Cloning and verification of the Lactococcus lactis pyrG gene and characterization of the gene product, CTP synthase

Steen Lyders Lerche Wadskov-Hansen, M. Willemoës, Jan Martinussen, Karin Hammer, J. Neuhard, S. Larsen

    Research output: Contribution to journalJournal articleResearchpeer-review

    Abstract

    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.
    Original languageEnglish
    JournalJournal of Biological Chemistry
    Volume276
    Issue number41
    Pages (from-to)38002-38009
    ISSN0021-9258
    Publication statusPublished - 2001

    Keywords

    • gram

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