CMP Kinase from Escherichia coli Is Structurally Related to Other Nucleoside Monophosphate Kinases

Abstract

CMP kinase from Escherichia coli is a monomeric protein of 225 amino acid residues. The protein exhibits little overall sequence similarities with other known NMP kinases. However, residues involved in binding of substrates and/or in catalysis were found conserved, and sequence comparison suggested conservation of the global fold found in adenylate kinases or in several CMP/UMP kinases. The enzyme was purified to homogeneity, crystallized, and analyzed for its structural and catalytic properties. The crystals belong to the hexagonal space group P6 3 , have unit cell parameters a ‫؍‬ b ‫؍‬ 82.3 Å and c ‫؍‬ 60.7 Å, and diffract x-rays to a 1.9 Å resolution. The bacterial enzyme exhibits a fluorescence emission spectrum with maximum at 328 nm upon excitation at 295 nm, which suggests that the single tryptophan residue (Trp 30 ) is located in a hydrophobic environment. Substrate specificity studies showed that CMP kinase from E. coli is active with ATP, dATP, or GTP as donors and with CMP, dCMP, and arabinofuranosyl-CMP as acceptors. This is in contrast with CMP/UMP kinase from Dictyostelium discoideum, an enzyme active on CMP or UMP but much less active on the corresponding deoxynucleotides. Binding of CMP enhanced the affinity of E. coli CMP kinase for ATP or ADP, a particularity never described in this family of proteins that might explain inhibition of enzyme activity by excess of nucleoside monophosphate.

Structural and Functional Characterization of Escherichia coli UMP Kinase in Complex with Its Allosteric Regulator GTP

“…Taking in account that these two Gly and Glu residues are well conserved in bacterial UMPKs, we suggest that some structural rearrangement induced by GTP and probably involving Asp-93 would favor the particular orientation of Arg-62 side chain that is not compatible with an efficient binding of UTP. The substitution of Arg-62 in UMPKeco by a histidine resulted in a far less active enzyme, which was no more inhibited by excess of UMP (29). In addition, the dimer-dimer contacts induced by GTP could also intervene in UTP release.…”

“…In particular, the side chains of Thr-144 and Asp-201 (residues conserved in bacterial UMP kinases) have a common orientation in the UMPKeco structures with UMP, UDP, or GTP but flip to a different orientation in the UMPKeco-UTP complex. Asp-201 variants of the enzyme have been studied in two Gram-negative bacteria: a D201N variant in E. coli (29) and a D201G variant in Salmonella typhimurium (30). Both variants were still inhibited by UTP but no more activated by GTP.…”

Use of pyrimidine pathway to produce mutant plants that fail to respond to wound induction

“…It seems that the binding of one substrate affects the affinity of these mutant enzymes for the other substrate. The similar case has been found in CMP kinase from E. coli (Bucurenci et al 1996). The E coli CMP kinase shows little overall sequence similarities with other known NMP kinases, but it contained the conserved sequences involved in substrate binding and catalysis.…”

“…Whereas all eukaryotic enzymes have specificity for both UMP and CMP, the yeast enzyme also has specificity for AMP. Indeed, the yeast UMP kinase has such a high affinity for .AMP that the ura6 gene has been isolated as multicopy suppressors of yeast deficient in adenylate kinase (Schricker et al, 1992 In addition to studies of tfie eukaryotic enzyme, the prokaryotic enzyme has also received much attention (Yamanaka et al, 1992;Valentin-Hansen, 1978;Serina et al, 1995Serina et al, , 1996. Because of this, significant differences between the enzymatic activities of the prokaryotic and eukaryotic enzymes have been identified.…”

“…GTP functions to stimulate the enzyme when there is an overabundance of purine triphosphates, and UTP down regulates the enzyme when pyrimidine triphosphates have accumulated to a high level. In addition, UMP kinase is further regulated by divalent metal ions in a novel mechanism related to metal free UTP binding (Serina et al, 1996). The binding of metal-free UTP causes a gelsol transition that affects the state of UMP kinase aggregation and subsequently the enzyme activity.…”