Pectin-associated carbohydrate binding modules (CBMs)

Currently, there are 9 CBM families characterized with the binding affinity for pectin or monomeric components of pectin as listed in Appendix 2. Despite the importance of pectin in maintaining the cell wall structure in the majority of terrestrial plants, remarkably few pectin-targeting CBMs have been described to date, possibly because a large number of CBMs were studied using the cellulose- and glycan-degrading bacteria as model organisms (Cid et al., 2010). The carbohydrate binding domains of CAZymes often reflect the target substrate specificity of the enzymes, providing clues for the enzyme functions (Fujimoto et al., 2002). For example, arabinose-binding CBM42 family was in association with GH43, GH51, and GH54 families of α-L-arabinofuranosidases (Fujimoto et al., 2008; Miyanaga et al., 2004). Similarly, the CBM13 domain appended to the endo-β-1,4-D xylanase (GH10) of Streptomyces olivaceoviridis showed a preferential binding affinity for xylose or xylo- oligosaccharides from plant hemicellulose (Fujimoto et al., 2002). CBM13 of S. olivaceoviridis was also shown to be capable of binding single units of galactose and lactose in its xylan-binding site, as it shared the conserved polar residues Asp, Gln, Asn, and Tyr with the galactose binding sites of Ricinus communis (castor-oil plant) (Fujimoto et al., 2002). Similar capacity to bind galactose and the

galactose moiety of lactose was demonstrated using the CBM32 appended to the sialidase of Micromonospora viridifaciens (Newstead et al., 2005). CBM32s from Clostridium perfringens and Cladobotryum dendroides have also been assigned with similar galactose-binding functions (Abbott et al., 2007). On the other hand, the periplasmic CBM32 of Yersinia enterocolitica (YeCBM32)shared only ~20 % amino acid sequence similarities with other CBM32s, and possessed a structurally unique binding site architecture in which only a single active site residue (Trp) was conserved (Abbott et al., 2007). The unique binding site topology of YeCBM32 led to the alkalinisation of the surface to accommodate the internal binding of acidic oligosaccharides, resulting in the non-typical binding specificity to polymerized galacturonic acid residues, potato RG-I, and potato pectin galactan (Abbott et al., 2007c). YeCBM32 distinguishes itself from other characterized CBM32s in that it stands as an independent protein, and not as an accessory module of a larger catalytic protein (Abbott et al., 2007c). Apart from the cellulose-binding CBM of CttA protein in the cellulosome of R. flavefaciens, and the Sus proteins of B. thetaiotaomicron, CBMs appended within non-catalytic polypeptides have been rarely reported (Flint et al., 2008; Abbott et al., 2007c; Cameron et al., 2012). Ligand binding prior to intracellular transport and the prolonged retention of the substrate close to the bacterial cell surface have been proposed as potential functions of independent CBM domains (Abbott et al., 2007c). Substrate binding affinity for unsaturated Δ4,5-GalpA was reported from CBM35 domains appended to PL10 (Pel-CBM35), CE12 (Rhe-CBM35), and GH10 xylanase (Xyl-CBM35) from an environmental isolate, R. thermocellum, and C. japonicus, respectively (Montanier et al., 2009). All CBM35s were tested positive for binding the resultant unsaturated pectin product of β-elimination, but showed no affinity for pectin pre-digested with glycoside hydrolases, indicating CBM35

recognized Δ4,5-GalpA (Montanier et al., 2009). While Xyl-CBM35 may target the GlcA side chains present in some xylans, its binding to degraded pectic structure may play a role in recruiting xylanases at the sites of active plant degradation (Montanier et al., 2009). CBM62 appended to the GH5 endo-β- 1,4-xylanase shows a similar CBM-dependent targeting of catalytic modules into close proximity with the degrading complex polysaccharides (Montanier et al., 2011). The CBM62 fromthe cellulosomal GH5 endo-β-1,4-xylanase of C. thermocellum (CtCBM62) is currently the only constituting member of family CBM62 (Montanier et al., 2011). Although CtCBM62 showed binding affinity with

complex polysaccharides such as galactomannan, arabinogalactan, and xyloglucan, the binding is mediated by single ligand-binding sites which recognize single units of D-galactose and L- arabinopyranose decorating the target glycans (Montanier et al., 2011). CtCBM62 showed a significantly augmented binding affinity with complex polysaccharides in the presence of calcium ions which play important roles during the oligomerization of CtCBM62, resulting in the formation of multivalent ligand recognition sites (Montanier et al.,2011). CBM60 and CBM61 both displayed binding capacity for β-1,4-galactans (Montanier et al., 2011; Cid et al., 2010). CBM60 from GH11 xylanase binds tightly to 4 – 6 sugar residues of both β-1,4-galactans and xylan, although CBM60 was almost always associated with a catalytic module of xylanases, and xylan was usually selected as a preferred substrate for binding in the primary cell walls of tobacco stems (Montanier et al., 2010). CBM61 was first described in the functionally obscure ~160 amino acid region of GH53 endo-β-1,4- galactanase from Thermotoga maritima (Cid et al., 2010). CBM61 showed the greatest binding affinity to pectin and β-galactan motifs present in complex polysaccharides (Cid et al., 2010). Using glycan microarray and immunofluorescence microscopy, similar substrate binding profile was observed between CBM61 and anti-β-1,4-galactan monoclonal antibody LM5, although CBM61 showed a greater efficiency at accessing a wide range of cell wall sections of Arabidopsis thaliana stem, indicating CBM61 was able to tolerate glycan decorations present on the target polysaccharide (Cid et al., 2010). In Streptomyces avermitilis, CBM67 appended to GH78 α-L-rhamnosidase showed binding to L-rhamnose, and a reduced affinity for L-mannose (Fujimoto et al., 2013). CBM67 activity is calcium-dependent, and the EDTA-induced chelation of calcium ions led to a significantly reduced enzyme activity of GH78 against gum arabic, a rhamnose-containing complex polysaccharide (Fujimoto et al., 2013). CBM67 domains are also found in PL1 of Sorangium cellulosum and α-L- rhamnosidase of Paenibacillus sp., and the calcium binding motif within the ligand recognition sites was conserved in both CBM67s (Fujimoto et al., 2013). CBM77 was recently discovered in a

cellulosomal enzyme which possessed bifunctional pectate lyase families 1 and 9 from R. flavefaciens (Venditto et al., 2016). CBM77PL1/9 exclusively targeted low methylated homogalacturonan with a degree of polymerization > 7 or 8 GalpA residues (Venditto et al., 2016). Despite the mandatory requirements for divalent cations in pectate lyases, CBM77 activity was not affected by the use of

EDTA (Venditto et al., 2016). Currently, CtCBM62 and CBM77PL1/9 are the only cellulosomal CBM domains from which the target specificities for pectic polysaccharides have been demonstrated.