Nomchit Kaewthai | Rajamangala University of Technology Srivijaya (original) (raw)

Papers by Nomchit Kaewthai

Cover illustration: Confocal microscopy showing Arabidopsis thaliana root labeled with different ... more Cover illustration: Confocal microscopy showing Arabidopsis thaliana root labeled with different probes. Green: Xyloglucan probed with CCRC-M1 antibody. Yellow: in situ xyloglucan endo-hydrolase activity probed with XXXG-Res substrate. Red: in situ xyloglucan endo-transglycosylase activity probed with XXXG-SR substrate.

FEBS Journal, Dec 8, 2008

Xyloglucan transglycosylases ⁄ hydrolases (XTHs) are members of the glycoside hydrolase family 16... more Xyloglucan transglycosylases ⁄ hydrolases (XTHs) are members of the glycoside hydrolase family 16 (GH16) [1] and are widely distributed in plants. Enzymes that fall within the XTH classification can have xyloglucan endotransglycosylase (XET) activity or both XET and xyloglucan endohydrolase (XEH) activities [2-6]. The

Selective lowering of amyloid-β levels with small-molecule γ-secretase inhibitors is a promising ... more Selective lowering of amyloid-β levels with small-molecule γ-secretase inhibitors is a promising therapeutic approach for Alzheimer's disease. In this work, we developed a high throughput assay for screening of γ-secretase inhibitors with endogenous γ-secretase and a fluorogenic substrate. The IC 50 values of known γ-secretase inhibitors generated with this method were comparable with reported values obtained by other methods. The assay was optimized and applied to a small-scale screening of 1,280 compounds. The discovery of several new inhibitors warrants further investigation. This assay was also proven to be easily adopted to test compounds for drosophila and mouse γsecretase, which could be very useful to assess compounds activity against γ-secretase from different species before the in vivo test in animal models.

Plant Physiology, Oct 25, 2012

Journal of Experimental Botany, Aug 22, 2010

Xyloglucan endotransglucosylase/hydrolases (XTHs) are cell wall enzymes that are able to graft xy... more Xyloglucan endotransglucosylase/hydrolases (XTHs) are cell wall enzymes that are able to graft xyloglucan chains to oligosaccharides or to other available xyloglucan chains and/or to hydrolyse xyloglucan chains. As they are involved in the modification of the load-bearing cell-wall components, they are believed to be very important in the regulation of growth and development. Given the large number (33) of XTH genes in Arabidopsis and the overlapping expression patterns, specific enzymic properties may be expected. Five predominantly root-expressed Arabidopsis thaliana XTHs belonging to subgroup I/II were analysed here. These represent two sets of closely related genes: AtXTH12 and 13 on the one hand (trichoblast-enriched) and AtXTH17, 18, and 19 on the other (expressed in nearly all cell types in the root). They were all recombinantly produced in the yeast Pichia pastoris and partially purified by ammonium sulphate precipitation before they were subsequently all subjected to a series of identical in vitro tests. The kinetic properties of purified AtXTH13 were investigated in greater detail to rule out interference with the assays by contaminating yeast proteins. All five proteins were found to exhibit only the endotransglucosylase (XET; EC 2.4.1.207) activity towards xyloglucan and non-detectable endohydrolytic (XEH; EC 3.2.1.151) activity. Their endotransglucosylase activity was preferentially directed towards xyloglucan and, in some cases, water-soluble cellulose acetate, rather than to mixed-linkage b-glucan. Isoforms differed in optimum pH (5.0-7.5), in temperature dependence and in acceptor substrate preferences.

Group III-A XTH genes encode predominant xyloglucan endo hydrolase active in expanding tissues of... more Group III-A XTH genes encode predominant xyloglucan endo hydrolase active in expanding tissues of Arabidopsis thaliana

... KTH, School of Biotechnology (BIO), Glycoscience). Ibatullin, Farid M. Ezcurra, Ines (KTH, Sc... more ... KTH, School of Biotechnology (BIO), Glycoscience). Ibatullin, Farid M. Ezcurra, Ines (KTH, School of Biotechnology (BIO), Glycoscience). Bhalerao, Rishikesh P. Brumer, Harry (KTH, School of Biotechnology (BIO), Glycoscience). ...

