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Family 16 CBMs are found essentially in bacteria (with the exception of some CBM16 members in archaea). They are also found associated with catalytic modules belonging mainly to 4 families of CAZymes: GH5 mannanases [1, 2], GH16 kappa carrageenases [3, 4, 5], GH18 chitinases [6] and PL18 alginate lyases [7, 8]. Binding to glucomannan and kappa-carrageenan has been demonstrated [1, 2, 5]. CBM16 binding to glucomannan (mixed β-1,4-linked polymer contains both glucose and mannose) has been studied by ITC (isothermal titration calorimetry) analyses and X-ray crystallography of complexes with pentomannan and pentoglucan [1, 2]. Conversely, binding to kappa-carrageenan has been shown by a double-blind approach using polysaccharide microarrays [5].
Structural Features
Figure 1. The structure of CBM16-1 of Caldanaerobius polysaccharolyticus ManA, in complex with cellopentaose, PDB ID 2zex. Four key residues of the binding cleft are highlighted.
CBM16 is a type B CBM family, with a characteristic concave cleft, allowing the binding of ligand longer than triose. The ligand binding cleft shows some promiscuity as it can accommodate pentoses containing glucose and mannose, but only in the context of planar polymers like β-1,4-glucans and not helical β-1,3-glucans [1]. The crystallographic structure determination of both CBMs from Caldanaerobius polysaccharolyticus (formerly Thermoanaerobacterium polysaccharolyticum) ManA revealed the importance of two aromatic residues in the binding cleft as well as two stretches of polar residues on both sides of the cleft, PDB ID 2zew [1]. Affinity studies of targeted mutants for the predicted key residues confirmed the importance of two tryptophans (Trp-20 and Trp-125), and two glutamines (Gln-81 and Gln-93) [2] (see Figure 1).
Based on sequence similarity and conservation of secondary structure elements it has been proposed that, along with the CBM4, CBM17, CBM22 and CBM27 families, they form a superfamily [9].
Functionalities
In the Man5A of Caldanaerobius polysaccharolyticus, the deletion of both its CBM16s severely impairs the ability of the catalytic module (GH5) to bind cellulose [10].
In the case of CgkA from Zobellia galactanivorans, the presence of the CBM16 is not required for the enzymatic activity on kappa-carrageenan, but has been shown to take part in the processive mechanism of the catalytic module (GH16) [4].
Even if frequently encoded within the gene for alginate lyase from family PL18, it is absent in the mature form of the enzyme, and no role in alginate degradation has been found up to now [8]. A chaperone function of this N-terminal module has been proposed after observation that its deletion hindered the correct folding and activity of the catalytic module [7].
Family Firsts
First Identified
Cloning of Man5A GH5 by Cann et al. in 1999 reaveled the presence of two tandem CBM16s on the C-terminal end. Their deletion resulted in failure of the catalytic module to bind to a cellulose column, and significant loss of both mannanase and carboxy methylcellulase activities [10].
First Structural Characterization
In 2008 Bae et al. solved the first structures of the CBM16 family: both modules of Caldanaerobius polysaccharolyticus Man5A, PDB ID 2zew, and two complexes of CBM16-1, one with cellopentaose PDB ID 2zex and one with mannopentaose PDB ID 2zey[1].
