Carbohydrate Binding Module Family 32

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

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CAZy DB link

http://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 [13]. 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) [5, 9, 10]. 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 [7, 8, 9, 10]. 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 [5, 7, 8, 9, 10]; 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 [2], 2J7M [9], 4A45 [8], 4A6O [8], and 2W1U [10], to name just a few.

Functionalities

CBM32 modules are thought to target the catalytic modules to their respective substrates. CBM32s from the Gram positive pathogen C. perfringens may well have a dual role as many of the enzymes have an LPXTG motif at their C-terminal end; this signals for 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 [8, 18]. Other modules that may be associated in the same enzymes are different families of CBMs, FNIII domains, and cohesin and dockerin domains[8, 19, 20, 21].

There are now examples of CBM32s that are independant of a catalytic module, such as the YeCBM32 periplasmic-binding protein; 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 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

The first identification of the galactose carbohydrate-binding function of a CBM32 was from the fungus Fusarium graminearum (previously Dactylium dendroides)[1].

First Structural Characterization

The first native crystal structure of a CBM32 from the fungus Fusarium graminearum was determined in 1991, although it had not yet been identified as a CBM [22].

References

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 |
2. Gaskell A, Crennell S, and Taylor G. (1995). The three domains of a bacterial sialidase: a beta-propeller, an immunoglobulin module and a galactose-binding jelly-roll. Structure. 1995;3(11):1197-205. DOI:10.1016/s0969-2126(01)00255-6 |
3. Newstead SL, Watson JN, Bennet AJ, and Taylor G. (2005). Galactose recognition by the carbohydrate-binding module of a bacterial sialidase. Acta Crystallogr D Biol Crystallogr. 2005;61(Pt 11):1483-91. DOI:10.1107/S0907444905026132 |
4. Centeno MS, Goyal A, Prates JA, Ferreira LM, Gilbert HJ, and Fontes CM. (2006). Novel modular enzymes encoded by a cellulase gene cluster in Cellvibrio mixtus. FEMS Microbiol Lett. 2006;265(1):26-34. DOI:10.1111/j.1574-6968.2006.00464.x |
5. Mizutani K, Fernandes VO, Karita S, Luís AS, Sakka M, Kimura T, Jackson A, Zhang X, Fontes CM, Gilbert HJ, and Sakka K. (2012). Influence of a mannan binding family 32 carbohydrate binding module on the activity of the appended mannanase. Appl Environ Microbiol. 2012;78(14):4781-7. DOI:10.1128/AEM.07457-11 |
6. Abbott DW, Hrynuik S, and Boraston AB. (2007). Identification and characterization of a novel periplasmic polygalacturonic acid binding protein from Yersinia enterolitica. J Mol Biol. 2007;367(4):1023-33. DOI:10.1016/j.jmb.2007.01.030 |
7. Boraston AB, Ficko-Blean E, and Healey M. (2007). Carbohydrate recognition by a large sialidase toxin from Clostridium perfringens. Biochemistry. 2007;46(40):11352-60. DOI:10.1021/bi701317g |
8. Ficko-Blean E, Stuart CP, Suits MD, Cid M, Tessier M, Woods RJ, and Boraston AB. (2012). Carbohydrate recognition by an architecturally complex α-N-acetylglucosaminidase from Clostridium perfringens. PLoS One. 2012;7(3):e33524. DOI:10.1371/journal.pone.0033524 |
9. Ficko-Blean E and Boraston AB. (2006). The interaction of a carbohydrate-binding module from a Clostridium perfringens N-acetyl-beta-hexosaminidase with its carbohydrate receptor. J Biol Chem. 2006;281(49):37748-57. DOI:10.1074/jbc.M606126200 |
10. Ficko-Blean E and Boraston AB. (2009). N-acetylglucosamine recognition by a family 32 carbohydrate-binding module from Clostridium perfringens NagH. J Mol Biol. 2009;390(2):208-20. DOI:10.1016/j.jmb.2009.04.066 |

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 |
14. Hashimoto H (2006). Recent structural studies of carbohydrate-binding modules. Cell Mol Life Sci. 2006;63(24):2954-67. DOI:10.1007/s00018-006-6195-3 |
15. Shoseyov O, Shani Z, and Levy I. (2006). Carbohydrate binding modules: biochemical properties and novel applications. Microbiol Mol Biol Rev. 2006;70(2):283-95. DOI:10.1128/MMBR.00028-05 |
16. Guillén D, Sánchez S, and Rodríguez-Sanoja R. (2010). Carbohydrate-binding domains: multiplicity of biological roles. Appl Microbiol Biotechnol. 2010;85(5):1241-9. DOI:10.1007/s00253-009-2331-y |
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 |
18. Abbott DW, Eirín-López JM, and Boraston AB. (2008). Insight into ligand diversity and novel biological roles for family 32 carbohydrate-binding modules. Mol Biol Evol. 2008;25(1):155-67. DOI:10.1093/molbev/msm243 |
19. Ficko-Blean E, Gregg KJ, Adams JJ, Hehemann JH, Czjzek M, Smith SP, and Boraston AB. (2009). Portrait of an enzyme, a complete structural analysis of a multimodular {beta}-N-acetylglucosaminidase from Clostridium perfringens. J Biol Chem. 2009;284(15):9876-84. DOI:10.1074/jbc.M808954200 |
20. Chitayat S, Gregg K, Adams JJ, Ficko-Blean E, Bayer EA, Boraston AB, and Smith SP. (2008). Three-dimensional structure of a putative non-cellulosomal cohesin module from a Clostridium perfringens family 84 glycoside hydrolase. J Mol Biol. 2008;375(1):20-8. DOI:10.1016/j.jmb.2007.10.031 |
21. Adams JJ, Gregg K, Bayer EA, Boraston AB, and Smith SP. (2008). Structural basis of Clostridium perfringens toxin complex formation. Proc Natl Acad Sci U S A. 2008;105(34):12194-9. DOI:10.1073/pnas.0803154105 |
22. Ito N, Phillips SE, Stevens C, Ogel ZB, McPherson MJ, Keen JN, Yadav KD, and Knowles PF. (1991). Novel thioether bond revealed by a 1.7 A crystal structure of galactose oxidase. Nature. 1991;350(6313):87-90. DOI:10.1038/350087a0 |