A yeast expression system for human galactose-1-phosphate uridylyltransferase.

Abstract

Galactose-1-phosphate uridylyltransferase (GALT) (UTP: alpha-D-hexose-1-phosphate uridylyltransferase, EC 2.7.7.10) is an essential enzyme of the Leloir pathway of galactose metabolism. Mutations in human GALT are associated with the potentially lethal disorder galactosemia, which affects 1 in 30,000-60,000 live-born infants. Although a number of base substitutions have been identified in the GALT alleles of galactosemia patients, the detailed biochemical impact of these mutations on GALT enzymatic activity remains obscure. Similarly, little is known about the sequence/structure/function relationships for wild-type human GALT. As a first step toward addressing these questions, we have developed a yeast-based expression system for the human enzyme. The wild-type human GALT coding sequence has been introduced into a strain of Saccharomyces cerevisiae that carries a disruption of the GALT-encoding GAL7 gene and, therefore, expresses no endogenous GALT. Transformants were tested for restoration of GALT activity both indirectly, by cell growth on galactose, and directly, by analysis of enzyme activity in cell extracts. The results of both tests were striking; wild-type human GALT functioned in yeast almost as well as the endogenous enzyme. In contrast, cells transformed with either human or yeast GALT sequences engineered to carry a common human GALT mutation, Q188R (changing Gln188 to Arg), exhibited essentially no detectable GALT activity and failed to grow on galactose. Lymphoblasts from patients homozygous for the Q188R mutation similarly exhibited essentially no detectable GALT activity in parallel assays. The results reported here establish the utility of the yeast-based expression system for human GALT and set the stage for more detailed studies of this important enzyme and its role in galactosemia.