Solubilization and characterization of a galacturonosyltransferase that synthesizes the pectic polysaccharide homogalacturonan

Doong, Ron Lou; Mohnen, Debra

doi:10.1046/j.1365-313x.1998.00042.x

Cited by 67 publications

(66 citation statements)

References 71 publications

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“…Both sequences are predicted to encode glycosyltransferases with type II membrane topology (23), C-terminal catalytic domains with consensus sequences clustered in CAZy glycosyltransferase family 8 (24), and to have basic isoelectric points. These characteristics are consistent with the known in vitro biochemical properties of GalATs (17,20). The predicted catalytic domains also contain DxD motifs, a conserved motif present in many well characterized glycosyltransferase families (25) and involved in the coordination of divalent cations (26).…”

supporting

confidence: 79%

“…Work in pea localized HG:GalAT activity to the luminal side of Golgi vesicles (13), the same location as pectin synthesis (14,15). The most extensive study of HG:GalAT activity was done in tobacco (16,17), and characteristics of the tobacco enzyme are comparable with the activity described in other plant species.…”

mentioning

confidence: 73%

“…Establishment of conditions to recover detergentsolubilized GalAT activity from membrane fractions (17) and in vitro studies using radiolabeled substrate (17) or fluorescently tagged (18,19) acceptors established that, in vitro, GalAT preferentially transfers GalA onto the nonreducing end (20) of HG oligosaccharide acceptors [oligogalacturonides (OGAs)] of a degree of polymerization (DP) Ͼ9 (17,18), although OGA acceptors as small as a trimer can be used (18,19). Polymeric pectins, such as poly-GalA and pectin, are less favorable substrates (21).…”

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confidence: 99%

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Functional identification of an Arabidopsis pectin biosynthetic homogalacturonan galacturonosyltransferase

Sterling

Atmodjo

Inwood

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

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Galacturonosyltransferases (GalATs) are required for the synthesis of pectin, a family of complex polysaccharides present in the cell walls of all land plants. We report the identification of a pectin GalAT (GAUT1) using peptide sequences obtained from Arabidopsis thaliana proteins partially purified for homogalacturonan (HG) ␣-1,4-GalAT activity. Transient expression of GAUT1 cDNA in the human embryonic kidney cell line HEK293 yielded uridine diphosphogalacturonic acid:GalAT activity. Polyclonal antibodies generated against GAUT1 immunoabsorbed HG ␣-1,4-GalAT activity from Arabidopsis solubilized membrane proteins. BLAST analysis of the Arabidopsis genome identified a family of 25 genes with high sequence similarity to GAUT1 and homologous genes in other dicots, in rice, and in Physcomitrella. Sequence alignment and phylogenetic Bayesian analysis of the Arabidopsis GAUT1-related gene family separates them into four related clades of GAUT and GAUT-like genes that are distinct from the other Arabidopsis members of glycosyltransferase family 8. The identification of GAUT1 as a HG GalAT and of the GAUT1-related gene family provides the genetic and biochemical tools required to study the function of these genes in pectin synthesis.biosynthesis ͉ cell wall ͉ glycosyltransferase ͉ polygalacturonic acid ͉ pectic polysaccharide

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supporting

confidence: 79%

mentioning

confidence: 73%

mentioning

confidence: 99%

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Functional identification of an Arabidopsis pectin biosynthetic homogalacturonan galacturonosyltransferase

Sterling

Atmodjo

Inwood

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

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312

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“…The addition to the incorporation reaction of (135)-linked ␣-l-arabino-oligosaccharides as acceptors stimulated the transfer of radiolabeled Ara onto these acceptor molecules, suggesting that it was the loss of endogenous acceptors that resulted in reduced incorporation. The success of using oligosaccharides as acceptor molecules in solubilized microsomal systems has also been reported for other transferases, including homogalacturonan galacturonosyltransferase (Doong and Mohnen, 1998), galactomannan galactosyltransferase (Edwards et al, 1999), xylan xylosyltransferase (Kuroyama and Tsumuraya, 2001), and xyloglucan xylosyltransferase (Faik et al, 2002). The addition of large polysaccharides such as debranched arabinan and galactan did not show a stimulation of incorporation using membranes prepared from mung bean hypocotyls.…”

Section: Discussionmentioning

confidence: 66%

Solubilization of an Arabinan Arabinosyltransferase Activity from Mung Bean Hypocotyls

