Cloning and verification of the Lactococcus lactis pyrG gene and characterization of the gene product, CTP synthase.
The pyrG gene of Lactococcus lactis subsp. cremoris, encoding CTP synthase, has been cloned and sequenced. It is flanked upstream by an open reading frame showing homology to several aminotransferases and downstream by an open reading frame of unknown function. L. lactis strains harboring disrupted pyrG alleles were constructed. These mutants required cytidine for growth, proving that in L. lactis, the pyrG product is the only enzyme responsible for the amination of UTP to CTP. In contrast to the situation in Escherichia coli, an L. lactis pyrG mutant could be constructed in the presence of a functional cdd gene encoding cytidine deaminase. A characterization of the enzyme revealed similar properties as found for CTP synthases from other organisms. However, unlike the majority of CTP synthases the lactococcal enzyme can convert dUTP to dCTP, although a half saturation concentration of 0.6 mm for dUTP makes it unlikely that this reaction plays a significant physiological role. As for other CTP synthases, the oligomeric structure of the lactococcal enzyme was found to be a tetramer, but unlike most of the other previously characterized enzymes, the tetramer was very stable even at dilute enzyme concentrations.
Wadskov-Hansen
S L
SL
Department of Microbiology, Technical University of Denmark, Building 301, DK-2800 Lyngby, Denmark.
Willemoës
M
M
Martinussen
J
J
Hammer
K
K
Neuhard
J
J
Larsen
S
S
D001483:Base Sequence; D019731:Carbon-Nitrogen Ligases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D003001:Cloning, Molecular; D017931:DNA Primers; D004926:Escherichia coli / Q000235:genetics; D005798:Genes, Bacterial; D007700:Kinetics; D013294:Lactococcus lactis / Q000201:enzymology / Q000235:genetics*; D008969:Molecular Sequence Data; D009154:Mutation; D020836:Protein Structure, Quaternary
D017931:DNA Primers; D019731:Carbon-Nitrogen Ligases; C019951:CTP synthetase