Fig. 4: Biochemical confirmation of PUL predictions.

a, Gene organization of a predicted mannan PUL identified in Prevotella sp. PREV32, which was selected for in-depth biochemical characterization. Gene abbreviations: epim., mannobiose-2-epimerase; GPH, glycoside-pentoside-hexuronide transporter; hypo., hypothetical. b, Purified proteins incubated with six plant polymers highlighted the differential use of plant polymers by glycoside hydrolases. The bars represent an average of three replicates (white diamonds), are coloured by plant polymer and represent reducing ends (glucose equivalents) recovered. c, Incubation of purified enzymes with carob galactomannan (CGM) identified peaks matching elution times of standards, including mannose (M), β-1,4-manno-oligosaccharides (M2–M3) and manno-oligosaccharides substituted with (X) galactose residues (Gal(X)M(X)). The spectra represent one run of three replicates. ‘CGM neg’ is a negative control with CGM and no enzymes. nC, nanocoulombs. d, A hypothetical model, based on predicted protein locations^70,71 and biochemical data, depicting a process in which galactomannan is bound via outer membrane lipoproteins (SusCD), hydrolysed via GH5B to shorter manno-oligomers (with and without galactosylations) and imported into the periplasm. Galactose residues could be removed by a GH36 (detected in proteomics and located in another part of the genome), providing manno-oligomers as substrate for the GH26 enzyme. Hydrolysed galactomannan products (mannobiose and mannose) could be further transported into the cytoplasm by the GPH transporter (trans). In the cytoplasm, the GH130 enzyme can process either mannobiose or mannosyl-glucose generated from the epimerase in the PUL. Based on the substrates identified for GH5A, it could have a role in paving the way for GH5B access to mannans in complex substrates. Other enzymes in this PUL (indicated by the asterisks) include CE7, which could contribute to the degradation of acetylated mannans, and GH26B, which could be complementing GH26A.