ESCHERICHIA-COLI UMP-KINASE, A MEMBER OF THE ASPARTOKINASE FAMILY, ISA HEXAMER REGULATED BY GUANINE-NUCLEOTIDES AND UTP

The pyrH gene, encoding UMP-kinase from Escherichia coli, was cloned u sing as a genetic probe the property of the carAB operon to be control led for its expression by the concentration of cytoplasmic UTP. The op en reading frame of the pyrH gene of 723 bp was found to be identical to that of the smbA gene [Yamanaka, K., et al. (1992) J. Bacteriol. 17 4, 7517-7526], previously described as being involved in chromosome pa rtitioning in E. coli. The bacterial UMP-kinase did not display signif icant sequence similarity to known nucleoside monophosphate kinases. O n the contrary, it exhibited similarity with three families of enzymes including aspartokinases, glutamate kinases, and Pseudomonas aerugino sa carbamate kinase. UMP-kinase overproduced in E. coli was purified t o homogeneity and analyzed for its structural and catalytic properties . The protein consists of six identical subunits, each of 240 amino ac id residues (the N-terminal methionine residue is missing in the expre ssed protein). Upon excitation at 295 nm, the bacterial enzyme exhibit s a fluorescence emission spectrum with maximum at 332 nm which indica tes that the single tryptophan residue of the protein (Trp119) is loca ted in a hydrophobic environment. Like other enzymes involved in the d e novo synthesis of pyrimidine nucleotides, UMP-kinase of E. coli is s ubject to regulation by nucleotides: GTP is an allosteric activator, w hereas UTP serves as an allosteric inhibitor. UTP and UDP, but none of the other nucleotides tested such as GTP, ATP, and UMP, enhanced the fluorescence of the protein. The sigmoidal shape of the dose-response curve indicated cooperativity in binding of UTP and UDP. A UMP-kinase mutant (D201N) recognized earlier as responsible for altered morpholog ical phenotype in E. coli (Yamanaka et al., 1992) was analyzed for its stability and kinetic properties. The protein exhibited 10% of the ac tivity of the wild-type enzyme and had altered stability and regulator y properties. This favors the hypothesis that UMP-kinase, i.e., SmbA p rotein, participates only indirectly in cell division.