Structural insight into the hydrolase and synthase activities of an alkaline α-galactosidase from Arabidopsis from complexes with substrate/product (2023)

Abstract

The alkaline α-galactosidase AtAkαGal3 from Arabidopsis thaliana catalyzes the hydrolysis of α-d-galactose from galacto-oligosaccharides under alkaline conditions. A phylogenetic analysis based on sequence alignment classifies AtAkαGal3 as more closely related to the raffinose family of oligosaccharide (RFO) synthases than to the acidic α-galactosidases. Here, thin-layer chromatography is used to demonstrate that AtAkαGal3 exhibits a dual function and is capable of synthesizing stachyose using raffinose, instead of galactinol, as the galactose donor. Crystal structures of complexes of AtAkαGal3 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 α-galactosidase. The structure of AtAkαGal3 comprises three domains: An N-Terminal domain with 13 antiparallel β-strands, a catalytic domain with an (α/β)8-barrel fold and a C-Terminal domain composed of β-sheets that form two Greek-key motifs. The WW box of the N-Terminal domain, which comprises the conserved residues FRSK75XW77W78 in the RFO synthases, contributes Trp77 and Trp78 to the +1 subsite to contribute to the substrate-binding ability together with the (α/β)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 α-galactosidase AtAkαGal3 and possibly in RFO synthases, but not in acidic α-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 α-galactosidases and RFO synthases, the structure of AtAkαGal3 might also serve a model for the study of RFO synthases, structures of which are lacking.

Original languageEnglish
Pages (from-to)154-167
Number of pages14
JournalActa Crystallographica Section D: Structural Biology
Volume79
DOIs
Publication statusPublished - 2023 Jan 20

All Science Journal Classification (ASJC) codes

  • Structural Biology

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Chuankhayan, P., Lee, R. H., Guan, H. H., Lin, C. C., Chen, N. C., Huang, Y. C., Yoshimura, M., Nakagawa, A., & Chen, C. J. (2023). Structural insight into the hydrolase and synthase activities of an alkaline α-galactosidase from Arabidopsis from complexes with substrate/product. Acta Crystallographica Section D: Structural Biology, 79, 154-167. https://doi.org/10.1107/S2059798323000037

Chuankhayan, Phimonphan ; Lee, Ruey Hua ; Guan, Hong Hsiang et al. / Structural insight into the hydrolase and synthase activities of an alkaline α-galactosidase from Arabidopsis from complexes with substrate/product. In: Acta Crystallographica Section D: Structural Biology. 2023 ; Vol. 79. pp. 154-167.

@article{f4cb7ca7a39240a980d29a4e8803e3ba,

title = "Structural insight into the hydrolase and synthase activities of an alkaline α-galactosidase from Arabidopsis from complexes with substrate/product",

abstract = "The alkaline α-galactosidase AtAkαGal3 from Arabidopsis thaliana catalyzes the hydrolysis of α-d-galactose from galacto-oligosaccharides under alkaline conditions. A phylogenetic analysis based on sequence alignment classifies AtAkαGal3 as more closely related to the raffinose family of oligosaccharide (RFO) synthases than to the acidic α-galactosidases. Here, thin-layer chromatography is used to demonstrate that AtAkαGal3 exhibits a dual function and is capable of synthesizing stachyose using raffinose, instead of galactinol, as the galactose donor. Crystal structures of complexes of AtAkαGal3 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 α-galactosidase. The structure of AtAkαGal3 comprises three domains: An N-Terminal domain with 13 antiparallel β-strands, a catalytic domain with an (α/β)8-barrel fold and a C-Terminal domain composed of β-sheets that form two Greek-key motifs. The WW box of the N-Terminal domain, which comprises the conserved residues FRSK75XW77W78 in the RFO synthases, contributes Trp77 and Trp78 to the +1 subsite to contribute to the substrate-binding ability together with the (α/β)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 α-galactosidase AtAkαGal3 and possibly in RFO synthases, but not in acidic α-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 α-galactosidases and RFO synthases, the structure of AtAkαGal3 might also serve a model for the study of RFO synthases, structures of which are lacking.",

author = "Phimonphan Chuankhayan and Lee, {Ruey Hua} and Guan, {Hong Hsiang} and Lin, {Chein Chih} and Chen, {Nai Chi} and Huang, {Yen Chieh} and Masato Yoshimura and Atsushi Nakagawa and Chen, {Chun Jung}",

note = "Funding Information: We are indebted to the staff of beamlines TPS 05A and TLS 15A at the National Synchrotron Radiation Research Center (NSRRC) in Taiwan, Eiki Yamashita at BL44XU and the staff of the Taiwan beamline BL12B2 at SPring-8 in Japan for technical assistance. We are grateful for travel support from the International Collaborative Research Program of the Institute for Protein Research, Osaka University. We are also grateful to Ming-Hong Lai for the preliminary crystallization conditions of AtAkαGal3 and to Chien-Ming Chiang for discussion. We thank Shinya Fushinobu for the online Cremer–Pople parameter calculator. Publisher Copyright: {\textcopyright} 2023 International Union of Crystallography. All rights reserved.",

year = "2023",

month = jan,

day = "20",

doi = "10.1107/S2059798323000037",

language = "English",

volume = "79",

pages = "154--167",

journal = "Acta Crystallographica Section D: Structural Biology",

issn = "0907-4449",

publisher = "John Wiley and Sons Inc.",

}

Chuankhayan, P, Lee, RH, Guan, HH, Lin, CC, Chen, NC, Huang, YC, Yoshimura, M, Nakagawa, A & Chen, CJ 2023, 'Structural insight into the hydrolase and synthase activities of an alkaline α-galactosidase from Arabidopsis from complexes with substrate/product', Acta Crystallographica Section D: Structural Biology, vol. 79, pp. 154-167. https://doi.org/10.1107/S2059798323000037

