In vivo and in vitro function of human UDP-galactose 4′-epimerase variants

Thomas J. McCorvie, Jamie Wasilenko, Ying Liu, Judith L. Fridovich-Keil, David J. Timson

    Research output: Contribution to journalArticlepeer-review


    Type III galactosemia results from reduced activity of the enzyme UDP-galactose 4′-epimerase. Five disease-associated alleles (G90E, V94M, D103G, N34S and L183P) and three artificial alleles (Y105C, N268D, and M284K) were tested for their ability to alleviate galactose-induced growth arrest in a Saccharomyces cerevisiae strain which lacks endogenous UDP-galactose 4′-epimerase. For all of these alleles, except M284K, the ability to alleviate galactose sensitivity was correlated with the UDP-galactose 4′-epimerase activity detected in cell extracts. The M284K allele, however, was able to substantially alleviate galactose sensitivity, but demonstrated near-zero activity in cell extracts. Recombinant expression of the corresponding protein in Escherichia coli resulted in a protein with reduced enzymatic activity and reduced stability towards denaturants in vitro. This lack of stability may result from the introduction of an unpaired positive charge into a bundle of three α-helices near the surface of the protein. The disparities between the in vivo and in vitro data for M284K-hGALE further suggest that there are additional, stabilising factors present in the cell. Taken together, these results reinforce the need for care in the interpretation of in vitro, enzymatic diagnostic tests for type III galactosemia.

    Original languageEnglish
    Pages (from-to)1747-1754
    Number of pages8
    Issue number10
    Publication statusPublished - 1 Oct 2011


    • Disease-associated mutation
    • GALE
    • Type III galactosemia
    • UDP-galactose 4′-epimerase
    • Yeast model


    Dive into the research topics of 'In vivo and in vitro function of human UDP-galactose 4′-epimerase variants'. Together they form a unique fingerprint.

    Cite this