Cloning and functional expression of alkaline α‐galactosidase from melon fruit: similarity to plant SIP proteins uncovers a novel family of plant glycosyl hydrolases

Abstract

SummaryRaf®nose 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 a-galactosidases (a-gal). We report the cloning and functional expression of the ®rst genes, CmAGA1 and CmAGA2, encoding for plant a-gals with alkaline pH optima from melon fruit (Cucumis melo L.), a raf®nose and stachyose translocating species. The alkaline a-gal genes show very high sequence homology with a family of unde®ned`seed imbibition proteins' (SIPs) which are present in a wide range of plant families. In order to con®rm the function of SIP proteins, a representative SIP gene, from tomato, was expressed and shown to have alkaline a-gal activity. Phylogenetic analysis based on amino acid sequences shows that the family of alkaline a-gals shares little homology with the known prokaryotic and eukaryotic a-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-speci®c a-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.

Cloning, Functional Expression, and Characterization of the Raffinose Oligosaccharide Chain Elongation Enzyme, Galactan:Galactan Galactosyltransferase, from Common Bugle Leaves

“…Phylogenetic analysis shows that GGT groups with acid a-Gals and clearly separates from raffinose synthases and stachyose synthases, which are more closely related to alkaline a-Gals/SIPs from family 36 of glycosylhydrolases (Carmi et al, 2003; Fig. 3).…”

“…3, subgroup I; 64% to 78% identical). It does not group with the recently defined alkaline a-Gals/SIPs (Carmi et al, 2003;Fig. 3, subgroup II;25% to 27% identical), nor with the galactinol-dependent RFO biosynthetic enzymes, raffinose synthases (Fig.…”

Nicotiana benthamiana α-galactosidase A1.1 can functionally complement human α-galactosidase A deficiency associated with Fabry disease

“…Based on primary and secondary structures, they are classified into the three glycoside hydrolase families 4, 27 and 36. Family 27 contains mainly eukaryotic α-galactosidases removing terminal α-galactosyl moieties from glycoconjugates (1)(2)(3). Plant α-galactosidases are so far known to participate in the catabolism of galactosyl-sucrose oligosaccharides, raffinose family of oligosaccharides and cell wall galactomannans primarily during germination of seeds (1,(4)(5)(6)(7).…”

“…A well-established classification of the plant enzymes is based on their pH optimum for enzymatic activity: acidic ones with broad pH optima from 4.5 to 6.5, and alkaline ones with pH optimum between 7.0 -7.5 (1,10). These two classes of plant α-galactosidases might differ in localization.…”

“…These two classes of plant α-galactosidases might differ in localization. The acidic α-galactosidases most likely locate inside the vacuoles and apoplast, whereas alkaline α-galactosidases act in the cytoplasm with a neutral pH, where they might catalyse removal of terminal galactose residues of substrates (1,10). One of the first plant α-galactosidases to be cloned and biochemically characterized was an enzyme from coffee beans (11).…”