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Jens M. Ekl?f Shaheen Shojania Mark Okon Lawrence P. McIntosh Harry Brumer 《The Journal of biological chemistry》2013,288(22):15786-15799
The large xyloglucan endotransglycosylase/hydrolase (XTH) gene family continues to be the focus of much attention in studies of plant cell wall morphogenesis due to the unique catalytic functions of the enzymes it encodes. The XTH gene products compose a subfamily of glycoside hydrolase family 16 (GH16), which also comprises a broad range of microbial endoglucanases and endogalactanases, as well as yeast cell wall chitin/β-glucan transglycosylases. Previous whole-family phylogenetic analyses have suggested that the closest relatives to the XTH gene products are the bacterial licheninases (EC 3.2.1.73), which specifically hydrolyze linear mixed linkage β(1→3)/β(1→4)-glucans. In addition to their specificity for the highly branched xyloglucan polysaccharide, XTH gene products are distinguished from the licheninases and other GH16 enzyme subfamilies by significant active site loop alterations and a large C-terminal extension. Given these differences, the molecular evolution of the XTH gene products in GH16 has remained enigmatic. Here, we present the biochemical and structural analysis of a unique, mixed function endoglucanase from black cottonwood (Populus trichocarpa), which reveals a small, newly recognized subfamily of GH16 members intermediate between the bacterial licheninases and plant XTH gene products. We postulate that this clade comprises an important link in the evolution of the large plant XTH gene families from a putative microbial ancestor. As such, this analysis provides new insights into the diversification of GH16 and further unites the apparently disparate members of this important family of proteins. 相似文献
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When [glucitol-3H]XXFGol (a NaB3H4-reduced xyloglucan nonasaccharide) was applied to excised shoots of pea (Pisum sativum L. cv. Progress) at the base of the epicotyl, it inhibited growth in the elongation zone, 4–5 cm distal. Experiments were
conducted to discover whether such 3H-oligosaccharides are translocated intact over this distance, or whether an intercellular second messenger would have to
be postulated. After 24 h, 3H from [glucitol-3H]XXFGol and [glucitol-3H]XXXGol showed U-shaped distributions, with most 3H at the base and apex of the stem. Radioactivity from [fucosyl-3H]XXFG and [xylosyl-3H]XXFG also moved acropetally, but did not concentrate at the apex, possibly owing to removal from the transpiration stream
of fucose and xylose formed by partial hydrolysis of XXFG en route. When 10−7 M [glucitol-3H]XXFGol was supplied, about 14 fmol · seedling–1 of apparently intact [3H]XXFGol was extractable from the elongation zone after 24 h. Larger amounts of degradation products were extractable (including
free [3H]glucitol) and some wall-bound 3H-hemicellulose was present (presumably formed by the oligosaccharides acting as acceptor substrates for transglycosylation).
We conclude that biologically active oligosaccharides of xyloglucan can move through the stem acropetally and that they are
maintained at low steady-state concentrations by both hydrolysis and transglycosylation.
Received: 1 April 1997 / Accepted: 28 May 1997 相似文献
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Biochemical and molecular characterisation of xyloglucan endotransglycosylase from ripe kiwifruit 总被引:4,自引:0,他引:4
Xyloglucan endotransglycosylase (XET) from the core tissue of ripe kiwifruit (Actinidia deliciosa [A. Chev.] C.F. Liang et A.R. Ferguson var. deliciosa cv. Hayward) was purified 3000-fold to homogeneity. The enzyme has a molecular weight of 34 kDa, is N-glycosylated, and is
active between pH 5.0 and 8.0, with an optimum between 5.5 and 5.8. The K
m was 0.6 mg · mL−1 for kiwifruit xyloglucan and 100 μM for [3H]XXXG-ol, a reduced heptasaccharide derived from kiwifruit xyloglucan. Kiwifruit core XET was capable of depolymerising xyloglucan
in the absence of [3H]XXXG-ol by hydrolysis, and in the presence of [3H]XXXG-ol by hydrolysis and endotransglycosylation. Six cDNA clones (AdXET1-6) with homology to other reported XETs were isolated
from ripe kiwifruit mRNA. The six cDNA clones share 93–99% nucleotide identity and appear to belong to a family of closely
related genes. Peptide sequencing indicated that ripe kiwifruit XET was encoded by AdXET6. Northern analysis indicated that
expression of the AdXET1-6 gene family was induced in ripening kiwifruit when endogenous ethylene production could first be
detected, and peaked in climacteric samples when fruit were soft. A full-length cDNA clone (AdXET5) was overexpressed in E. coli to produce a recombinant protein that showed endotransglycosylase activity when refolded.
Received: 2 June 1997 / Accepted: 17 June 1997 相似文献
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