Biochemical and molecular characterisation of xyloglucan endotransglycosylase from ripe kiwifruit.
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 Km was 0.6 mg.mL-1 for kiwifruit xyloglucan and 100 microM 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.
Schröder
R
R
Food Research Institute of New Zealand, Mt. Albert Research Centre, Auckland, New Zealand.
Atkinson
R G
RG
Langenkämper
G
G
Redgwell
R J
RJ
D000595:Amino Acid Sequence; D001483:Base Sequence; D015139:Blotting, Southern; D017931:DNA Primers; D018076:DNA, Complementary; D018744:DNA, Plant; D004926:Escherichia coli / Q000378:metabolism; D005638:Fruit / Q000201:enzymology*; D015870:Gene Expression; D017343:Genes, Plant; D005936:Glucans; D016695:Glycosyltransferases / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D007553:Isotope Labeling; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D011134:Polysaccharides / Q000378:metabolism / Q000494:pharmacology; D013379:Substrate Specificity; D014316:Tritium; D014990:Xylans
D017931:DNA Primers; D018076:DNA, Complementary; D018744:DNA, Plant; D005936:Glucans; D011134:Polysaccharides; D014990:Xylans; D014316:Tritium; C029353:xyloglucan; D016695:Glycosyltransferases; C073693:xyloglucan endotransglycosylase