located in the vicinity of the active site pocket and affects enzyme kinetic parameters as well as protein thermal stability.

• 5′(3′)-deoxyribonucleotidase
• alternative splicing
• crystal structure
• hydrolase
• mitochondria

Acknowledgements

The authors thank Dr. P. Šácha of IOCB AS CR for discussions and the gift of Clontech Quick-Clone cDNA libraries. Diffraction data were collected on BL14.1 and BL14.2 operated by the Helmholtz-Zentrum Berlin (HZB) at the BESSY II electron storage ring (Berlin-Adlershof, Germany)14.

Declaration of interest

The authors have no conflicts of interest to declare. This work was supported by the Grant Agency of the Czech Republic (research project No. GA 203/09/0820), the Ministry of Education of the Czech Republic – LK11205 (programme “NAVRAT”), and in part by research projects RVO 61388963 and 68378050 awarded by the Academy of Sciences of the Czech Republic.