Discovery of an alliinase association with a legumin-like storage protein in Allium species subgenus Melanocrommyum

The genus Allium with more than 800 species is one of the largest genera in the plant kingdom [1]. Only a few species thereof are of great economic importance, especially Allium cepa and Allium sativum due to their usage as a condiment. However, in Central Asia other species, especially A. stipitatum, belonging to subgenus Melanocrommyum, are consumed frequently. All Allium species express an alliinase which cleaves cysteine sulfoxides after cell disruption leading to the formation of aroma compounds [2]. Only few other proteins contained in Allium were investigated. In A. sativum, a mannose-binding lectin was discovered, happening to be associated with alliinase [3]. Besides lectin, no further storage proteins in Allium have been reported yet.

On an SDS-gel using reducing conditions, two intense protein spots can be seen between 20 and 30 kDa being characteristic in protein extracts of Melanocrommyum species. Alliinase is always associated with them. Further investigation revealed that those protein subunits are parts of a single protein connected to each other by disulfide bonds. N-terminal sequencing indicated a conserved domain of legumin-like storage proteins. Sequencing with mass spectrometry after trypsine digestion allowed the creation of primers and to obtain the middle part of the gene of that storage protein by PCR. The sequence was amplified to the flanking ends using the method of restriction enzyme site-directed amplification [4]. Thus knowledge of the DNA sequence of that storage protein was achieved. The resulting amino acid sequence has almost no analogy to known sequences. In the ongoing research, the question of modification of the alliinase function by this protein has to be answered.

References:

[1] Fritsch RM et al. Phyton (Horn, Austria) (2010) 49(2):145 – 220

[2] Stoll A, Seebeck E, Helvet Chim Acta. (1949) 32(1): 197 – 205

[3] Rabinkov A, Wilchek M, Mirelman D, Gycoconj j. (1995) 12(5):690 – 698

[4] Gonzáles-Ballester D et al. Anal Biochem. (2005) 340(2):330 – 5