Carbohydrate Binding Module Family 20

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• Author: ^^^Marie Sofie Møller^^^
• Responsible Curators: ^^^Birte Svensson^^^ and ^^^Stephan Janecek^^^

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CAZy DB link

http://www.cazy.org/CBM20.html

Ligand specificities

CBM20 binds starch granules (raw starch) its soluble components amylose and amylopectin as well as β-cyclodextrin, a small amylose mimicing cyclic carbohydrate.

Structural Features

• Fold: Beta sandwich.
• Type: Type B
• Features of ligand binding: At least one but more typically two binding sites have been found in modules having the CBM20 complexed with bound carbohydrate. Such complexes have been studied for modules originating from several amylolytic enzymes, e.g. GH13_2 CGTase from Bacillus circulans [1], GH14 β-amylase from Bacillus cereus [2] and GH15 glucoamylase from Aspergillus niger [3], as well as the human glucan phosphatase laforin [4]. The two binding sites of CBM20 have been best illustrated in the NMR structure of the isolated module from A. niger glucoamylase complexed with β-cyclodextrin [3] and the X-ray structure of the module of the intact B. circulans CGTase in complex with maltose [1]. Binding site 1, important for raw starch binding ability, is formed from two tryptophan residues (Trp543 and Trp590 in the glucoamylase and Trp616 and Trp662 in the CGTase) making a compact and rigid hydrophobic site exposed on the surface and well adapted to bind glucose residues in the cyclodextrin ligands, considered as starch mimics. This small and easily accessible site may function as the place where the starch is initially recognized and it in fact does not change conformation after β-cyclodextrin binding compared to the free CBM20 [5]. It is worth mentioning that both tryptophan residues make stacking interactions with glucose rings and are conserved in the sequence alignment of CBM20s [6]. This is not the case, however, for aromatic residues stacked against glucose rings in binding site 2, which may function to guide the starch chains to the active site and is thus more extended and flexible, undergoing a larger conformational rearrangement when binding the β-cyclodextrin [3]. While there are two tyrosines (Tyr527 and Tyr556) in the glucoamylase binding site 2, only one aromatic residue (Tyr633, corresponding to the Tyr556) is believed to play the analogous role in the CGTase. On the other hand, a third well-conserved tryptophan residue (Trp563 in glucoamylase and Trp636 in CGTase), although buried and thus not able to interact with β-cyclodextrin directly, was found to be involved in making contacts with several residues at binding site 2 [3].

Functionalities

• Functional role of CBM: The CBM20 from the Aspergillus niger glucoamylase has been shown not only to bind starch but also disrupting its surface, thereby enhancing the amylolytic rate [7]. A CBM20 from an auxiliary activities family AA13 starch polysaccharide monooxygenase was shown to be important for amylose binding and activity on amylose [8].
• Most Common Associated Modules: The enzymes, of which the CBM20 module constitutes a domain, have predominantly specificities from the ɑ-amylase family GH13 or enzymes from families GH70 and GH77, but can also belong to families GH14 β-amylases and GH15 glucoamylases [9]. Among other CAZy GH families, the CBM20 is in some cases found associated with enzymes from other CAZy families GH31, GH57, GH119 and the auxiliary activities family AA13. Furthermore, CBM20 modules have been recognised in enzymes of which the catalytic domain is not classified in CAZy. Examples are phosphoglucan, water dikinase, glycerophosphodiester phosphodiesterase-5, laforin, and genethonin-1 [6]. The modules of family CBM20 have commonly been found in a single copy and usually appear without SBDs from other CBM families within the same protein, although co-occurence has been observed with CBM25, CBM34, and CBM48 [6].
• Novel Applications:

Family Firsts

First Identified

The first CBM20 was recognised in the early 1980s at the C-termini of glucoamylases from Aspergillus awamori [10] and Aspergillus niger [11, 12, 13].

