Carbohydrate Binding Module Family 32

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• Author: ^^^Elizabeth Ficko-Blean^^^
• Responsible Curator: ^^^Al Boraston^^^

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CAZy DB link

https://www.cazy.org/CBM32.html

Ligand specificities

In 1994 the first CBM32 structure in complex with D-galactose was determined from a fungal galactose oxidase[1]. Following that, a CBM32 from a multi-modular sialidase produced by Micromonospora viridifaciens was shown to demonstrate galactose and lactose binding specificity [2, 3]. A CBM32 from a Cellvibrio mixtus family 16 glycoside hydrolase binds laminarin and pustulan [4] while a CBM32 from a Clostridium thermocellum mannanase has demonstrated binding on the non-reducing end of β-mannans and β-1,4-linked mannooligosaccharides[5]. A periplasmic-binding protein, YeCBM32, from Yersinia enterolitica shares sequence identity with the CBM32 family and binds the polygalaturonic acid components of pectin [6]. The Clostridium perfringens CBM32s have been well studied and many of their ligand specificities determined as follows: D-galactose, N-acetyl-D-galactosamine[7, 8, 9], D-galactose-β-1,4-N-acetyl-D-glucosamine (LacNAc), L-fucose-α-1,2-D-galactose-β-1,4-N-acetyl-D-glucosamine (type II blood group H-trisaccharide) [9] N-acetyl-D-glucosamine, N-acetyl-D-glucosamine-β-1,3-N-acetyl-D-galactosamine, N-acetyl-D-glucosamine-β-1,2-D-mannose, N-acetyl-D-glucosamine-β-1,3-D-mannose (non-biological) [10], N-acetyl-D-glucosamine-α-1,4-D-galactose[8]. Some members of the family 32 CBMs have demonstrated a degree of promiscuity in their binding, these include CpCBM32-2 from the NagH enzyme and CpCBM32C from the NagJ enzyme, both produced by Clostridium perfringens[9, 10].

Please see these references for an essential introduction to the CAZy classification system: [11, 12]. CBMs, in particular, have been extensively reviewed [13, 14, 15, 16].

Structural Features

The CBM32s share the common beta sandwich fold and have a bound structural metal ion most often attributed to be calcium. Most family members fall into the Type C category of lectin-like CBMs[17] and as such typically have fairly weak binding affinities (K_as in the mM^-1 and low μM^-1 range). These binding site are located at the terminal loop region within the CBM32 family. The binding sites are surface located and in some cases quite shallow and designed to bind monosaccharides or short oligosaccharides. Variability within the apical loop region within the family confers the different ligand specificities. In most cases the CBM32 members interact with the non-reducing end of oligosacchides; however, this is not always the case as demonstrated by the periplasmic-binding protein from Y. enterocolitica - which shares sequence identity with the CBM32 family - and binds polygalacturonic acid polymers[6]. Some structural examples of the complex oligosaccharide binding sites of the CBM32s can be found in the following pdbs: 1EUU, 2J7M, 4A6O and 4A45, to name just a few.

Functionalities

CBM32 modules are thought to target the catalytic modules to their respective substrates. In the Gram positive pathogen, C. perfringens, the CBM32s may well have a dual role as many of the enzymes have an LPXTG motif at their C-terminal end, signalling sortase-mediated anchoring to the bacterial cell wall. Thus, not only would the catalytic modules be targeted to substrate, but also the bacterium as a whole, suggesting an adhesin-like activity for these CBMs.

The types of catalytic modules that the CBM32 members are associated with vary widely and include sialidases, β-N-acetylglucosaminidases, α-N-acetylglucosaminidases, mannanases and galactose oxidases. In enteric bacteria the CBM32 motif may occur more than once in the same enzyme and they may or may not share the same ligand specifities suggesting the possibility of heterogenic multivalent binding events. Other modules that may be associated in the same enzymes are different families of CBMs, FNIII domains, and cohesin and dockerin domains.

There are now examples of CBM32s that are independant of a catalytic module, such as the periplasmic-binding protein from YeCBM32; however, under the strict definition of CBMs being appended to carbohydrate-active enzymes there is some debate as to whether the CBMs without covalently-bound carbohydrate-active enzymes are really CBMs. In any case, nature has found a way to use the CBM amino acid sequence, structural motif, modular character, and carbohydrate-binding functionality beyond the bounds of the current definition of a CBM.

Finally, the CBM32 family's "exotic" specificities for animal glycans suggest the possibility for novel application development, though to date none have been published.

Family Firsts

First Identified

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First Structural Characterization

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References

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1. Ito N, Phillips SE, Yadav KD, and Knowles PF. (1994). Crystal structure of a free radical enzyme, galactose oxidase. J Mol Biol. 1994;238(5):794-814. DOI:10.1006/jmbi.1994.1335 |

11. Davies, G.J. and Sinnott, M.L. (2008) Sorting the diverse: the sequence-based classifications of carbohydrate-active enzymes. Biochem. J. (BJ Classic Paper, online only). DOI: 10.1042/BJ20080382

12. Cantarel BL, Coutinho PM, Rancurel C, Bernard T, Lombard V, and Henrissat B. (2009). The Carbohydrate-Active EnZymes database (CAZy): an expert resource for Glycogenomics. Nucleic Acids Res. 2009;37(Database issue):D233-8. DOI:10.1093/nar/gkn663 |
13. Boraston AB, Bolam DN, Gilbert HJ, and Davies GJ. (2004). Carbohydrate-binding modules: fine-tuning polysaccharide recognition. Biochem J. 2004;382(Pt 3):769-81. DOI:10.1042/BJ20040892 |
17. Boraston AB, Notenboom V, Warren RA, Kilburn DG, Rose DR, and Davies G. (2003). Structure and ligand binding of carbohydrate-binding module CsCBM6-3 reveals similarities with fucose-specific lectins and "galactose-binding" domains. J Mol Biol. 2003;327(3):659-69. DOI:10.1016/s0022-2836(03)00152-9 |