Three highly acidic Equisetum XTHs differ from hetero-trans-β-glucanase in donor substrate specificity and are predominantly xyloglucan homo-transglucosylases

Holland, Claire; Simmons, Thomas J.; Meulewaeter, Frank; Hudson, Andrew; Fry, Stephen C.

doi:10.1016/j.jplph.2020.153210

Cited by 13 publications

(24 citation statements)

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“…For example, EfHTG of Equisetum fluviatile (water horsetail) catalyzes two additional types of transglycosylation: cleavage of mixed-linkage β(1,3;1,4)-glucan (or MLG) and reconnection to xyloglucan and cleavage of cellulose and reconnection to xyloglucan. This broader substrate specificity is not found in three closely related members (EfXTH-A, EfXTH-H and EfXTH-I) of the E. fluviatile XTH family ( Holland et al. 2020 ).…”

Section: Introductionmentioning

confidence: 82%

Cryogenian Origin and Subsequent Diversification of the Plant Cell-Wall Enzyme XTH Family

Shinohara

Nishitani

2021

Plant and Cell Physiology

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All land plants encode large multigene families of xyloglucan endotransglucosylase/hydrolases (XTHs), plant-specific enzymes that cleave and reconnect plant cell-wall polysaccharides. Despite the ubiquity of these enzymes, considerable uncertainty remains regarding the evolutionary history of the XTH family. Phylogenomic and comparative analyses in this study traced the non-plant origins of the XTH family to Alphaproteobacteria ExoKs, bacterial enzymes involved in loosening biofilms, rather than Firmicutes licheninases, plant biomass digesting enzymes, as previously supposed. The relevant horizontal gene transfer (HGT) event was mapped to the divergence of non-swimming charophycean algae in the Cryogenian geological period. This HGT event was the likely origin of charophycean EG16-2s, which are putative intermediates between ExoKs and XTHs. Another HGT event in the Cryogenian may have led from EG16-2s or ExoKs to fungal CRHs, enzymes that cleave and reconnect chitin and glucans in fungal cell walls. This successive transfer of enzyme-encoding genes may have supported the adaptation of plants and fungi to the ancient icy environment by facilitating their sessile lifestyles. Furthermore, several protein evolutionary steps, including coevolution of substrate-interacting residues and putative intra-family gene fusion, occurred in the land plant lineage and drove diversification of the XTH family. At least some of those events correlated with the evolutionary gain of broader substrate specificities, which may have underpinned the expansion of the XTH family by enhancing duplicated gene survival. Together, this study highlights the Precambrian evolution of life and the mode of multigene family expansion in the evolutionary history of the XTH family.

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Section: Introductionmentioning

confidence: 82%

Cryogenian Origin and Subsequent Diversification of the Plant Cell-Wall Enzyme XTH Family

Shinohara

Nishitani

2021

Plant and Cell Physiology

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“…Heterologous production of Ef HTG and Ef XTH-H using a recombinant pPICZaA vector system in Pichia pastoris strain SMD1168H was as described elsewhere ( Simmons et al., 2015 ). Both these Equisetum enzymes are highly acidic (p I of Ef HTG and Ef XTH-H ≈ 4.1 and 4.6, respectively); Ef XTH-H has XET but very little MXE and CXE activity ( Holland et al, 2020 ). Enzyme extraction from Equisetum followed the protocol of Fry et al., 2008a , Fry et al., 2008b .…”

Section: Methodsmentioning

confidence: 99%

Hetero-trans-β-Glucanase Produces Cellulose–Xyloglucan Covalent Bonds in the Cell Walls of Structural Plant Tissues and Is Stimulated by Expansin

