CRYSTAL-STRUCTURE OF BACTERIOPHAGE-T4 DEOXYNUCLEOTIDE KINASE WITH ITSSUBSTRATES DGMP AND ATP

NMP kinases catalyse the phosphorylation of the canonical nucleotides to the corresponding diphosphates using ATP as a phosphate donor. Bact eriophage T4 deoxynucleotide kinase (DNK) is the only member of this f amily of enzymes that recognizes three structurally dissimilar nucleot ides: dGMP, dTMP and 5-hydroxymethyl-dCMP while excluding dCMP and dAM P. The crystal structure of DNK with its substrate dGMP has been deter mined at 2.0 Angstrom resolution by single isomorphous replacement. Th e structure of the ternary complex with dGMP and ATP has been determin ed at 2.2 Angstrom resolution. The polypeptide chain of DNK is folded into two domains of equal size, one of which resembles the mononucleot ide binding motif with the glycine-rich P-loop. The second domain, con sisting of five alpha-helices, forms the NMP binding pocket. A hinge c onnection between the domains allows for large movements upon substrat e binding which are not restricted by dimerization of the enzyme. The mechanism of active centre formation via domain closure is described. Comparison with other P-loop-containing proteins indicates an induced- fit mode of NTP binding. Protein-substrate interactions observed at th e NMP and NTP sites provide the basis for understanding the principles of nucleotide discrimination.