Abstract
The hydrolysis of the plant cell wall by microbial glycoside hydrolases and esterases is the primary mechanism by which stored organic carbon is utilized in the biosphere, and thus these enzymes are of considerable biological and industrial importance. Plant cell wall-degrading enzymes in general display a modular architecture comprising catalytic and non-catalytic modules. The X4 modules in glycoside hydrolases represent a large family of non-catalytic modules whose function is unknown. Here we show that the X4 modules from a Cellvibrio japonicus mannanase (Man5C) and arabinofuranosidase (Abf62A) bind to polysaccharides, and thus these proteins comprise a new family of carbohydrate-binding modules (CBMs), designated CBM35. The Man5C-CBM35 binds to galactomannan, insoluble amorphous mannan, glucomannan, and manno-oligosaccharides but does not interact with crystalline mannan, cellulose, cello-oligosaccharides, or other polysaccharides derived from the plant cell wall. Man5C-CBM35 also potentiates mannanase activity against insoluble amorphous mannan. Abf62A-CBM35 interacts with unsubstituted oat-spelt xylan but not substituted forms of the hemicellulose or xylo-oligosaccharides, and requires calcium for binding. This is in sharp contrast to other xylan-binding CBMs, which interact in a calcium-independent manner with both xylo-oligosaccharides and decorated xylans.
Highlights
EXPERIMENTAL PROCEDURESThe recombinant plasmid pDH26, which encodes Man5C-CBM35-glycoside hydrolase family 5 (GH5), was generated by amplifying the region of man5C comprising nucleotides 589 –2495 using the primers 5Ј-GCGCATATGATGGCAGTACCGGAAGGC-3Ј and 5Ј-GCGGGATCCTTATTGCATCAGCGACCGG-3Ј, which contain NdeI and BamHI restriction sites, respectively
The hydrolysis of the plant cell wall by microbial glycoside hydrolases and esterases is the primary mechanism by which stored organic carbon is utilized in the biosphere, and these enzymes are of considerable biological and industrial importance
When the primary structure of Abf62A-CBM35 was used to query databases using BLAST, a number of sequences that display similarity to this sequence were identified in enzymes that modify carbohydrates, including glycoside hydrolases and lyases that attack the hemicellulosic and pectic polysaccharides, respectively, within the plant cell wall
Summary
The recombinant plasmid pDH26, which encodes Man5C-CBM35-GH5, was generated by amplifying the region of man5C comprising nucleotides 589 –2495 using the primers 5Ј-GCGCATATGATGGCAGTACCGGAAGGC-3Ј and 5Ј-GCGGGATCCTTATTGCATCAGCGACCGG-3Ј, which contain NdeI and BamHI restriction sites, respectively. To produce GST-Abf62A-CBM35, E. coli JM83 containing pDB2 was cultured as described above except that expression of the recombinant protein was induced at 30 °C for 4 h using a final IPTG concentration of 0.5 mM. The binding data was fitted by treating the CBM in the sample cell as the ligand and the polysaccharide as the acceptor This gives accurate values for KA and ⌬H, but not n (in this case the number of binding sites on each molecule of polysaccharide). Binding to Insoluble Polysaccharides—The binding of the CBM35 proteins to insoluble polysaccharides (acid-swollen cellulose, bacterial microcrystalline cellulose, DGM, ivory nut mannan, and insoluble oatspelt xylan) was determined qualitatively using SDS-PAGE. To determine the rate of mannohexaose hydrolysis, the hexasaccharide (0.6 mM) was incubated with 40 nM enzyme in PC buffer, pH 6.5 at 37 °C for up to 30 min in a total volume of 0.4 ml, and the release of mannotetraose was quantified as described above
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