et al. 2020

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Current cell-wall models assume no covalent bonding between cellulose and hemicelluloses such as xyloglucan or mixed-linkage β- d -glucan (MLG). However, Equisetum hetero-trans-β-glucanase (HTG) grafts cellulose onto xyloglucan oligosaccharides (XGOs) – and, we now show, xyloglucan polysaccharide – in vitro , thus exhibiting CXE (cellulose:xyloglucan endotransglucosylase) activity. In addition, HTG also catalyzes MLG-to-XGO bonding (MXE activity). In this study, we explored the CXE action of HTG in native plant cell walls and tested whether expansin exposes cellulose to HTG by disrupting hydrogen bonds. To quantify and visualize CXE and MXE action, we assayed the sequential release of HTG products from cell walls pre-labeled with substrate mimics. We demonstrated covalent cellulose–xyloglucan bonding in plant cell walls and showed that CXE and MXE action was up to 15% and 60% of total transglucanase action, respectively, and peaked in aging, strengthening tissues: CXE in xylem and cells bordering intercellular canals and MXE in sclerenchyma. Recombinant bacterial expansin (EXLX1) strongly augmented CXE activity in vitro . CXE and MXE action in living Equisetum structural tissues potentially strengthens stems, while expansin might augment the HTG-catalyzed CXE reaction, thereby allowing efficient CXE action in muro . Our methods will enable surveys for comparable reactions throughout the plant kingdom. Furthermore, engineering similar hetero-polymer formation into angiosperm crop plants may improve certain agronomic traits such as lodging tolerance.

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“…Ef XTH‐H does not exhibit appreciable CXE activity (Holland et al ., 2020 ; Figure S1 a), but it was able to catalyse detectable XyG‐to‐[ 3 H]XGO (XET) transglycosylation in XyG‐impregnated paper (Figure 2 b). The highest XET activity thus detected was on papers that had been infiltrated with 0.05% (w/v) XyG (and, as above, washed free of loosely bound XyG).…”

Section: Resultsmentioning

confidence: 99%

“…Production of EfHTG and EfXTH-H in Pichia pastoris strain SMD1168H was done as described before (Simmons et al, 2015;Holland et al, 2020). Enzyme extraction from Equisetum followed the protocol of Fry et al (2008).…”

Section: Heterologous Protein Production and Extractionmentioning

confidence: 99%

Defining natural factors that stimulate and inhibit cellulose:xyloglucan hetero‐transglucosylation

et al. 2021

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Certain transglucanases can covalently graft cellulose and mixed-linkage b-glucan (MLG) as donor substrates onto xyloglucan as acceptor substrate and thus exhibit cellulose:xyloglucan endotransglucosylase (CXE) and MLG:xyloglucan endotransglucosylase (MXE) activities in vivo and in vitro. However, missing information on factors that stimulate or inhibit these hetero-transglucosylation reactions limits our insight into their biological functions. To explore factors that influence hetero-transglucosylation, we studied Equisetum fluviatile hetero-trans-b-glucanase (EfHTG), which exhibits both CXE and MXE activity, exceeding its xyloglucan:xyloglucan homo-transglucosylation (XET) activity. Enzyme assays employed radiolabelled and fluorescently labelled oligomeric acceptor substrates, and were conducted in vitro and in cell walls (in situ). With whole denatured Equisetum cell walls as donor substrate, exogenous EfHTG (extracted from Equisetum or produced in Pichia) exhibited all three activities (CXE, MXE, XET) in competition with each other. Acting on pure cellulose as donor substrate, the CXE action of Pichia-produced EfHTG was up to approximately 300% increased by addition of methanol-boiled Equisetum extracts; there was no similar effect when the same enzyme acted on soluble donors (MLG or xyloglucan). The methanol-stable factor is proposed to be expansin-like, a suggestion supported by observations of pH dependence. Screening numerous low-molecular-weight compounds for hetero-transglucanase inhibition showed that cellobiose was highly effective, inhibiting the abundant endogenous CXE and MXE (but not XET) action in Equisetum internodes. Furthermore, cellobiose retarded Equisetum stem elongation, potentially owing to its effect on hetero-transglucosylation reactions. This work provides insight and tools to further study the role of cellulose hetero-transglucosylation in planta by identifying factors that govern this reaction.

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Three highly acidic Equisetum XTHs differ from hetero-trans-β-glucanase in donor substrate specificity and are predominantly xyloglucan homo-transglucosylases

Abstract: Three highly acidic Equisetum XTHs differ from hetero-trans-glucanase in donor substrate specificity and are predominantly xyloglucan homo-transglucosylases

Cited by 13 publications

References 68 publications

Cryogenian Origin and Subsequent Diversification of the Plant Cell-Wall Enzyme XTH Family

Cryogenian Origin and Subsequent Diversification of the Plant Cell-Wall Enzyme XTH Family

Hetero-trans-β-Glucanase Produces Cellulose–Xyloglucan Covalent Bonds in the Cell Walls of Structural Plant Tissues and Is Stimulated by Expansin

Defining natural factors that stimulate and inhibit cellulose:xyloglucan hetero‐transglucosylation

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