Abstract
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.
MeSH terms
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Adenine / analogs & derivatives*
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Adenine / chemistry
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Adenine / metabolism
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Adenosine / analogs & derivatives
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Adenosine / chemistry
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Adenosine / metabolism
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Adenosine Monophosphate / analogs & derivatives
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Adenosine Monophosphate / chemistry
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Adenosine Monophosphate / metabolism
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Amino Acid Substitution
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Antiviral Agents / chemistry
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Antiviral Agents / metabolism
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Binding Sites
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Catalytic Domain
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Crystallography, X-Ray
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Enzyme Inhibitors / chemistry
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Enzyme Inhibitors / metabolism
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Herpesvirus 1, Human / chemistry*
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Herpesvirus 1, Human / isolation & purification
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Herpesvirus 1, Human / metabolism
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Mutation
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Nucleosides / chemistry
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Nucleosides / metabolism*
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Organophosphonates*
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Prodrugs / chemistry
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Prodrugs / metabolism
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Protein Structure, Tertiary
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Stereoisomerism
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Substrate Specificity
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Thymidine / chemistry
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Thymidine / metabolism
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Thymidine Kinase / antagonists & inhibitors
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Thymidine Kinase / chemistry*
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Thymidine Kinase / metabolism
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Viral Proteins / chemistry
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Viral Proteins / isolation & purification
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Viral Proteins / metabolism
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Water / metabolism
Substances
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9-(2-hydroxypropyl)adenine
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Antiviral Agents
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Enzyme Inhibitors
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Nucleosides
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Organophosphonates
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Prodrugs
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Viral Proteins
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Water
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Adenosine Monophosphate
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adefovir
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Thymidine Kinase
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Adenine
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Adenosine
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Thymidine