Carbohydrate Binding Module Family 92

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• Author: Xuanwei Mei and Lauren McKee

• Responsible Curator: Yaoguang Chang

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CAZy DB link

http://www.cazy.org/CBM92.html

Ligand specificities

The first published CBM92 domain, and founding member of the CBM92 family, is the Cgk16A-CBM92 from the marine bacterium Wenyingzhuangia aestuarii OF219 [1]. The CBM92 bound specifically to the red algal polysaccharide carrageenan, a substrate of the appended GH16 enzyme domain. It was incapable of binding to other polysaccharide components in red algae including agarose, porphyran, and funoran [1]. Meanwhile, the CBM92 displayed no affinity to several anionic polysaccharides, namely pectin, chondroitin sulfates, dermatan sulfate, and sulfated fucans [1]. The Cgk16A-CBM92 showed no significant difference in the affinity to κ- and ι-carrageenan.

An earlier paper had demonstrated carbohydrate binding by a so-called Bacterial Fascin-like Domain (BFLD) within the GH16 β−1,3-glucanase LamC from a myxobacterial Corallococcus species. Affinity gel electrophoresis showed that the domain, now recognized as a CBM92 domain could bind to β−1,3-glucans [2]. Later, Lu et al showed binding to β-1,6-glucan by a CBM92 domain found within CpGlu30A, a GH30 β-1,6-glucanase [3]. Binding to the Glc- β-1,6-Glc structure found in pustulan, laminarin, scleroglucan, and yeast β-glucan has since been demonstrated for 12 additional members of family CBM92, none of which were able to bind carrageenan [4].

Structural Features

Figure 1. A consensus sequence logo generated from an alignment of 818 CBM92 domain sequences, including all carrageenan- and β-glucan binders characterised as of May 2024.

Several conserved residues (e.g., Phe-70, Arg-72, and Phe-75) were discovered through the multiple sequence alignments of Cgk16A-CBM92 and its close homologs [1], which were suggested to be critical for the ligand binding of that CBM. Phylogenetic analysis by Hao et al later showed that the homologs of Cgk16A-CBM92 represent a small sub-group within the CBM92 family (Fig. 1) [4]. In most other members of CBM92, there are three apparent binding sites identified by a conserved WExF sequence motif. The Trp of this motif is the principal sugar-binding residue, as evidenced by a site-directed mutagenesis study that could abolish binding by altering these residues to Ala [4]. The same paper also showed that wild-type proteins with fewer Trp-containing binding sites generally showed weaker binding to polysaccharide ligand [4]. A conserved CNR motif is found just to the N-terminal of the WExF sequence (Fig. 1). Structural analysis revealed that the Arg of this trio contributes to ligand binding, although it is absent in some binding sites in some proteins [4].

Figure 2. a Overall structure of CpCBM92B with the subdomains distinctly coloured and its ligand binding Trp and Glu residues shown as sticks. b The β-subdomain of CpCBM92B in complex with glucose. c Overlay of the CpCBM92A and -B subdomains showing sequence conservation within all putative binding sites. Single letter residue codes are colored based on the subdomains shown in panel a , and are labelled for subdomains ⍺/β/γ, in that order, with the CpCBM92A codes shown above those for CpCBM92B. Figure generated by Scott Mazurkewich

The crystal structures of CpCBM92A and CpCBM92B reveal a β-trefoil structure comprised of 12 β-strands arranged into 3 subdomains (⍺, β, and γ), like the β-trefoil domains found in Fascin and CBM13 proteins (Fig.2). Soaking experiments of CpCBM92B crystals with glucose, gentiobiose, and sophorose revealed a binding cleft within each subdomain comprising a Trp-Glu binding pair, contributed by the WExF motifs [4]. Ligand-bound structures suggest the potential for end-on binding to glucose and glucan oligo- or polysaccharides of potentially any linkage, but also reveal the possibility for extensions from both the O1 and O6 positions, enabling the observed mid-chain binding to a β−1,6-glucan such as pustulan, or to β-1,6-linked glucosyl substitutions in scleroglucan or laminarin.

