title: Distinct catalytic capacities of two aluminium-repressed Arabidopsis thaliana xyloglucan endotransglucosylase/hydrolases, XTH15 and XTH31, heterologously produced in Pichia.
abstract: Xyloglucan plays an important structural role in primary cell walls, possibly tethering adjacent microfibrils and restraining cell expansion. There is therefore considerable interest in understanding the role of xyloglucan endotransglucosylase/hydrolases (XTHs), which are encoded in Arabidopsis by a 33-member gene family. We compared the key catalytic properties of two very different Arabidopsis XTHs (heterologously produced in Pichia), both of which are aluminium-repressed. Reductively tritiated oligosaccharides of xyloglucan were used as model acceptor substrates. Untransformed Pichia produced no xyloglucan-acting enzymes; therefore purification of the XTHs was unnecessary. XTH15, a classical group-I/II XTH, had high XET and undetectable XEH activity in vitro; its XET Km values were 31 μM XXXGol (acceptor substrate) and 2.9 mg/ml xyloglucan (donor substrate). In contrast, XTH31, a group-III-A XTH, showed predominant XEH activity and only slight XET activity in vitro; its XET Km was 86μM XXXGol (acceptor), indicating a low affinity of this predominantly hydrolytic protein for a transglycosylation acceptor substrate. The Km of XTH31's XEH activity was 1.6 mg/ml xyloglucan. For both proteins, the preferred XET acceptor substrate, among five cellotetraitol-based oligosaccharides tested, was XXXGol. XTH31's XET activity was strongly compromised when the second Xyl residue was galactosylated. XTH15's XET activity, in contrast, tolerated substitution at the second Xyl residue. The two enzymes also showed different pH preferences, XTH31 exhibiting an unusually low pH optimum and XTH15 an unusually broad optimum. XTH31's hydrolase activity increased almost linearly with decreasing pH in the apoplastic range, 6.2-4.5, consistent with a possible role in 'acid growth'. In conclusion, these two Al(3+)-repressed XTHs differ, in several important enzymic features, from other members of the Arabidopsis XTH family.
authors.affiliation: College of Environment and Resource Sciences, Zhejiang University, Hangzhou 310058, China; Edinburgh Cell Wall Group, Institute of Molecular Plant Sciences, The University of Edinburgh, Edinburgh EH9 3JH, UK; Tea Research Institute, Chinese Academy of Agricultural Sciences, Hangzhou 310008, China.
authors.forename: Yuan Zhi
authors.initials: YZ
authors.lastname: Shi
authors.affiliation: College of Environment and Resource Sciences, Zhejiang University, Hangzhou 310058, China.
authors.forename: Xiao Fang
authors.initials: XF
authors.lastname: Zhu
authors.affiliation: Edinburgh Cell Wall Group, Institute of Molecular Plant Sciences, The University of Edinburgh, Edinburgh EH9 3JH, UK.
authors.forename: Janice G
authors.initials: JG
authors.lastname: Miller
authors.affiliation: Edinburgh Cell Wall Group, Institute of Molecular Plant Sciences, The University of Edinburgh, Edinburgh EH9 3JH, UK.
authors.forename: Timothy
authors.initials: T
authors.lastname: Gregson
authors.affiliation: College of Environment and Resource Sciences, Zhejiang University, Hangzhou 310058, China.
authors.forename: Shao Jian
authors.initials: SJ
authors.lastname: Zheng
authors.affiliation: Edinburgh Cell Wall Group, Institute of Molecular Plant Sciences, The University of Edinburgh, Edinburgh EH9 3JH, UK. Electronic address: s.fry@ed.ac.uk.
authors.forename: Stephen C
authors.initials: SC
authors.lastname: Fry
mesh_terms: D000535:Aluminum / Q000494:pharmacology*; D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000201:enzymology* / Q000235:genetics*; D029681:Arabidopsis Proteins / Q000235:genetics* / Q000378:metabolism*; D055162:Biocatalysis; D015536:Down-Regulation / Q000187:drug effects; D015870:Gene Expression; D006026:Glycoside Hydrolases / Q000235:genetics* / Q000378:metabolism*; D006031:Glycosylation; D016695:Glycosyltransferases / Q000235:genetics* / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007700:Kinetics; D010843:Pichia / Q000235:genetics*; D013379:Substrate Specificity
chemical_list: D029681:Arabidopsis Proteins; D000535:Aluminum; D016695:Glycosyltransferases; C000603581:XTH15 protein, Arabidopsis; C000603582:XTH31 protein, Arabidopsis; C473049:xyloglucan - xyloglucosyltransferase; D006026:Glycoside Hydrolases
keywords: Acid growth; Arabidopsis thaliana; Plant cell wall; XEH; XET; XTH15; XTH31; Xyloglucan