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Deoxycytidylate deaminase from Bacillus subtilis. Purification, characterization, and physiological function.

H Møllgaard, J Neuhard

    The Journal of Biological Chemistry
    |May 25, 1978
    PubMed
    Summary

    Bacillus subtilis dCMP deaminase requires specific cofactors for activity and stability. The enzyme is essential for only 45% of dTTP synthesis, as alternative pathways support growth.

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    Area of Science:

    • Biochemistry
    • Enzymology
    • Molecular Biology

    Background:

    • Deoxycytidine monophosphate (dCMP) deaminase is a key enzyme in pyrimidine nucleotide metabolism.
    • Understanding its catalytic properties and physiological role is crucial for comprehending DNA synthesis pathways.
    • Bacillus subtilis provides a model system for studying bacterial enzyme function and regulation.

    Purpose of the Study:

    • To purify and characterize dCMP deaminase from Bacillus subtilis.
    • To investigate the enzyme's cofactor requirements, kinetic properties, and stability.
    • To determine the contribution of dCMP deaminase to thymidine nucleotide synthesis in vivo.

    Main Methods:

    • Enzyme purification using standard biochemical techniques.
    • Enzyme kinetics studies involving varying substrate and cofactor concentrations.

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  • Enzyme stability assays under different conditions (temperature, presence of cofactors).
  • Isolation and characterization of a dCMP deaminase deficient (dcd) mutant.
  • Analysis of pyrimidine nucleotide pools in parent and mutant strains.

    • Main Results:

    • Purified dCMP deaminase exhibited 700-fold enrichment and required dCTP, Zn2+, and 2-mercaptoethanol.
    • Enzyme kinetics revealed hyperbolic dCMP saturation and sigmoidal dCTP saturation, dependent on Zn2+ and dCMP.
    • The enzyme demonstrated instability without thiols but was protected by Zn2+ and dCTP against heat inactivation.
    • A dcd mutant showed normal growth, indicating that alternative pathways supply sufficient dUMP for thymidine synthesis.

    Conclusions:

    • Bacillus subtilis dCMP deaminase possesses unique cofactor requirements and regulatory properties.
    • The enzyme is relatively unstable but can be stabilized by its cofactors.
    • dCMP deaminase is dispensable for growth in Bacillus subtilis, as uridine nucleotide reduction alone adequately supports dUMP production for thymidine synthesis.