Functional studies on tandem carbohydrate-binding modules of a multimodular enzyme possessing two catalytic domains

Although functional studies on carbohydrate-binding module (CBM) have been carried out extensively, the role of tandem CBMs in the enzyme containing multiple catalytic domains (CDs) is unclear. Here, we identified a multidomain enzyme (Lc25986) with a novel modular structure from lignocellulolytic bacterial consortium. It consists of a mannanase domain, two CBM65 domains (LcCBM65-1/LcCBM65-2), and an esterase domain. To investigate CBM function and domain interactions, full-length Lc25986 and its variants were constructed and used for enzymatic activity, binding, and bioinformatic analyses. The results showed that LcCBM65-1 and LcCBM65-2 both bind mannan and xyloglucan but not cellulose or β-1,3-1,4-glucan, which differs from the ligand specificity of reported CBM65s. Compared to LcCBM65-2, LcCBM65-1 showed a stronger ligand affinity and a preference for acetylation sites. Both CBM65s stimulated the enzymatic activities of their respective neighboring CDs against acetylated mannan, but did not contribute to the activities of the distal CDs. The time course of mannan hydrolysis indicated that the full-length Lc25986 was more effective in the complete degradation of mixed acetyl/non-acetyl substrates than the mixture of single-CD mutants. When acting on complex substrates, LcCBM65-1 not only improved the enzymatic activity of the mannanase domain, but also directed the esterase domain to the acetylated polysaccharides. LcCBM65-2 adopted a low affinity to reduce interference with the catalysis of the mannanase domain. These results demonstrate the importance of CBMs for the synergism between the two CDs of a multidomain enzyme and suggest that they contribute to the adequate degradation of complex substrates such as plant cell walls.