Life Science Group, Scientific Research Division, National Synchrotron Radiation Research Cente, Hsinchu 30076, Taiwan.
Institute of Tropical Plant Sciences and Microbiology, National Cheng Kung University, Tainan City 701, Taiwan.
Acta Crystallogr D Struct Biol. 2023 Feb 1;79(Pt 2):154-167. doi: 10.1107/S2059798323000037. Epub 2023 Jan 20.
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)-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 FRSKXWW in the RFO synthases, contributes Trp77 and Trp78 to the +1 subsite to contribute to the substrate-binding ability together with the (α/β) 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.
拟南芥的碱性α-半乳糖苷酶 AtAkαGal3 在碱性条件下催化半乳糖寡糖中α-D-半乳糖的水解。基于序列比对的系统发育分析将 AtAkαGal3 归类为与棉子糖家族寡糖（RFO）合酶更密切相关，而不是与酸性α-半乳糖苷酶相关。在这里，薄层层析用于证明 AtAkαGal3 具有双重功能，能够使用棉子糖而不是半乳糖醇作为半乳糖供体来合成水苏糖。AtAkαGal3 及其 D383A 突变体与各种底物和产物（包括半乳糖、半乳糖醇、棉子糖、水苏糖和蔗糖）的复合物的晶体结构作为碱性α-半乳糖苷酶的第一个代表结构被报道。AtAkαGal3 的结构包括三个结构域：包含 13 个反平行β-链的 N 端结构域、具有（α/β）桶折叠的催化结构域和由形成两个希腊钥匙基序的β-片层组成的 C 端结构域。N 端结构域的 WW 盒包含 RFO 合酶中保守的 FRSKXWW 残基，将色氨酸 77 和色氨酸 78 贡献到+1亚位点，与催化结构域的（α/β）桶一起有助于底物结合能力。C 端结构域可能参与结构稳定性。与各种底物和产物（特别是天然底物/产物水苏糖）形成复合物的 D383A 突变体的结构揭示了催化位点上的四个完整亚位点（-1 至+3）。存在于碱性α-半乳糖苷酶 AtAkαGal3 中，可能存在于 RFO 合酶中，但不存在于酸性α-半乳糖苷酶中的功能环（残基 329-352）稳定+2 和+3 亚位点的水苏糖，并扩展了用于转移酶机制的催化口袋。考虑到碱性α-半乳糖苷酶和 RFO 合酶在氨基酸序列、催化结构域和活性方面的相似性，AtAkαGal3 的结构也可能作为 RFO 合酶的研究模型，因为缺乏其结构。
Acta Crystallogr D Struct Biol. 2023-2-1
J Agric Food Chem. 2020-6-3
J Biol Chem. 2003-5-30
Methods Enzymol. 1997
Nucleic Acids Res. 2015-7-1
Acta Crystallogr D Biol Crystallogr. 2014-9
Nucleic Acids Res. 2014-4-21
Acta Crystallogr D Biol Crystallogr. 2011-4
Acta Crystallogr D Biol Crystallogr. 2011-4
Zotero 插件
