ABSTRACT
Fucosylated oligosaccharides present a predominant group of free oligosaccharides found in human milk. Here, a microbial conversion of lactose, D-glucose and L-fucose to fucosylated lacto-N-tetraose by growing Escherichia coli cultures is presented. The recombinant expression of genes encoding for the ß1,3-N-acetylglucosaminyltransferase (LgtA) and the ß1,3-galactosyltransferase (WbgO) enables the whole-cell biotransformation of lactose to lacto-N-tetraose. By the additional expression of a recombinant GDP-L-fucose salvage pathway together with a bacterial fucosyltransferase, lacto-N-tetraose is further converted into the respective fucosylated compounds. The expression of a gene encoding the α1,2-fucosyltransferase (FutC) in the lacto-N-tetraose producing E. coli strain led to the formation of lacto-N-fucopentaose I, whereas the expression of a gene encoding the α1,4-fucosyltransferase (FucT14) mainly led to the conversion of lacto-N-tetraose to lacto-N-difucohexaose II.
Subject(s)
Oligosaccharides/biosynthesis , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Escherichia coli/enzymology , Escherichia coli/metabolism , Fucosyltransferases/metabolism , Galactosyltransferases/genetics , Galactosyltransferases/metabolism , Glycosylation , Humans , Lactose/metabolism , Magnetic Resonance Spectroscopy , Milk, Human/metabolism , N-Acetylglucosaminyltransferases/genetics , N-Acetylglucosaminyltransferases/metabolism , Oligosaccharides/chemistryABSTRACT
For optimal proteolytic function, the central core of the proteasome (core particle (CP) or 20S) has to associate with activators. We investigated the impact of the yeast activator Blm10 on proteasomal peptide and protein degradation. We found enhanced degradation of peptide substrates in the presence of Blm10 and demonstrated that Blm10 has the capacity to accelerate proteasomal turnover of the unstructured protein tau-441 in vitro. Mechanistically, proteasome activation requires the opening of a closed gate, which allows passage of unfolded proteins into the catalytic chamber. Our data indicate that gate opening by Blm10 is achieved via engagement of its C-terminal segment with the CP. Crucial for this activity is a conserved C-terminal YYX motif, with the penultimate tyrosine playing a preeminent role. Thus, Blm10 utilizes a gate opening strategy analogous to the proteasomal ATPases HbYX-dependent mechanism. Because gating incompetent Blm10 C-terminal point mutants confers a loss of function phenotype, we propose that the cellular function of Blm10 is based on CP association and activation to promote the degradation of proteasome substrates.
