7exf
Crystal structure of wild-type from Arabidopsis thaliana complexed with GalactoseCrystal structure of wild-type from Arabidopsis thaliana complexed with Galactose
Structural highlights
FunctionRFS6_ARATH Transglycosidase operating by a ping-pong reaction mechanism. Involved in the synthesis of raffinose, a major soluble carbohydrate in seeds, roots and tubers (By similarity). Publication Abstract from PubMedThe alkaline alpha-galactosidase AtAkalphaGal3 from Arabidopsis thaliana catalyzes the hydrolysis of alpha-D-galactose from galacto-oligosaccharides under alkaline conditions. A phylogenetic analysis based on sequence alignment classifies AtAkalphaGal3 as more closely related to the raffinose family of oligosaccharide (RFO) synthases than to the acidic alpha-galactosidases. Here, thin-layer chromatography is used to demonstrate that AtAkalphaGal3 exhibits a dual function and is capable of synthesizing stachyose using raffinose, instead of galactinol, as the galactose donor. Crystal structures of complexes of AtAkalphaGal3 and its D383A mutant with various substrates and products, including galactose, galactinol, raffinose, stachyose and sucrose, are reported as the first representative structures of an alkaline alpha-galactosidase. The structure of AtAkalphaGal3 comprises three domains: an N-terminal domain with 13 antiparallel beta-strands, a catalytic domain with an (alpha/beta)(8)-barrel fold and a C-terminal domain composed of beta-sheets that form two Greek-key motifs. The WW box of the N-terminal domain, which comprises the conserved residues FRSK(75)XW(77)W(78) in the RFO synthases, contributes Trp77 and Trp78 to the +1 subsite to contribute to the substrate-binding ability together with the (alpha/beta)(8) barrel of the catalytic domain. The C-terminal domain is presumably involved in structural stability. Structures of the D383A mutant in complex with various substrates and products, especially the natural substrate/product stachyose, reveal four complete subsites (-1 to +3) at the catalytic site. A functional loop (residues 329-352) that exists in the alkaline alpha-galactosidase AtAkalphaGal3 and possibly in RFO synthases, but not in acidic alpha-galactosidases, stabilizes the stachyose at the +2 and +3 subsites and extends the catalytic pocket for the transferase mechanism. Considering the similarities in amino-acid sequence, catalytic domain and activity between alkaline alpha-galactosidases and RFO synthases, the structure of AtAkalphaGal3 might also serve a model for the study of RFO synthases, structures of which are lacking. Structural insight into the hydrolase and synthase activities of an alkaline alpha-galactosidase from Arabidopsis from complexes with substrate/product.,Chuankhayan P, Lee RH, Guan HH, Lin CC, Chen NC, Huang YC, Yoshimura M, Nakagawa A, Chen CJ Acta Crystallogr D Struct Biol. 2023 Feb 1;79(Pt 2):154-167. doi: , 10.1107/S2059798323000037. Epub 2023 Jan 20. PMID:36762861[1] From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. References
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