The FEBS journal, 2009

A family 16 glycoside hydrolase, xyloglucan xyloglucosyl transferase (EC 2.4.1.207), also known a... more 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 subst...

Plant Biotechnology, 2010

Heterologous expression of plant genes, particularly those encoding carbohydrate-active enzymes s... more Heterologous expression of plant genes, particularly those encoding carbohydrate-active enzymes such as glycoside hydrolases and glycosyl transferases, continues to be a major hurdle in the functional analysis of plant proteomes. Presently, there are few convenient systems for the production of recombinant plant enzymes in active form and at adequate levels for biochemical and structural characterization. The methylotrophic yeast Pichia pastoris is an attractive expression host due to its ease of manipulation and its capacity to perform post-translational protein modifications, such as Nglycosylation [Daly and Hearn (2005) J Mol Recognit 18: 119-138]. Here, we demonstrate the utility of the P. pastoris SMD1168H/pPICZ-alpha C system for the expression of a range of xyloglucan endo-transglycosylase/hydrolase (XTH) cDNAs from barley (Hordeum vulgare). Although stable transformants were readily obtained by positive selection for vector-induced antibiotic resistance for all of the nine constructs tested, only five isoforms were secreted as soluble proteins into the culture medium, four in active form. Furthermore, production levels of these five isoforms were found to be variable, depending on the transformant, which further underscores the necessity of screening multiple clones for expression of active enzyme. Failure to express certain XTH isoforms in P. pastoris could not be correlated with any conserved gene or protein sequence properties, and this precluded using rational sequence engineering to enhance heterologous expression of the cDNAs. Thus, while significant advances are reported here, systems for the heterologous production of plant proteins require further development.

PLANT PHYSIOLOGY, 2013

The molecular basis of primary wall extension endures as one of the central enigmas in plant cell... more The molecular basis of primary wall extension endures as one of the central enigmas in plant cell morphogenesis. Classical cell wall models suggest that xyloglucan endo-transglycosylase activity is the primary catalyst (together with expansins) of controlled cell wall loosening through the transient cleavage and religation of xyloglucan-cellulose cross links. The genome of Arabidopsis (Arabidopsis thaliana) contains 33 phylogenetically diverse XYLOGLUCAN ENDO-TRANSGLYCOSYLASE/ HYDROLASE (XTH) gene products, two of which were predicted to be predominant xyloglucan endohydrolases due to clustering into group III-A. Enzyme kinetic analysis of recombinant AtXTH31 confirmed this prediction and indicated that this enzyme had similar catalytic properties to the nasturtium (Tropaeolum majus) xyloglucanase1 responsible for storage xyloglucan hydrolysis during germination. Global analysis of Genevestigator data indicated that AtXTH31 and the paralogous AtXTH32 were abundantly expressed in expanding tissues. Microscopy analysis, utilizing the resorufin b-glycoside of the xyloglucan oligosaccharide XXXG as an in situ probe, indicated significant xyloglucan endohydrolase activity in specific regions of both roots and hypocotyls, in good correlation with transcriptomic data. Moreover, this hydrolytic activity was essentially completely eliminated in AtXTH31/AtXTH32 double knockout lines. However, single and double knockout lines, as well as individual overexpressing lines, of AtXTH31 and AtXTH32 did not demonstrate significant growth or developmental phenotypes. These results suggest that although xyloglucan polysaccharide hydrolysis occurs in parallel with primary wall expansion, morphological effects are subtle or may be compensated by other mechanisms. We hypothesize that there is likely to be an interplay between these xyloglucan endohydrolases and recently discovered apoplastic exo-glycosidases in the hydrolytic modification of matrix xyloglucans.