Nunan¹,

Scheller²

2003

Plant Physiology

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The biosynthesis of polysaccharides destined for the plant cell wall and the subsequent assembly of the cell wall are poorly understood processes that are currently the focus of much research. Arabinan, a component of the pectic polysaccharide rhamnogalacturonan I, is composed of arabinosyl residues connected via various glycosidic linkages, and therefore, the biosynthesis of arabinan is likely to involve more than one arabinosyltransferase. We have studied the transfer of [ 14 C]arabinose (Ara) from UDP-l-arabinopyranose onto polysaccharides using microsomal membranes isolated from mung bean (Vigna radiata) hypocotyls. [ 14 C]arabinosyl and [ 14 C]xylosyl residues were incorporated into endogenous products due to the presence of UDP-Xyl-4-epimerase activity. Enzymatic digestion of endogenous products with endo-arabinanase released very little radiolabeled sugars, whereas digestion with arabinofuranosidase released some [ 14 C]Ara. Microsomal membranes solubilized with the detergent octyl glucoside were able to add a single [ 14 C]Ara residue onto (135)-linked ␣-l-arabino-oligosaccharide acceptors. The reaction had a pH optimum of 6.5 and a requirement for manganese ions. However, enzymatic digestion of the radiolabeled oligosaccharides with endo-arabinanase and arabinofuranosidases could not fully release the radiolabeled Ara residue, indicating that the [ 14 C]Ara residue was not a (132)-, (133)-, or (135)-linked ␣-l-arabinofuranosyl residue. Rather, mild acid treatment of the product suggested that the radiolabeled Ara residue was in a pyranose conformation, and this result was confirmed by thin-layer chromatography of radiolabeled partially methylated sugars. Using microsomal membranes separated on a discontinuous sucrose gradient, the arabinosyltransferase activity appears to be mainly localized to Golgi membranes.Pectic polysaccharides are major components of primary plant cell walls and middle lamella. Three main types of pectic polysaccharides have been identified, homogalacturonan, rhamnogalacturonan I (RG I), and rhamnogalacturonan II (RG II). RG I is composed of a backbone of alternating [34-␣-d-GalA-(132)-␣-l-Rha- (13] in which some of the rhamnose residues are substituted with complex side chains containing Ara and Gal residues. The composition and size of these side chains varies depending on the plant species, tissue type, and stage of development (O'Neill et al., 1990;Carpita and Gibeaut, 1993). In general, the arabinan side chains consist of a linear chain of (135)-linked ␣-l-arabinofuranosyl residues that may be substituted at the O-3 and/or O-2 with additional arabinofuranosyl residues (Carpita and Gibeaut, 1993;Schols and Voragen, 2002).The study of the biosynthesis of plant cell wall polysaccharides in plants is a growing field, but to date, very few genes encoding glycosyltransferases have been characterized (Edwards et al., 1999;Perrin et al., 1999;Faik et al., 2002). Notably, many different transferases must be involved in pectin biosynthesis, but until very recently, none had been isola...

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“…Only a single Gal residue is transferred per acceptor molecule, even when the acceptor oligosaccharides are sufficiently long to accommodate repositioning of the acceptor relative to the recognition sequence. Detergent solubilization causes the processivity of an ␣-galacturonyltransferase involved in pectic homogalacturonan biosynthesis to be lost (Doong and Mohnen, 1998). Thus, it is possible that detergent solubilization may have disrupted any specific association between the MS and GMGT proteins that might impose single-chain action.…”

Section: Discussionmentioning

confidence: 99%

Transfer Specificity of Detergent-Solubilized Fenugreek Galactomannan Galactosyltransferase

et al. 2002

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The current experimental model for galactomannan biosynthesis in membrane-bound enzyme systems from developing legume-seed endosperms involves functional interaction between a GDP-mannose (Man) mannan synthase and a UDPgalactose (Gal) galactosyltransferase. The transfer specificity of the galactosyltransferase to the elongating mannan chain is critical in regulating the distribution and the degree of Gal substitution of the mannan backbone of the primary biosynthetic product. Detergent solubilization of the galactosyltransferase of fenugreek (Trigonella foenum-graecum) with retention of activity permitted the partial purification of the enzyme and the cloning and sequencing of the corresponding cDNA with proof of functional identity. We now document the positional specificity of transfer of ( 14 C)Gal from UDP-( 14 C)Gal to manno-oligosaccharide acceptors, chain lengths 5 to 8, catalyzed by the detergent-solubilized galactosyltransferase. Enzymatic fragmentation analyses of the labeled products showed that a single Gal residue was transferred per acceptor molecule, that the linkage was (136)-␣, and that there was transfer to alternative Man residues within the acceptor molecules. Analysis of the relative frequencies of transfer to alternative Man residues within acceptor oligosaccharides of different chain length allowed the deduction of the substrate subsite recognition requirement of the galactosyltransferase. The enzyme has a principal recognition sequence of six Man residues, with transfer of Gal to the third Man residue from the nonreducing end of the sequence. These observations are incorporated into a refined model for enzyme interaction in galactomannan biosynthesis.Galactomannans are the principal polysaccharide component of the walls of endosperm cells of legume seeds (Reid, 1985). Their structures are relatively simple, consisting of a linear (134)-␤-linked d-mannan backbone with single-unit (136)-linked ␣-d-galactopyranosyl side chains. The Man-to-Gal (Man/Gal) ratio is variable between 1.1 (high Gal) and about 3.4 (low Gal), and is taxonomically significant (Reid and Meier, 1970;Bailey, 1971). The galactomannans are multifunctional molecules, serving as the molecular basis of a water imbibition and drought avoidance mechanism before and during germination, and then as a source of storage carbohydrate for the developing seedling (Reid and Bewley, 1979). When isolated, galactomannans are water soluble, with solution properties that have industrial applications. For example, the galactomannans from guar (Cyamopsis tetragonoloba) and locust bean (Ceratonia siliqua) are used in the food industry as viscosifiers, stabilizers, and gelling agents, alone and in combination with other polysaccharides (Dea and Morrison, 1975;. The Man/Gal ratio is an important determinant of commercial functionality, with high Man/Gal ratios (low Gal substitution) favoring valuable mixed-polymer interactions (Dea et al., 1977).Galactomannan biosynthesis is catalyzed in vitro by membrane preparations isolated from the developin...

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Solubilization and characterization of a galacturonosyltransferase that synthesizes the pectic polysaccharide homogalacturonan

Cited by 67 publications

References 71 publications

Functional identification of an Arabidopsis pectin biosynthetic homogalacturonan galacturonosyltransferase

Functional identification of an Arabidopsis pectin biosynthetic homogalacturonan galacturonosyltransferase

Solubilization of an Arabinan Arabinosyltransferase Activity from Mung Bean Hypocotyls

Transfer Specificity of Detergent-Solubilized Fenugreek Galactomannan Galactosyltransferase

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