Subproject A4 – Structure and specificity of enzymes involved in the degradation of alpha-mannans from diatoms

Responsible: Jan-Hendrik Hehemann, MARUM, Bremen University

Subproject A4 aims to functionally characterize carbohydrate-active enzymes of marine Bacteroidetes that specialize in the utilization of mannose-rich algal polysaccharides.

Biochemical and structural studies of enzymes that degrade algal polysaccharides are key to better understand the marine carbon cycle. Furthermore, the characterization of marine CAZymes enables the discovery of new biocatalysts with high potential for the bioeconomy. In the second funding phase, we study the specificity and structure of enzymes from putative mannan PULs of bloom associated Bacteroidetes. This work focuses on the investigation of endo-acting mannanases, exo-acting mannosidases and sulfatases. Recent discoveries of putative alpha-mannan degrading PULs in bloom associated bacteria by the POMPU consortium partners and the isolation of alpha—mannans from marine diatoms point towards an important connection between heterotrophic bacteria and diatom derived alpha-mannans in the marine carbon cycle. This hypothesis remains unexplored. The work proposed here will address this hypothesis by functionally characterizing alpha-mannan degrading PULs in marine bacteria and testing their activity on diatom derived alpha-mannan.

Functional characterization of a cytochrome P450 monooxygenase that uses an oxidative mechanism to demethylate 6-O-methyl-galactose from the polysaccharides agar and porphyrin (Reisky et al., 2018, Nature Chemical Biology 14: 342–344). A: 3D crystal structure of P450ZoGa in complex of 6-O-methyl-d-galactose. The 2Fo-Fc map at 1 sigma is shown in blue along with the adjacent residues of the active site. Ligand coordination in the complex of 6-O-methyl-d-galactose and P450ZoGa. B: Overall view of catalytic site architecture with the relevant secondary structure elements and the residue ranges labeled. The iron is represented as a brown colored sphere. C: The possible electrostatic interactions found within the catalytic site that coordinate the ligand. Hydrogen bonds are shown in black and the C-H…π interaction is shown in purple with the distances in