Structural analysis of the plant glycoside hydrolase family 116 glucosylceramidase AtGCD3 by cryogenic electron microscopy
Issued Date
2026-03-01
Resource Type
eISSN
18790003
Scopus ID
2-s2.0-105032789132
Pubmed ID
41720384
Journal Title
International Journal of Biological Macromolecules
Volume
350
Rights Holder(s)
SCOPUS
Bibliographic Citation
International Journal of Biological Macromolecules Vol.350 (2026) , 150988
Suggested Citation
Choknud S., Arthanareeswaran K., Rungsarityotin W., Beagbandee C., Sanram S., Kamonsutthipaijit N., Mo-Mai P., Lai R.Y., Suginta W., Wangkanont K., Ounjai P., Ketudat Cairns J.R. Structural analysis of the plant glycoside hydrolase family 116 glucosylceramidase AtGCD3 by cryogenic electron microscopy. International Journal of Biological Macromolecules Vol.350 (2026) , 150988. doi:10.1016/j.ijbiomac.2026.150988 Retrieved from: https://repository.li.mahidol.ac.th/handle/123456789/115787
Title
Structural analysis of the plant glycoside hydrolase family 116 glucosylceramidase AtGCD3 by cryogenic electron microscopy
Corresponding Author(s)
Other Contributor(s)
Abstract
Glucosylceramide (GlcCer) is a common glycosphingolipid that accumulates in cells in response to Gaucher disease, diabetes, and skin disorders in humans and is also found in plants. In animals, GlcCer is catabolized by glucosylcerebrosidase 1 and 2 (GBA1 and GBA2). GBA1 is a lysosomal enzyme in glycoside hydrolase (GH) family GH30, while GBA2 is a membrane-associated cytoplasmic protein in family GH116. Currently, there are no experimental structures of eukaryotic GH116 homologues. Although the bacterial TxGH116 β-glucosidase structure was determined by X-ray crystallography, TxGH116 does not hydrolyze glucosylceramides, unlike the animal and plant enzymes. Therefore, we have investigated the structure of plant GH116 (AtGCD3) by cryogenic electron microscopy (Cryo-EM) single-particle analysis. The recombinant AtGCD3 protein was produced in Escherichia coli and purified by immobilized-metal affinity chromatography followed by size-exclusion chromatography. The Cryo-EM structure revealed a unique hexameric arrangement, composed of a dimer of trimers. Hydrophobic interactions and hydrogen bonds stabilize each trimer at the trimer interface. The two trimers stack face-to-face with a slight twist, with salt bridges and hydrogen bonding at their interface. Two α-helices not found in previously described GH116 structures cover the active site, forming two hydrophobic channels that may be involved in glucosylceramide binding. Molecular dynamics simulations showed that glucosylceramide can bind stably in the active site with its lipid tails in these channels. This first eukaryotic structure of a GH116 enzyme generates a template for improved modeling of human GBA2, with implications for treating human diseases, such as Gaucher disease and hereditary spastic paraplegia.