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

Nadia Bucurenci,Hiroshi Sakamoto,Pierre Briozzo,Nicolae Palibroda,Lidia Serina,Robert S Sarfati,Gilles Labesse,Gilbert Briand,Antoine Danchin,Octavian Bârzu,Anne-Marie Gilles

doi:10.1074/jbc.271.5.2856

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 A and c = 60.7 A, and diffract x-rays to a 1.9 A 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 (Trp30) 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.

Highlights

Nucleoside monophosphate (NMP)1 kinases represent an b Present address: Institut Cantacuzene, Bucharest, Romania. d Present address: Institut National Agronomique Paris-Grignon, 78850 Thiverval-Grignon, France. e Present address: Institut de Technologie Isotopique et Moleculaire, Cluj-Napoca, Romania. j To whom correspondence should be addressed: Unitede Biochimie des Regulations Cellulaires, Institut Pasteur, 28, rue du Docteur Roux, 75724 Paris Cedex 15, France
An insertion of 32–36 amino acids between helices ␣2Ј and ␣3 was present in CMP kinase from E. coli, B. subtilis, and M. leprae
CMP kinase from E. coli has a single Cys residue (Cys21) that is conserved in adenylate kinase 1 and UMP kinase from D. discoideum but not in CMP kinase from B. subtilis and M. leprae

Summary

EXPERIMENTAL PROCEDURES

Chemicals—Adenine, cytidine, and uridine nucleotides, restriction enzymes, T4DNA ligase, and coupling enzymes were from Boehringer Mannheim. Ant-dATP, Ant-dADP, Ant-dAMP, and Ant-dCMP were synthesized according to published procedures (Hiratsuka, 1982; Sarfati et al, 1990) from isatoic anhydride and the corresponding deoxynucleoside phosphates. Bacterial Strains, Plasmids, Growth Conditions, and DNA Manipulations—The cmk/mssA gene encoding CMP kinase was amplified by polymerase chain reaction using chromosomal DNA from the E. coli strain NM554 (Raleigh et al, 1988) as the matrix. Fluorescence Measurements—The emission spectrum of CMP kinase (␭exc ϭ 295 nm; bandwidth, 5 nm) was recorded from 305 to 400 nm using a Perkin-Elmer LS-5B luminescence spectrometer thermostated at 25 °C using a 1 ϫ 1-cm UV-grade quartz cuvette (sample volume, 2 ml). The N-terminal amino acid sequence of the protein from the excised band was determined by a protein sequencer (Applied Biosystems Inc.)

RESULTS

UMP dUMP

TABLE III

DISCUSSION