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

dCMP deaminase from Bacillus subtilis has been purified 700-fold. In addition to the substrate, dCMP, the enzyme requires dCTP, Zn2+, and 2-mercaptoethanol, Mg2+ cannot substitute for Zn2+. The dCMP saturation curve is hyperbolic in the presence of saturating concentrations of dCTP and Zn2+. The dCTP saturation curve is sigmoidal, the sigmoidicity being dependent on the Zn2+ and dCMP concentrations. The molecular weight as determined by gel filtration is 170,000 both in the presence and in the absence of dCTP and Zn2+. In the absence of thiols, the enzyme is highly unstable. At 0 degrees, the half-life of the enzyme activity is 30 min. Addition of Zn2+ and dCTP protects against this inactivation. In the presence of a thiol, dCTP and Zn2+ protect the enzyme against heat inactivation at 50 degrees. A mutant lacking dCMP deaminase (dcd) was isolated. Labeling of the pyrimidine nucleotide pools reveals that in the parent strain, 45% of the dTTP pool is derived via dCMP deamination, the residual 55% being derived via reduction of a uridine nucleotide. Since the dcd mutant grows with the same doubling time as the parent strain, we conclude that uridine nucleotide reduction alone is capable of supplying sufficient dUMP for normalthymidine nucleotide synthesis.