Tài liệu Báo cáo khoa học: Functional studies of active-site mutants from Drosophila melanogaster

Functional studies of active-site mutants from Drosophila

melanogaster deoxyribonucleoside kinase

Investigations of the putative catalytic glutamate–arginine pair and

of residues responsible for substrate specificity

Louise Egeblad-Welin1,2,*, Yonathan Sonntag1,*, Hans Eklund3 and Birgitte Munch-Petersen1

1 Department of Science, Systems and Models, Roskilde University, Denmark

2 Department of Molecular Biosciences, Swedish University of Agricultural Sciences, Uppsala Biomedical Centre, Sweden

3 Department of Molecular Biology, Swedish University of Agricultural Sciences, Uppsala Biomedical Centre, Sweden

Drosophila melanogaster deoxyribonucleoside kinase

(Dm-dNK) phosphorylates the four natural deoxyribo￾nucleosides, thymidine, deoxycytidine, deoxyadenosine

and deoxyguanosine, which is a crucial step in the bio￾synthesis of DNA precursors via the salvage pathway.

In addition, Dm-dNK phosphorylates a number of

important nucleoside analogue pro-drugs [1,2], making

it a potential candidate for use in suicide gene therapy.

Keywords

catalytic mechanism; deoxyribonucleoside

kinase; dTTP; enzyme kinetics; nucleoside

analogues

Correspondence

B. Munch-Petersen, Department of Science,

Systems and Models, Roskilde University,

Box 260, DK 4000 Roskilde, Denmark

Fax: +45 46743011

Tel: +45 46742418

E-mail: [email protected]

L. Egeblad-Welin, Department of Molecular

Biosciences, Swedish University of

Agricultural Sciences, Box 575, Biomedical

Center, S-751 25 Uppsala, Sweden

Fax: +46 18536971

Tel. +46 184714192

E-mail: [email protected]

*These authors contributed equally to this

work

(Received 2 November 2006, revised 4

January 2007, accepted 16 January 2007)

doi:10.1111/j.1742-4658.2007.05701.x

The catalytic reaction mechanism and binding of substrates was investi￾gated for the multisubstrate Drosophila melanogaster deoxyribonucleoside

kinase. Mutation of E52 to D, Q and H plus mutations of R105 to K and

H were performed to investigate the proposed catalytic reaction mech￾anism, in which E52 acts as an initiating base and R105 is thought to sta￾bilize the transition state of the reaction. Mutant enzymes (E52D, E52H

and R105H) showed a markedly decreased kcat, while the catalytic activity

of E52Q and R105K was abolished. The E52D mutant was crystallized

with its feedback inhibitor dTTP. The backbone conformation remained

unchanged, and coordination between D52 and the dTTP–Mg complex

was observed. The observed decrease in kcat for E52D was most likely due

to an increased distance between the catalytic carboxyl group and 5¢-OH of

deoxythymidine (dThd) or deoxycytidine (dCyd). Mutation of Q81 to N

and Y70 to W was carried out to investigate substrate binding. The muta￾tions primarily affected the Km values, whereas the kcat values were of the

same magnitude as for the wild-type. The Y70W mutation made the

enzyme lose activity towards purines and negative cooperativity towards

dThd and dCyd was observed. The Q81N mutation showed a 200- and

100-fold increase in Km, whereas kcat was decreased five- and twofold for

dThd and dCyd, respectively, supporting a role in substrate binding. These

observations give insight into the mechanisms of substrate binding and

catalysis, which is important for developing novel suicide genes and drugs

for use in gene therapy.

Abbreviations

ACV, 9-(2-hydroxyethoxymethyl)-guanine; AraA, 9-(b-D-arabinofuranosyl)-adenine; AraC, 1-(b-D-arabinofuranosyl)-cytosine; AraT,

1-(b-D-arabinofuranosyl)-thymine; BVDU, (E)-bromvinyl-2¢-deoxyuridine; CdA, 2-chloro-2¢-deoxyadenosine; dAdo, deoxyadenosine; dCK,

cytosolic deoxycytidine kinase; dCyd, deoxycytidine; dGK, deoxyguanosine kinase; dGuo, deoxyguanosine; Dm-dNK, Drosophila

melanogaster deoxyribonucleoside kinase; dThd, deoxythymidine; F-AraA, 2-flouro-9-(b-D-arabinofuranosyl)-adenine; FdUrd, 5-flouro-2¢-

deoxyuridine; HSV1-TK, Herpes simplex virus Type 1 thymidine kinase; TK, thymidine kinase.

1542 FEBS Journal 274 (2007) 1542–1551 ª 2007 The Authors Journal compilation ª 2007 FEBS