First Structural Characterization

References

1. Penninga D, van der Veen BA, Knegtel RM, van Hijum SA, Rozeboom HJ, Kalk KH, Dijkstra BW, and Dijkhuizen L. (1996). The raw starch binding domain of cyclodextrin glycosyltransferase from Bacillus circulans strain 251. J Biol Chem. 1996;271(51):32777-84. DOI:10.1074/jbc.271.51.32777 |
2. Mikami B, Adachi M, Kage T, Sarikaya E, Nanmori T, Shinke R, and Utsumi S. (1999). Structure of raw starch-digesting Bacillus cereus beta-amylase complexed with maltose. Biochemistry. 1999;38(22):7050-61. DOI:10.1021/bi9829377 |
3. Sorimachi K, Le Gal-Coëffet MF, Williamson G, Archer DB, and Williamson MP. (1997). Solution structure of the granular starch binding domain of Aspergillus niger glucoamylase bound to beta-cyclodextrin. Structure. 1997;5(5):647-61. DOI:10.1016/s0969-2126(97)00220-7 |
4. Raththagala M, Brewer MK, Parker MW, Sherwood AR, Wong BK, Hsu S, Bridges TM, Paasch BC, Hellman LM, Husodo S, Meekins DA, Taylor AO, Turner BD, Auger KD, Dukhande VV, Chakravarthy S, Sanz P, Woods VL Jr, Li S, Vander Kooi CW, and Gentry MS. (2015). Structural mechanism of laforin function in glycogen dephosphorylation and lafora disease. Mol Cell. 2015;57(2):261-72. DOI:10.1016/j.molcel.2014.11.020 |
5. Sorimachi K, Jacks AJ, Le Gal-Coëffet MF, Williamson G, Archer DB, and Williamson MP. (1996). Solution structure of the granular starch binding domain of glucoamylase from Aspergillus niger by nuclear magnetic resonance spectroscopy. J Mol Biol. 1996;259(5):970-87. DOI:10.1006/jmbi.1996.0374 |
6. Janeček Š, Mareček F, MacGregor EA, and Svensson B. (2019). Starch-binding domains as CBM families-history, occurrence, structure, function and evolution. Biotechnol Adv. 2019;37(8):107451. DOI:10.1016/j.biotechadv.2019.107451 |
7. Southall SM, Simpson PJ, Gilbert HJ, Williamson G, and Williamson MP. (1999). The starch-binding domain from glucoamylase disrupts the structure of starch. FEBS Lett. 1999;447(1):58-60. DOI:10.1016/s0014-5793(99)00263-x |
8. Vu VV, Hangasky JA, Detomasi TC, Henry SJW, Ngo ST, Span EA, and Marletta MA. (2019). Substrate selectivity in starch polysaccharide monooxygenases. J Biol Chem. 2019;294(32):12157-12166. DOI:10.1074/jbc.RA119.009509 |
9. Janeček Š, Svensson B, and MacGregor EA. (2011). Structural and evolutionary aspects of two families of non-catalytic domains present in starch and glycogen binding proteins from microbes, plants and animals. Enzyme Microb Technol. 2011;49(5):429-40. DOI:10.1016/j.enzmictec.2011.07.002 |

10. Hayashida, S., Kunisaki, S., Nakao, M. and Flor, P.Q. (1982) Evidence for raw starch-affinity site on Aspergillus awamori glucoamylase I. Agric. Biol. Chem., vol. 46, pp. 83-89.

11. Svensson, B., Pedersen, T.G., Svendsen, I., Sakai, T. and Ottesen, M. (1982) Characterization of two forms of glucoamylase from Aspergillus niger. Carlsb. Res. Commun. vol. 47, pp. 55-69.

12. Svensson, B., Larsen, K., Svendsen, I., and Boel, E. (1983) The complete amino acid sequence of the glycoprotein, glucoamylase G1, from Aspergillus niger. Carlsb. Res. Commun. vol. 48, pp. 529-544.

13. Boel E, Hjort I, Svensson B, Norris F, Norris KE, and Fiil NP. (1984). Glucoamylases G1 and G2 from Aspergillus niger are synthesized from two different but closely related mRNAs. EMBO J. 1984;3(5):1097-102. DOI:10.1002/j.1460-2075.1984.tb01935.x |
14. 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 |

15. Davies, G.J. and Sinnott, M.L. (2008) Sorting the diverse: the sequence-based classifications of carbohydrate-active enzymes. The Biochemist, vol. 30, no. 4., pp. 26-32. Download PDF version.

16. 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. 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 |
18. 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 |
19. 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 |