Structure-function analysis of a bacterial deoxyadenosine kinase reveals the basis for substrate specificity.
Deoxyribonucleoside kinases (dNKs) catalyze the transfer of a phosphoryl group from ATP to a deoxyribonucleoside (dN), a key step in DNA precursor synthesis. Recently structural information concerning dNKs has been obtained, but no structure of a bacterial dCK/dGK enzyme is known. Here we report the structure of such an enzyme, represented by deoxyadenosine kinase from Mycoplasma mycoides subsp. mycoides small colony type (Mm-dAK). Superposition of Mm-dAK with its human counterpart's deoxyguanosine kinase (dGK) and deoxycytidine kinase (dCK) reveals that the overall structures are very similar with a few amino acid alterations in the proximity of the active site. To investigate the substrate specificity, Mm-dAK has been crystallized in complex with dATP and dCTP, as well as the products dCMP and dCDP. Both dATP and dCTP bind to the enzyme in a feedback-inhibitory manner with the dN part in the deoxyribonucleoside binding site and the triphosphates in the P-loop. Substrate specificity studies with clinically important nucleoside analogs as well as several phosphate donors were performed. Thus, in this study we combine structural and kinetic data to gain a better understanding of the substrate specificity of the dCK/dGK family of enzymes. The structure of Mm-dAK provides a starting point for making new anti bacterial agents against pathogenic bacteria.
Welin
Martin
M
Department of Molecular Biology, Swedish University of Agricultural Sciences, Box 590, Biomedical Center, S-751 24 Uppsala, Sweden.
Wang
Liya
L
Eriksson
Staffan
S
Eklund
Hans
H
D000595:Amino Acid Sequence; D000596:Amino Acids; D001426:Bacterial Proteins / Q000032:analysis / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D001665:Binding Sites; D017124:Conserved Sequence; D018360:Crystallography, X-Ray; D003838:Deoxyadenine Nucleotides / Q000378:metabolism; D003845:Deoxycytosine Nucleotides / Q000378:metabolism; D019281:Dimerization; D015195:Drug Design; D006801:Humans; D006860:Hydrogen Bonding; D008024:Ligands; D008958:Models, Molecular; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009176:Mycoplasma mycoides / Q000201:enzymology; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000032:analysis / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D011485:Protein Binding; D017433:Protein Structure, Secondary; D017386:Sequence Homology, Amino Acid; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
D000596:Amino Acids; D001426:Bacterial Proteins; D003838:Deoxyadenine Nucleotides; D003845:Deoxycytosine Nucleotides; D008024:Ligands; C024107:2'-deoxycytidine 5'-triphosphate; D017853:Phosphotransferases (Alcohol Group Acceptor); C020005:deoxyadenosine kinase; C026600:2'-deoxyadenosine triphosphate