Cloning and functional expression of alkaline alpha-galactosidase from melon fruit: similarity to plant SIP proteins uncovers a novel family of plant glycosyl hydrolases.
Raffinose and stachyose are ubiquitous galactosyl-sucrose oligosaccharides in the plant kingdom which play major roles, second only to sucrose, in photoassimilate translocation and seed carbohydrate storage. These sugars are initially metabolised by alpha-galactosidases (alpha-gal). We report the cloning and functional expression of the first genes, CmAGA1 and CmAGA2, encoding for plant alpha-gals with alkaline pH optima from melon fruit (Cucumis melo L.), a raffinose and stachyose translocating species. The alkaline alpha-gal genes show very high sequence homology with a family of undefined 'seed imbibition proteins' (SIPs) which are present in a wide range of plant families. In order to confirm the function of SIP proteins, a representative SIP gene, from tomato, was expressed and shown to have alkaline alpha-gal activity. Phylogenetic analysis based on amino acid sequences shows that the family of alkaline alpha-gals shares little homology with the known prokaryotic and eukaryotic alpha-gals of glycosyl hydrolase families 27 and 36, with the exception of two cross-family conserved sequences containing aspartates which probably function in the catalytic step. This previously uncharacterised, plant-specific alpha-gal family of glycosyl hydrolases, with optimal activity at neutral-alkaline pH likely functions in key processes of galactosyl-oligosaccharide metabolism, such as during seed germination and translocation of RFO photosynthate.
Carmi
Nir
N
Institute of Field and Garden Crops, ARO, the Volcani Center, Bet Dagan 50250, Israel.
Zhang
Genfa
G
Petreikov
Marina
M
Gao
Zhifang
Z
Eyal
Yoram
Y
Granot
David
D
Schaffer
Arthur A
AA
D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D028462:Cucumis / Q000145:classification / Q000201:enzymology / Q000235:genetics*; D015971:Gene Expression Regulation, Enzymologic; D018506:Gene Expression Regulation, Plant; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D018551:Solanum lycopersicum / Q000201:enzymology / Q000235:genetics; D008969:Molecular Sequence Data; D005810:Multigene Family; D010802:Phylogeny; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D000519:alpha-Galactosidase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism
D010940:Plant Proteins; D000519:alpha-Galactosidase