Sigrid Rombouts
Katholieke Universiteit Leuven
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Featured researches published by Sigrid Rombouts.
Biochemical Journal | 2007
Ellen Fierens; Sigrid Rombouts; Kurt Gebruers; Hans Goesaert; Kristof Brijs; Johnny Beaugrand; Guido Volckaert; Steven Van Campenhout; Paul Proost; Christophe M. Courtin; Jan A. Delcour
Wheat (Triticum aestivum) contains a previously unknown type of xylanase (EC 3.2.1.8) inhibitor, which is described in the present paper for the first time. Based on its >60% similarity to TLPs (thaumatin-like proteins) and the fact that it contains the Prosite PS00316 thaumatin family signature, it is referred to as TLXI (thaumatin-like xylanase inhibitor). TLXI is a basic (pI> or =9.3 in isoelectric focusing) protein with a molecular mass of approx. 18-kDa (determined by SDS/PAGE) and it occurs in wheat with varying extents of glycosylation. The TLXI gene sequence encodes a 26-amino-acid signal sequence followed by a 151-amino-acid mature protein with a calculated molecular mass of 15.6-kDa and pI of 8.38. The mature TLXI protein was expressed successfully in Pichia pastoris, resulting in a 21-kDa (determined by SDS/PAGE) recombinant protein (rTLXI). Polyclonal antibodies raised against TLXI purified from wheat react with epitopes of rTLXI as well as with those of thaumatin, demonstrating high structural similarity between these three proteins. TLXI has a unique inhibition specificity. It is a non-competitive inhibitor of a number of glycoside hydrolase family 11 xylanases, but it is inactive towards glycoside hydrolase family 10 xylanases. Progress curves show that TLXI is a slow tight-binding inhibitor, with a K(i) of approx. 60-nM. Except for zeamatin, an alpha-amylase/trypsin inhibitor from maize (Zea mays), no other enzyme inhibitor is currently known among the TLPs. TLXI thus represents a novel type of inhibitor within this group of proteins.
Biochemical Journal | 2008
Elien Vandermarliere; Tine M. Bourgois; Sigrid Rombouts; Steven Van Campenhout; Guido Volckaert; Sergei V. Strelkov; Jan A. Delcour; Anja Rabijns; Christophe M. Courtin
GH 11 (glycoside hydrolase family 11) xylanases are predominant enzymes in the hydrolysis of heteroxylan, an abundant structural polysaccharide in the plant cell wall. To gain more insight into the protein-ligand interactions of the glycone as well as the aglycone subsites of these enzymes, catalytically incompetent mutants of the Bacillus subtilis and Aspergillus niger xylanases were crystallized, soaked with xylo-oligosaccharides and subjected to X-ray analysis. For both xylanases, there was clear density for xylose residues in the -1 and -2 subsites. In addition, for the B. subtilis xylanase, there was also density for xylose residues in the -3 and +1 subsite showing the spanning of the -1/+1 subsites. These results, together with the observation that some residues in the aglycone subsites clearly adopt a different conformation upon substrate binding, allowed us to identify the residues important for substrate binding in the aglycone subsites. In addition to substrate binding in the active site of the enzymes, the existence of an unproductive second ligand-binding site located on the surface of both the B. subtilis and A. niger xylanases was observed. This extra binding site may have a function similar to the separate carbohydrate-binding modules of other glycoside hydrolase families.
Proteins | 2010
Elien Vandermarliere; Willem Lammens; Jan-Wolfgang Schoepe; Sigrid Rombouts; Ellen Fierens; Kurt Gebruers; Guido Volckaert; Anja Rabijns; Jan A. Delcour; Sergei V. Strelkov; Christophe M. Courtin
Crystal structure of the noncompetitive xylanase inhibitor TLXI, member of the small thaumatin-like protein family Elien Vandermarliere, Willem Lammens, Jan Schoepe, Sigrid Rombouts, Ellen Fierens, Kurt Gebruers, Guido Volckaert, Anja Rabijns, Jan A. Delcour, Sergei V. Strelkov, and Christophe M. Courtin* 1Department of Pharmaceutical Sciences, Laboratory for Biocrystallography, Leuven 3000, Belgium 2Department of Biology, Laboratory of Molecular Plant Physiology, Leuven 3001, Belgium 3Department of Biosystems, Laboratory of Gene Technology, Leuven 3001, Belgium 4Department of Molecular and Microbial Systems, Laboratory of Food Chemistry and Biochemistry, Leuven 3001, Belgium
Journal of Enzyme Inhibition and Medicinal Chemistry | 2009
Sigrid Rombouts; Ellen Fierens; Elien Vandermarliere; Arnout Voet; Kurt Gebruers; Johnny Beaugrand; Christophe M. Courtin; Jan A. Delcour; Marc De Maeyer; Anja Rabijns; Steven Van Campenhout; Guido Volckaert
Recently, a novel wheat thaumatin-like protein, TLXI, which inhibits microbial glycoside hydrolase family (GH) 11 xylanases has been identified. It is the first xylanase inhibitor that exerts its inhibition in a non-competitive way. In the present study we gained insight into the interaction between TLXI and xylanases via combined molecular modeling and mutagenic approaches. More specifically, site-specific mutation of His22, situated on a loop which distinguishes TLXI from other, non-inhibiting, thaumatin-like proteins, and subsequent expression of the mutant in Pichia pastoris resulted in a protein lacking inhibition capacity. The mutant protein was unable to form a complex with GH11 xylanases. Based on these findings, the interaction of TLXI with GH11 xylanases is discussed.
Biochemical and Biophysical Research Communications | 2007
Steven Van Campenhout; Annick Pollet; Tine M. Bourgois; Sigrid Rombouts; Johnny Beaugrand; Kurt Gebruers; Evelien De Backer; Christophe M. Courtin; Jan A. Delcour; Guido Volckaert
Biochemical and Biophysical Research Communications | 2005
Gert Raedschelders; Katleen Fierens; Stefaan Sansen; Sigrid Rombouts; Kurt Gebruers; Johan Robben; Anja Rabijns; Christophe M. Courtin; Jan A. Delcour; Steven Van Campenhout; Guido Volckaert
Journal of Biotechnology | 2007
Tine M. Bourgois; Dung V. Nguyen; Stefaan Sansen; Sigrid Rombouts; Tim Beliën; Katleen Fierens; Gert Raedschelders; Anja Rabijns; Christophe M. Courtin; Jan A. Delcour; Steven Van Campenhout; Guido Volckaert
Journal of Molecular Recognition | 2007
Tim Beliën; Steven Van Campenhout; An Vanden Bosch; Tine M. Bourgois; Sigrid Rombouts; Johan Robben; Christophe M. Courtin; Jan A. Delcour; Guido Volckaert
Enzyme and Microbial Technology | 2007
Karolien Moers; Tine M. Bourgois; Sigrid Rombouts; Tim Beliën; Steven Van Campenhout; Guido Volckaert; Johan Robben; Kristof Brijs; Jan A. Delcour; Christophe M. Courtin
Archive | 2008
Ellen Fierens; Sigrid Rombouts; Kurt Gebruers; Christophe Courtin; Jan Delcour