Pasteurella multocida thymidine kinase 1 efficiently activates pyrimidine nucleoside analogs

Area of Science:

• Microbiology
• Molecular Biology
• Biochemistry

Background:

• The Pasteurella multocida genome contains a single gene encoding a putative deoxyribonucleoside kinase, identified as thymidine kinase 1 (PmTK1).
• Understanding the substrate specificity and enzymatic activity of PmTK1 is crucial for exploring its potential applications.

Purpose of the Study:

• To characterize the Pasteurella multocida thymidine kinase 1 (PmTK1) enzyme.
• To investigate the substrate specificity of PmTK1 and its potential as a drug target or activator.

Main Methods:

• Sub-cloning of the PmTK1 gene into Escherichia coli.
• Over-expression and purification of PmTK1 using two different affinity tags.
• Sensitization of E. coli to various nucleoside analogs, including gemcitabine (dFdC), AZT, and 5F-dU.

Main Results:

• The expressed PmTK1 sensitized E. coli to gemcitabine, AZT, and 5F-dU, indicating its kinase activity.
• Purified PmTK1 demonstrated a preference for deoxyuridine (dU) as a substrate over thymidine (dT).

Conclusions:

• Pasteurella multocida thymidine kinase 1 (PmTK1) is a functional deoxyribonucleoside kinase.
• PmTK1's substrate preference suggests its potential utility as a species-specific activator for uracil-based nucleoside antibiotics.