Poster: Cell walls

Abs # 625: Specificity of fungal endo-xylogalacturonan hydrolase expressed in Pichia pastoris

Presenter:	Naran, Radnaa , naran@biochem.okstate.edu
Authors	Naran, Radnaa  (A)   Pierce, Margaret L. (A)   Mort, Andrew J. (A)
Affiliations:	(A): Oklahoma State University

Xylogalacturonans (XGA) are a subunit of plant cell wall pectic polymers made of a homogalacturonan backbone substituted with β-xylose at the C-3 position. Little work has been done on comparing the structure of XGA in various plant species. Fungal enzymes cloned in Pichia pastoris can be readily prepared free from contaminating activities (1) and are therefore valuable tools for structural study of plant cell wall polysaccharides. Knowledge of the specificity and activity of such enzymes are essential for their application. The specificity of Aspergillus fumigatus endo-xylogalacturonan hydrolase cloned in Pichia pastoris has been studied by using an endopolygalacturonase-resistant alkali extract of cotton suspension cell walls, which are rich in XGA. Individual oligomers produced by the endo-xylogalacturonan hydrolase digestion of cotton XGA were separated and purified by gel filtration and anion-exchange chromatography. The oligomers were profiled by CZE with LIF-detection and their structures were elucidated by NMR-spectroscopy and MALDI-TOF mass spectrometry. The study revealed that all XGA oligomers produced had a xylose residue at the reducing end GalA while the non-reducing end GalA of the oligomers is not necessarily substituted with xylose. The smallest oligomer produced by the enzyme action is GalA₂Xyl with Xyl at the reducing end GalA. GA₂Xyl₂ was found in large amounts, suggesting that cotton suspension cell wall XGA might be rather heavily substituted. (1). Fu, J., R. Prade and A. Mort. 2001. Carbohydr. Res. 330: 73-81