Escherichia coli UMP-kinase, a member of the aspartokinase family, is a hexamer regulated by guanine nucleotides and UTP.
The pyrH gene, encoding UMP-kinase from Escherichia coli, was cloned using as a genetic probe the property of the carAB operon to be controlled for its expression by the concentration of cytoplasmic UTP. The open 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. 174, 7517-7526], previously described as being involved in chromosome partitioning in E. coli. The bacterial UMP-kinase did not display significant sequence similarity to known nucleoside monophosphate kinases. On the contrary, it exhibited similarity with three families of enzymes including aspartokinases, glutamate kinases, and Pseudomonas aeruginosa carbamate kinase. UMP-kinase overproduced in E. coli was purified to homogeneity and analyzed for its structural and catalytic properties. The protein consists of six identical subunits, each of 240 amino acid residues (the N-terminal methionine residue is missing in the expressed protein). Upon excitation at 295 nm, the bacterial enzyme exhibits a fluorescence emission spectrum with maximum at 332 nm which indicates that the single tryptophan residue of the protein (Trp119) is located in a hydrophobic environment. Like other enzymes involved in the de novo synthesis of pyrimidine nucleotides, UMP-kinase of E. coli is subject to regulation by nucleotides: GTP is an allosteric activator, whereas 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.(ABSTRACT TRUNCATED AT 250 WORDS)
Serina
L
L
Unité de Biochimie des Régulations Cellulaires, Institut Pasteur, Paris, France.
Blondin
C
C
Krin
E
E
Sismeiro
O
O
Danchin
A
A
Sakamoto
H
H
Gilles
A M
AM
Bârzu
O
O
D000595:Amino Acid Sequence; D001222:Aspartate Kinase; D001483:Base Sequence; D003001:Cloning, Molecular; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D006150:Guanine Nucleotides / Q000378:metabolism*; D006160:Guanosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009703:Nucleoside-Phosphate Kinase / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D010802:Phylogeny; D013050:Spectrometry, Fluorescence; D014544:Uridine Triphosphate / Q000378:metabolism*
D006150:Guanine Nucleotides; C037756:2-(guanylformylmethoxy)-3-(triphospho)propanal; D006160:Guanosine Triphosphate; D001222:Aspartate Kinase; C023276:uridine monophosphate kinase; D009703:Nucleoside-Phosphate Kinase; D014544:Uridine Triphosphate