Structural and biochemical insight into a modular β-1,4-galactan synthase in plants

Abstract

Rhamnogalacturonan I (RGI) is a structurally complex pectic polysaccharide with a backbone of alternating rhamnose and galacturonic acid residues substituted with arabinan and galactan side chains. Galactan synthase 1 (GalS1), transfers galactose and arabinose to either extend or cap the β-1,4 galactan side chains of RGI, respectively. Here we report the structure of GalS1 from Populus trichocarpa, showing a modular protein consisting of an N-terminal domain that represents the founding member of a new family of carbohydrate-binding module, CBMXX (number denoted as 'XX" assigned upon publication), and a C-terminal glycosyltransferase family 92 (GT92) catalytic domain that adopts a GT-A fold. GalS1 exists as a dimer in vitro, with stem domains interacting across the chains in a 'handshake' orientation that is essential for maintaining stability and activity. In addition to understanding the enzymatic mechanism of GalS1, we gained insight into the donor and acceptor substrate binding sites using deep evolutionary analysis, molecular simulations, and biochemical studies. Combining all the results, a mechanism for GalS1 catalysis and a new model for pectic galactan side chain addition are proposed.

The plant cell wall—dynamic, strong, and adaptable—is a natural shapeshifter

“…The identification of the β-1,4 galactan synthases (GALS) (GT92) that extend the galactose side branches ( Liwanag et al 2012 ; Ebert et al 2018 ; Laursen et al 2018 ) was an important step forward and provided catalysts for study of type II galactan synthesis and structure. Interestingly, GALS1 was demonstrated to be a bifunctional enzyme that also can add arabinose to β-1,4-galactan chains, an activity hypothesized to function in chain termination ( Laursen et al 2018 ; Prabhakar et al 2023 ). The 3D crystal structure of GalS1 from P. trichocarpa revealed a modular protein structure with an N-terminal CBM95 RG-I backbone binding domain, C-terminal GT92 GT-A fold catalytic domain, and stem region involved in homodimer formation ( Prabhakar et al 2023 ).…”

“…Interestingly, GALS1 was demonstrated to be a bifunctional enzyme that also can add arabinose to β-1,4-galactan chains, an activity hypothesized to function in chain termination ( Laursen et al 2018 ; Prabhakar et al 2023 ). The 3D crystal structure of GalS1 from P. trichocarpa revealed a modular protein structure with an N-terminal CBM95 RG-I backbone binding domain, C-terminal GT92 GT-A fold catalytic domain, and stem region involved in homodimer formation ( Prabhakar et al 2023 ). Mutagenesis informed by comparison of GT-A fold sequences to the GT92 family identified a novel histidine as the catalytic base and other amino acids involved in β-1,4-galactan synthesis and acceptor binding.…”

Enzymes in 3D: Synthesis, remodelling, and hydrolysis of cell wall (1,3;1,4)-β-glucans

“…Returning to higher plant enzymes that are involved in wall synthesis, the following structures have been solved: a solubilized (transmembrane anchor removed) Arabidopsis family GT37 xyloglucan fucosyltransferase ( At FUT1; Protein Data Bank-PDB-accession 5kop) ( Rocha et al 2016a , 2016b ; Urbanowicz et al 2017 ), an Arabidopsis family GT34 xyloglucan xylosyltransferase ( At XXT1; PDB accession 6bsu) ( Culbertson et al 2018 ), and a Populus trichocarpa family GT92 (1,4)-β-galactan synthase (GalS1; PDB accession 8d3t) ( Prabhakar et al 2023 ). These structures have provided valuable insights into the mechanisms of xyloglucan and pectic polysaccharide synthesis.…”