Purification and characterization of stachyose synthase from lentil (Lens culinaris) seeds: galactopinitol and stachyose synthesis.

Stachyose synthase (STS) (EC 2.4.1.67) was purified 313-fold from mature seeds of lentil. The final preparation had a specific activity of 9.09 nkat stachyose formed per milligram of protein. The enzyme was a monomeric protein with a molecular mass of 88.6 kDa (SDS-PAGE) and an isoelectric point of 4.8 (chromatofocusing). Western analysis revealed cross-reactivity of polyclonal antibodies raised against STS from adzuki bean with the lentil enzyme. The purified enzyme catalyzed a range of different galactosyl transfer reactions. In addition to the genuine STS reaction (raffinose + galactinol --> stachyose + myo-inositol), the enzyme catalyzed the reversible galactosyl transfer from galactinol to d-pinitol (1d-3-O-methyl-chiro-inositol), yielding galactopinitol A (O-alpha-d-galactopyranosyl-(1 --> 2)-4-O-methyl-d-chiro-inositol) and myo-inositol. Galactopinitol A could be further galactosylated by STS to give ciceritol (O-alpha-d-galactopyranosyl-(1 --> 6)-O-alpha-d-galactopyranosyl-(1 --> 2)-4-O-methyl-d-chiro-inositol). Enzymatic synthesis of galactopinitol A and ciceritol is a new observation. However, STS was not only able to utilize galactopinitol A as galactosyl acceptor, but also as galactosyl donor to form stachyose from raffinose. The role of STS in the metabolism of galactosyl cyclitols and oligosaccharides in plant seeds is discussed.