Structural insight into the hydrolase and synthase activities of an alkaline α-galactosidase from Arabidopsis from complexes with substrate/product. / Chuankhayan, Phimonphan; Lee, Ruey Hua; Guan, Hong Hsiang et al.
In: Acta Crystallographica Section D: Structural Biology, Vol. 79, 20.01.2023, p. 154-167.

Research output: Contribution to journalArticlepeer-review

TY - JOUR

T1 - Structural insight into the hydrolase and synthase activities of an alkaline α-galactosidase from Arabidopsis from complexes with substrate/product

AU - Chuankhayan, Phimonphan

AU - Lee, Ruey Hua

AU - Guan, Hong Hsiang

AU - Lin, Chein Chih

AU - Chen, Nai Chi

AU - Huang, Yen Chieh

AU - Yoshimura, Masato

AU - Nakagawa, Atsushi

AU - Chen, Chun Jung

N1 - Funding Information:We are indebted to the staff of beamlines TPS 05A and TLS 15A at the National Synchrotron Radiation Research Center (NSRRC) in Taiwan, Eiki Yamashita at BL44XU and the staff of the Taiwan beamline BL12B2 at SPring-8 in Japan for technical assistance. We are grateful for travel support from the International Collaborative Research Program of the Institute for Protein Research, Osaka University. We are also grateful to Ming-Hong Lai for the preliminary crystallization conditions of AtAkαGal3 and to Chien-Ming Chiang for discussion. We thank Shinya Fushinobu for the online Cremer–Pople parameter calculator.Publisher Copyright:© 2023 International Union of Crystallography. All rights reserved.

PY - 2023/1/20

Y1 - 2023/1/20

N2 - The alkaline α-galactosidase AtAkαGal3 from Arabidopsis thaliana catalyzes the hydrolysis of α-d-galactose from galacto-oligosaccharides under alkaline conditions. A phylogenetic analysis based on sequence alignment classifies AtAkαGal3 as more closely related to the raffinose family of oligosaccharide (RFO) synthases than to the acidic α-galactosidases. Here, thin-layer chromatography is used to demonstrate that AtAkαGal3 exhibits a dual function and is capable of synthesizing stachyose using raffinose, instead of galactinol, as the galactose donor. Crystal structures of complexes of AtAkαGal3 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 α-galactosidase. The structure of AtAkαGal3 comprises three domains: An N-Terminal domain with 13 antiparallel β-strands, a catalytic domain with an (α/β)8-barrel fold and a C-Terminal domain composed of β-sheets that form two Greek-key motifs. The WW box of the N-Terminal domain, which comprises the conserved residues FRSK75XW77W78 in the RFO synthases, contributes Trp77 and Trp78 to the +1 subsite to contribute to the substrate-binding ability together with the (α/β)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 α-galactosidase AtAkαGal3 and possibly in RFO synthases, but not in acidic α-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 α-galactosidases and RFO synthases, the structure of AtAkαGal3 might also serve a model for the study of RFO synthases, structures of which are lacking.

AB - The alkaline α-galactosidase AtAkαGal3 from Arabidopsis thaliana catalyzes the hydrolysis of α-d-galactose from galacto-oligosaccharides under alkaline conditions. A phylogenetic analysis based on sequence alignment classifies AtAkαGal3 as more closely related to the raffinose family of oligosaccharide (RFO) synthases than to the acidic α-galactosidases. Here, thin-layer chromatography is used to demonstrate that AtAkαGal3 exhibits a dual function and is capable of synthesizing stachyose using raffinose, instead of galactinol, as the galactose donor. Crystal structures of complexes of AtAkαGal3 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 α-galactosidase. The structure of AtAkαGal3 comprises three domains: An N-Terminal domain with 13 antiparallel β-strands, a catalytic domain with an (α/β)8-barrel fold and a C-Terminal domain composed of β-sheets that form two Greek-key motifs. The WW box of the N-Terminal domain, which comprises the conserved residues FRSK75XW77W78 in the RFO synthases, contributes Trp77 and Trp78 to the +1 subsite to contribute to the substrate-binding ability together with the (α/β)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 α-galactosidase AtAkαGal3 and possibly in RFO synthases, but not in acidic α-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 α-galactosidases and RFO synthases, the structure of AtAkαGal3 might also serve a model for the study of RFO synthases, structures of which are lacking.

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Chuankhayan P, Lee RH, Guan HH, Lin CC, Chen NC, Huang YC et al. Structural insight into the hydrolase and synthase activities of an alkaline α-galactosidase from Arabidopsis from complexes with substrate/product. Acta Crystallographica Section D: Structural Biology. 2023 Jan 20;79:154-167. doi: 10.1107/S2059798323000037

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