Identification of Residues Involved in the Specificity and Regulation of the Highly Efficient Multisubstrate Deoxyribonucleoside Kinase from Drosophila melanogaster Wolfgang Knecht 1 *, Birgitte Munch-Petersen 2 and Jure Pis Ï kur 1 1 Department of Microbiology Building 301, Technical University of Denmark DK 2800, Lyngby, Denmark 2 Department of Life Sciences and Chemistry, Roskilde University, P.O.Box 260 DK 4000, Roskilde, Denmark In contrast to all known deoxyribonucleoside kinases, a single highly ef®- cient deoxyribonucleoside kinase from Drosophila melanogaster (Dm-dNK) is able to phosphorylate all precursor nucleosides for DNA synthesis. Dm-dNK was mutated in vitro by high-frequency random mutagenesis, expressed in the thymidine kinase-de®cient Escherichia coli strain KY895 and clones were selected for sensitivity to the nucleoside analogs 1-beta- D-arabinofuranosylcytosine (AraC, Cytarabine), 3 0 -azido-2 0 ,3 0 -dideoxythy- midine (AZT, Zidovudine, Retrovir 1 ), 2 0 ,3 0 -dideoxyadenosine (ddA) and 2 0 ,3 0 -dideoxycytidine (ddC, Zalcitabine, Hivid 1 ). Thirteen mutants with increased sensitivity compared to the wild-type Dm-dNK were isolated from a relatively small pool of less than 10,000 clones. Eight mutant Dm- dNKs increased the sensitivity of KY895 to more than one analog, and two of these mutants even to all four nucleoside analogs. Surprisingly, the mutations did not map to the ®ve regions which are highly con- served among deoxyribonucleoside kinases. The molecular background of improved sensitivity was characterized for the double-mutant MuD (N45D, N64D), where the LD 100 value of transformed KY895 decreased 316-fold for AZT and more than 11-fold for ddC when compared to wild-type Dm-dNK. Puri®ed recombinant MuD displayed higher K m values for the native substrates than wild-type Dm-dNK and the V max values were substantially lower. On the other hand, the K m and V max values for AZT and the K m value for ddC were nearly unchanged between MuD and wild-type Dm-dNK. Additionally, a decrease in feed- back inhibition of MuD by thymidine triphosphate (TTP) was found. This study demonstrates how high-frequency mutagenesis combined with a parallel selection for desired properties provides an insight into the structure-function relationships of the multisubstrate kinase from D. melanogaster. At the same time these mutant enzymes exhibit proper- ties useful in biotechnological and medical applications. # 2000 Academic Press Keywords: deoxyribonucleoside kinase; directed protein evolution; hypermutagenesis; gene therapy; nucleoside analogs *Corresponding author E-mail address of the corresponding author: imwk@pop.dtu.dk Abbreviations used: ACV, acyclic guanosine; (d)Ado, (deoxy)adenosine; AraA, 9-beta-D-arabinofuranosyladenosine; AraC, 1-beta-D-arabinofuranosylcytosine; AraT, 1-beta-D-arabinofuranosylthymine; AZT, 3 0 -azido-2 0 ,3 0 - dideoxythymidine; AZTMP, 3 0 -azido-2 0 ,3 0 -dideoxythymidine monophosphate; AZTTP, 3 0 -azido-2 0 ,3 0 -dideoxythymidine triphosphate; BVDU, 5-bromo-vinyl-deoxyuridine; CdA, 2-chloro-deoxyadenosine; (d)Cyd, (deoxy)cytidine; D4T, 2 0 ,3 0 - didehydro-3 0 -deoxythymidine, 3 0 -dA, 3 0 -deoxyadenosine; dAK, deoxyadenosine kinase; dCK, deoxycytidine kinase; ddCyd, 2 0 ,3 0 -dideoxycytidine; dGK, deoxyguanosine kinase; (d)dNTP, (di)deoxynucleoside triphosphate; Dm-dNK, Drosophila melanogaster deoxyribonucleoside kinase; (d)Guo, (deoxy)guanosine; dThd, thymidine; (d)Urd, (deoxy)uridine; ddAdo, ddCyd, ddGuo and ddThd, 2 0 ,3 0 -dideoxyribonucleosides; FdUrd, 5-¯uorodeoxyuridine; HSV, herpes simplex virus; Mu, mutant; ORF, open reading frame; PCR, polymerase chain reaction; rDm-dNK, recombinant Drosophila melanogaster deoxyribonucleoside kinase; SD, standard deviation; TK, thymidine kinase; TK2, mitochondrial thymidine kinase; TMP, thymidine monophosphate; TMPK, TMP kinase. doi:10.1006/jmbi.2000.3990 available online at http://www.idealibrary.com on J. Mol. Biol. (2000) 301, 827±837 0022-2836/00/040827±11 $35.00/0 # 2000 Academic Press