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

Area of Science:

• Biochemistry
• Genetics
• Molecular Biology

Background:

• Galactose-1-phosphate uridylyltransferase (GALT) is crucial for galactose metabolism via the Leloir pathway.
• Mutations in human GALT cause galactosemia, a severe infant disorder affecting 1 in 30,000-60,000 newborns.
• The biochemical impact of GALT mutations and its sequence-structure-function relationships are poorly understood.

Purpose of the Study:

• To establish a yeast-based expression system for human GALT.
• To investigate the functional consequences of wild-type and mutated human GALT.
• To provide a platform for future studies on GALT and galactosemia.

Main Methods:

• Developed a Saccharomyces cerevisiae strain with a disrupted GAL7 gene (lacking endogenous GALT).
• Introduced wild-type and Q188R-mutated human GALT coding sequences into the yeast system.
• Assessed GALT activity indirectly via cell growth on galactose and directly via enzyme assays in cell extracts.
• Analyzed lymphoblasts from galactosemia patients homozygous for the Q188R mutation.

Main Results:

• Wild-type human GALT expressed in yeast restored enzyme activity and supported cell growth on galactose.
• Human and yeast GALT with the Q188R mutation showed no detectable activity and failed to rescue growth.
• Lymphoblasts from Q188R galactosemia patients also exhibited negligible GALT activity.

Conclusions:

• The yeast expression system is a validated tool for studying human GALT function.
• The Q188R mutation severely impairs GALT enzymatic activity, consistent with galactosemia.
• This system facilitates further research into GALT's role in galactosemia and enzyme mechanisms.