title: Substrate specificity and catalytic mechanism of a xyloglucan xyloglucosyl transferase HvXET6 from barley (Hordeum vulgare L.).
abstract: A family 16 glycoside hydrolase, xyloglucan xyloglucosyl transferase (EC 2.4.1.207), also known as xyloglucan endotransglycosylase (XET), and designated isoenzyme HvXET6, was purified approximately 400-fold from extracts of young barley seedlings. The complete amino acid sequence of HvXET6 was deduced from the nucleotide sequence of a near full-length cDNA, in combination with tryptic peptide mapping. An additional five to six isoforms or post-translationally modified XET enzymes were detected in crude seedling extracts of barley. The HvXET6 isoenzyme was expressed in Pichia pastoris, characterized and compared with the previously purified native HvXET5 isoform. Barley HvXET6 has a similar apparent molecular mass of 33-35 kDa to the previously purified HvXET5 isoenzyme, but the two isoenzymes differ in their isoelectric points, pH optima, kinetic properties and substrate specificities. The HvXET6 isoenzyme catalyses transfer reactions between xyloglucans and soluble cellulosic substrates, using oligo-xyloglucosides as acceptors, but at rates that are significantly different from those observed for HvXET5. No hydrolytic activity could be detected with either isoenzyme. Comparisons of the reaction rates using xyloglucan or hydroxyethyl cellulose as donors and a series of cellodextrins as acceptors indicated that the acceptor site of HvXET can accommodate five glucosyl residues. Molecular modelling supported this conclusion and further confirmed the ability of the enzyme's active site to accommodate xyloglucan and cellulosic substrates. The two HvXETs followed a ping-pong (Bi, Bi) rather than a sequential reaction mechanism.
authors.affiliation: School of Agriculture, Food and Wine, Australian Centre for Plant Functional Genomics, University of Adelaide, Australia. maria.hrmova@adelaide.edu.au
authors.forename: Maria
authors.initials: M
authors.lastname: Hrmova
authors.forename: Vladimir
authors.initials: V
authors.lastname: Farkas
authors.forename: Andrew J
authors.initials: AJ
authors.lastname: Harvey
authors.forename: Jelle
authors.initials: J
authors.lastname: Lahnstein
authors.forename: Bente
authors.initials: B
authors.lastname: Wischmann
authors.forename: Nomchit
authors.initials: N
authors.lastname: Kaewthai
authors.forename: Inés
authors.initials: I
authors.lastname: Ezcurra
authors.forename: Tuula T
authors.initials: TT
authors.lastname: Teeri
authors.forename: Geoffrey B
authors.initials: GB
authors.lastname: Fincher
mesh_terms: D000595:Amino Acid Sequence; D055162:Biocatalysis; D004586:Electrophoresis; D016695:Glycosyltransferases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D001467:Hordeum / Q000201:enzymology* / Q000235:genetics; D007527:Isoenzymes / Q000737:chemistry / Q000145:classification / Q000302:isolation & purification / Q000378:metabolism; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010802:Phylogeny; D017434:Protein Structure, Tertiary; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D021241:Spectrometry, Mass, Electrospray Ionization; D013379:Substrate Specificity; D013997:Time Factors
chemical_list: D007527:Isoenzymes; D016695:Glycosyltransferases; C073693:xyloglucan endotransglycosylase