Structural basis for substrate specificity of the human mitochondrial deoxyribonucleotidase.
The human mitochondrial deoxyribonucleotidase catalyzes the dephosphorylation of thymidine and deoxyuridine monophosphates and participates in the regulation of the dTTP pool in mitochondria. We present seven structures of the inactive D41N variant of this enzyme in complex with thymidine 3'-monophosphate, thymidine 5'-monophosphate, deoxyuridine 5'-monophosphate, uridine 5'-monophosphate, deoxyguanosine 5'-monophosphate, uridine 2'-monophosphate, and the 5'-monophosphate of the nucleoside analog 3'-deoxy 2'3'-didehydrothymidine, and we draw conclusions about the substrate specificity based on comparisons with enzyme activities. We show that the enzyme's specificity for the deoxyribo form of nucleoside 5'-monophosphates is due to Ile-133, Phe-49, and Phe-102, which surround the 2' position of the sugar and cause an energetically unfavorable environment for the 2'-hydroxyl group of ribonucleoside 5'-monophosphates. The close binding of the 3'-hydroxyl group of nucleoside 5'-monophosphates to the enzyme indicates that nucleoside analog drugs that are substituted with a bulky group at this position will not be good substrates for this enzyme.
Walldén
Karin
K
Department of Biochemistry and Biophysics, Stockholm University, SE-106 91 Stockholm, Sweden.
Ruzzenente
Benedetta
B
Rinaldo-Matthis
Agnes
A
Bianchi
Vera
V
Nordlund
Pär
P
D015720:5'-Nucleotidase / Q000737:chemistry*; D001665:Binding Sites; D018360:Crystallography, X-Ray; D015226:Dinucleoside Phosphates / Q000737:chemistry; D004583:Electrons; D006801:Humans; D007532:Isoleucine / Q000737:chemistry; D007700:Kinetics; D008928:Mitochondria / Q000201:enzymology* / Q000378:metabolism; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009705:Nucleosides / Q000737:chemistry; D011485:Protein Binding; D012694:Serine / Q000737:chemistry; D013379:Substrate Specificity; D013936:Thymidine / Q000737:chemistry; D013938:Thymidine Monophosphate / Q000737:chemistry
D015226:Dinucleoside Phosphates; D009705:Nucleosides; D007532:Isoleucine; D013938:Thymidine Monophosphate; D012694:Serine; D015720:5'-Nucleotidase; D013936:Thymidine