Metabolism of galactosyl-oligosaccharides in Stellaria media – Discovery of stellariose synthase, a novel type of galactosyltransferase

Abstract

The raffinose family oligosaccharides (RFOs), including raffinose (Gal-α(1 → 6)-Glc-α(1 → 2)β-Fru), stachyose (Gal-α(1 → 6)-Gal-α(1 → 6)-Glc-α(1 → 2)β-Fru) and higher degree of polymerization RFOs are the most widespread galactosyl-oligosaccharides (GOS) in the plant kingdom. Stellaria media is a typical representative of the Caryophyllaceae, a plant family lacking stachyose and the typical galactosyl extensions of stachyose. During cold treatment raffinose, lychnose (Gal-α(1 → 6)-Glc-α(1 → 2)β-Fru-α(1 → 1)-Gal) and stellariose (Gal-α(1 → 6)-[Gal-α(1 → 4)]-Glc-α(1 → 2)β-Fru-α(1 → 1)-Gal) were found to accumulate in S. media stems. Next to these prominent oligosaccharides, two extra GOS were discovered. Biochemical analyses (enzymatic incubations and mild acid hydrolysis) and mass spectrometry identified the first, most abundant oligosaccharide as Glc-α(1 → 2)β-Fru-α(1 → 1)-Gal, a breakdown product of lychnose. The structure of this trisaccharide was confirmed by full NMR characterization. The second, less abundant compound (termed mediose) was identified as Gal-α(1 → 6)-[Gal-α(1 → 4)]Glc-α(1 → 2)β-Fru after biochemical analyses. By partial enzyme purification the presence of discrete lychnose synthase (raffinose:raffinose 1 Fru galactosyltransferase) and stellariose synthase (raffinose:lychnose 4 Glc galactosyltransferase) activities were shown. A model is presented explaining the structural diversity of GOS in S. media. In the absence of stachyose, raffinose is further elongated by lychnose synthase and stellariose synthase to produce lychnose, mediose and stellariose. Most likely, these compounds are also subject to partial trimming by endogenous α-galactosidases.