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Deoxyadenosine/deoxycytidine kinase from Bacillus subtilis. Purification, characterization, and physiological

H Møllgaard

    The Journal of Biological Chemistry
    |September 10, 1980
    PubMed
    Summary

    Bacillus subtilis possesses distinct deoxyribonucleoside kinases. A purified deoxyadenosine/deoxycytidine kinase requires Mg2+ and GTP, with dATP/dCTP acting as inhibitors, and a mutant lacking this activity shows drug resistance.

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    Area of Science:

    • Biochemistry
    • Molecular Biology
    • Microbiology

    Background:

    • Deoxyribonucleoside kinases are crucial enzymes in DNA synthesis and salvage pathways.
    • Understanding these enzymes in bacteria like Bacillus subtilis provides insights into microbial metabolism and potential drug targets.

    Purpose of the Study:

    • To identify and characterize deoxyribonucleoside kinases in Bacillus subtilis.
    • To purify and determine the kinetic properties of the deoxyadenosine/deoxycytidine kinase.
    • To investigate the enzyme's substrate specificity, cofactor requirements, and inhibition patterns.

    Main Methods:

    • Enzyme purification using blue Sepharose CL-6B chromatography.
    • Polyacrylamide gel electrophoresis (PAGE) and gel filtration for molecular weight determination.

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  • Enzyme kinetics assays to determine Km and Vmax values.
  • Characterization of substrate specificity and cofactor requirements.
  • Analysis of a drug-resistant mutant lacking specific kinase activity.

    • Main Results:

    • Three deoxyribonucleoside kinases were identified, with specificities for thymidine, deoxyguanosine, and deoxyadenosine/deoxycytidine.
    • The deoxyadenosine/deoxycytidine kinase was purified 950-fold, exhibiting a molecular weight of 47,000 Da.
    • The enzyme phosphorylated deoxyadenosine and deoxycytidine with similar kinetics (Km ~5 µM) using GTP as the preferred phosphate donor.
    • Guanosine triphosphate (GTP) was the most effective phosphate donor, while deoxyadenosine triphosphate (dATP) and deoxycytidine triphosphate (dCTP) acted as inhibitors.
    • Enzyme activity required a divalent metal ion, with Mg2+ yielding the highest activity.
    • A spontaneous mutant resistant to 5-fluorodeoxycytidine lacked both deoxycytidine and deoxyadenosine kinase activities.

    Conclusions:

    • Bacillus subtilis possesses distinct deoxyribonucleoside kinases with specialized roles.
    • The characterized deoxyadenosine/deoxycytidine kinase is a monomeric enzyme with specific substrate and cofactor requirements.
    • The enzyme's properties and the characteristics of the resistant mutant provide valuable information for understanding DNA metabolism in bacteria.