Rhamnogalacturonan-I oligogalacturonide profiling, a tool to elucidate the mode of action of RGI-degrading enzymes and RGI structure

Pectins play a major role in the control of plant development and are widely used as hydrocolloids in the food industry. Yet, the fine structure of rhamnogalacturonan-I (RGI) pectic domain is difficult to determine owing to its chemical and structural complexity. In this study, we developed a sensitive analytical method based on the chromatographic separation of oligosaccharides derived from RGI, combined with accurate determination of their sizes and side-chains patterns using high resolution (HR) and tandem (MS/MS) mass spectrometry. This method revealed the structure of RGI from various sources following its hydrolysis by three enzymes, two rhamnogalacturonan hydrolase (RHG and RHG B) and one rhamnogalacturonan lyase (RGL) from A. Aculeatinus, that were heterologously expressed. We first defined the biochemical specificities of the recombinant RGI-ase, using enzymatic assays and NMR analyses, to ascertain their specific activities towards RGI. Finally, employing a specific library of >420 RGI-oligos, built from chromatography and mass spectrometry data, we assessed their mode of action by the analysis of the degradation products. Altogether, our results highlight that this method is of prime importance for analysis of RGI fine structure and to reveal differences in the enzyme's specificities related to peculiar structural motifs.