Vogt J, Perozzo R, Pautsch A, Prota A, Schelling P, Pilger B, Folkers G, Scapozza L, Schulz G E
Institut für Organische Chemie und Biochemie, Albert-Ludwigs-Universität, Freiburg im Breisgau, Germany.
Proteins. 2000 Dec 1;41(4):545-53. doi: 10.1002/1097-0134(20001201)41:4<545::aid-prot110>3.0.co;2-8.
The crystal structures of the full-length Herpes simplex virus type 1 thymidine kinase in its unligated form and in a complex with an adenine analogue have been determined at 1.9 A resolution. The unligated enzyme contains four water molecules in the thymidine pocket and reveals a small induced fit on substrate binding. The structure of the ligated enzyme shows for the first time a bound adenine analogue after numerous complexes with thymine and guanine analogues have been reported. The adenine analogue constitutes a new lead compound for enzyme-prodrug gene therapy. In addition, the structure of mutant Q125N modifying the binding site of the natural substrate thymidine in complex with this substrate has been established at 2.5 A resolution. It reveals that neither the binding mode of thymidine nor the polypeptide backbone conformation is altered, except that the two major hydrogen bonds to thymidine are replaced by a single water-mediated hydrogen bond, which improves the relative acceptance of the prodrugs aciclovir and ganciclovir compared with the natural substrate. Accordingly, the mutant structure represents a first step toward improving the virus-directed enzyme-prodrug gene therapy by enzyme engineering.