Functionalities

Figure 3. Domain architecture of the κ-carrageenase Cgk16A. The enzyme consists of a signal peptide (1-20 amino acids), a GH16 domain (21-347 amino acids), a CBM92 domain (viz., Cgk16A-CBM92; 378-490 amino acids) and a C-terminal Sorting domain (516-581 amino acids).

In the natural context, Cgk16A-CBM92 is a component of the κ-carrageenase Cgk16A [5] (Fig. 3). It thus might maintain the enzyme near its substrate to improve the enzymatic activity via the proximity effect. The same observation was made for a β-1,6-glucan-binding CBM92 found appended to a GH30 β-1,6-glucanase enzyme from the environmental bacterium Chitinophaga pinensis [3].

Members of the CBM92 family are present in different glycoside hydrolase (GH) family sequences, e.g., GH16_17, GH5_46, GH5_54, GH18, GH19, GH30, and GH95. According to the CAZy database, these GH families comprise enzymes with various substrate specificities, including κ-carrageenase (GH16_17), chitinase (GH19 and GH18), fucosidase (GH95), β-1,6-glucanase (GH30), β-1,3-glucanase (GH16), and galactosidase (GH95).

To evaluate the feasibility of Cgk16A-CBM92 as a tool in the in situ investigation of carrageenan, a fluorescent probe was constructed by fusing Cgk16A-CBM92 with a green fluorescent protein. The in situ visualization of carrageenan in red alga Kappaphycus alvarezii was realized by utilizing the fluorescent probe [1].

Family Firsts

First Identified

The first published CBM92 member [1] is a component of the κ-carrageenase Cgk16A [5], which was discovered from a marine bacterium Wenyingzhuangia aestuarii OF219.

First Structural Characterization

The structures of CpCBM92A and CpCBM92B were solved by Scott Mazurkewich using X-ray crystallography [4]. These CBM92 domains flank a GH18 chitinase [6] within a large multi-modular enzyme from Chitinophaga pinensis that also contains a weakly-acting GH5_46 β-1,6-glucanase [3].

References

1. Mei X, Chang Y, Shen J, Zhang Y, Han J, and Xue C. (2022). Characterization of a Novel Carrageenan-Specific Carbohydrate-Binding Module: a Promising Tool for the In Situ Investigation of Carrageenan. J Agric Food Chem. 2022;70(29):9066-9072. DOI:10.1021/acs.jafc.2c03139 |
2. Zhou J, Li Z, Wu J, Li L, Li D, Ye X, Luo X, Huang Y, Cui Z, and Cao H. (2017). Functional Analysis of a Novel β-(1,3)-Glucanase from Corallococcus sp. Strain EGB Containing a Fascin-Like Module. Appl Environ Microbiol. 2017;83(16). DOI:10.1128/AEM.01016-17 |
3. Lu Z, Rämgård C, Ergenlioğlu İ, Sandin L, Hammar H, Andersson H, King K, Inman AR, Hao M, Bulone V, and McKee LS. (2023). Multiple enzymatic approaches to hydrolysis of fungal β-glucans by the soil bacterium Chitinophaga pinensis. FEBS J. 2023;290(11):2909-2922. DOI:10.1111/febs.16720 |
4. Hao MS, Mazurkewich S, Li H, Kvammen A, Saha S, Koskela S, Inman AR, Nakajima M, Tanaka N, Nakai H, Brändén G, Bulone V, Larsbrink J, and McKee LS. (2024). Structural and biochemical analysis of family 92 carbohydrate-binding modules uncovers multivalent binding to β-glucans. Nat Commun. 2024;15(1):3429. DOI:10.1038/s41467-024-47584-y |
5. Shen J, Chang Y, Chen F, and Dong S. (2018). Expression and characterization of a κ-carrageenase from marine bacterium Wenyingzhuangia aestuarii OF219: A biotechnological tool for the depolymerization of κ-carrageenan. Int J Biol Macromol. 2018;112:93-100. DOI:10.1016/j.ijbiomac.2018.01.075 |
6. Li H, Lu Z, Hao MS, Kvammen A, Inman AR, Srivastava V, Bulone V, and McKee LS. (2023). Family 92 carbohydrate-binding modules specific for β-1,6-glucans increase the thermostability of a bacterial chitinase. Biochimie. 2023;212:153-160. DOI:10.1016/j.biochi.2023.04.019 |