Functional analysis of carbohydrate-active enzymes

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Subproject A2 aims to functionally characterize carbohydrate-active enzymes of marine Bacteroidetes that specialize in polysaccharide degradation. In the first POMPU phase, we found that xylan-specific PULs and their protein functions are repeatedly detectable in the bacterioplankton of microalgal spring blooms. Since the function of the encoded enzymes was largely unknown, we used the second funding phase to investigate PULs related to marine xylan degradation and their corresponding enzyme cascades. Specifically, we could biochemically and functionally characterize two different xylan-specific PULs of a marine Bacteroidetes strain. Furthermore, we could show that formaldehyde produced by the action of a P450 monooxygenase on 6-O-methyl-D-galactose is detoxified via the RuMP pathway. Finally, we discovered an alternative pathway for the degradation of ulvan, which includes a novel dehydratase activity. In the third funding phase of POMPU we will focus on a marine PUL type, which was abundantly detected in diatom-driven phytoplankton blooms. Our preliminary bioinformatic analyses indicated that this PUL type is responsible for the utilization of so far unknown sulfated xylans. In order to reach this goal, we have defined the following objectives: (i) functional analysis of the PUL encoded sulfatases for their activity on sulfated xylans isolated from diatoms, (ii) biochemical characterization of new xylan-degrading CAZymes, (iii) elucidation of the substrate specificity of SusD-like carbohydrate binding proteins in xylan utilization. Finally, we will support the consortium partners in the functional analysis of marine arabinogalactan- and ß-glucan-specific CAZymes. All experiments will include recombinant expression and purification of the enzymes/proteins including in-depth biochemical analyses and structural biological characterizations of selected CAZymes together with a range of partners within the POMPU consortium.