Carbohydrate Binding Module Family 21

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• Authors: Birte Svensson and Stefan Janecek
• Responsible Curators: Birte Svensson and Stefan Janecek

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CAZy DB link

https://www.cazy.org/CBM21.html

Ligand specificities

Modules from family CBM21 bind to the α-glucan starch and oligosaccharides derived from starch or related to such oligosaccharides that contain α-1,4-linked glucose and/or α-1,6-linked glucose including maltose through maltoheptaose, β- and γ-cyclodextrins, isomaltotriose and isomaltotetraose [1, 2, 3, 4]. CBM21 also interacts with amylose and alters its ultrastructure as demonstrated by atomic force microscopy [5]. Circular permutation enhanced the affinity for amylose [6]. The domain has been described as providing mainly glucoamylase and α-amylase with the ability to bind onto raw-starch (starch granules) [7, 8, 9, 10, 11].

Structural Features

Structures of CBM21 have been determined by NMR and X-ray crystallography both in free and in carbohydrate complexed form. They adopt a common β-sandwich fold and have two binding sites accommodating carbohydrate ligands similarly to other starch binding domains. The binding sites contain aromatic side chains participating in carbohydrate interaction. Initially it was described by modelling using an NMR structure of a CBM20 as template [1] and thereafter by docking onto the NMR structure determined for CBM21 from the family GH15 Rhizopus oryzae glucoamylase [3]. The crystal structure determined for this CBM21 in complex with β-cyclodextrin or maltoheptaose identified W47, Y83 and Y94 interacting at site I and Y32 and F58 at site II for both ligands [4]. Site I requires ligands with DP > 3 for binding [2]. A CBM21-like domain was identified in the crystal structure of barley family GH13 limit dextrinase [12].

Functionalities

CBM21s are mainly associated with some fungal glucoamylases and α-amylases of the family GH15 [7, 8, 9] and GH13 [10, 11], respectively. This was confirmed by an exhaustive evolutionary analysis of 85 fungal genomes [13]. In both cases, i.e. in GH15 glucoamylases and GH13 α-amylases, the CBM21 precedes the catalytic domain [14]. The CBM21 is present also as a part of the regulatory subunit of Ser/Thr-specific protein phosphatases that directs the protein phosphatase to glycogen [15]. Recently, a structural comparison identified an N-terminal CBM21-like domain in the barley GH13 limit dextrinase [12], where it is followed by the module from the family CBM48 succeeded by the catalytic TIM-barrel.

Family Firsts

First Identified

Family CBM21 was first observed as an N-terminally located domain in glucoamylase from Rhizopus oryzae [7]. The function was ascribed based on comparison of multiple forms of the glucoamylase [7, 16] and amino acid sequence alignment [17]. The CBM21 sequence was revealed as related to that of the starch binding domain of Aspergillus niger glucoamylase [18], which has been assigned the family CBM20 [14, 19, 20].

First Structural Characterization

The first CBM21 three-dimensional structure was determined by NMR for the module from the family GH15 glucoamylase from Rhizopus oryzae [3]. The first CBM21 complex structures were determined by X-ray crystallography for that domain binding to β-cyclodextrin or maltoheptaose [4].

Novel Applications

The CBM21 from Rhizopus oryzae glucoamylase has been introduced as a novel affinity purification tag [21].

References

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7. Ashikari T, Nakamura N, Tanaka Y, Kiuchi N, Shibano Y, Tanaka T, Amachi T, and Yoshizumi H. “Rhizopus raw-starch-degrading glucoamylase: its cloning and expression in yeast.” Agric. Biol. Chem. 1986; 50: 957-64.

12. Møller MS, Abou Hachem M, Svensson B, and Henriksen A. (2012). Structure of the starch-debranching enzyme barley limit dextrinase reveals homology of the N-terminal domain to CBM21. Acta Crystallogr Sect F Struct Biol Cryst Commun. 2012;68(Pt 9):1008-12. DOI:10.1107/S1744309112031004 |

17. Tanaka Y, Ashikari T, Nakamura N, Kiuchi N, Shibano Y, Amachi T, and Yoshizumi H. Comparison of amino acid sequences of three glucoamylases and their structure-function relationships. Agric. Biol. Chem. 1986; 50: 965-9.

18. Svensson B, Jespersen H, Sierks MR, and MacGregor EA. (1989). Sequence homology between putative raw-starch binding domains from different starch-degrading enzymes. Biochem J. 1989;264(1):309-11. DOI:10.1042/bj2640309 |
19. Machovic M and Janecek S. (2006). Starch-binding domains in the post-genome era. Cell Mol Life Sci. 2006;63(23):2710-24. DOI:10.1007/s00018-006-6246-9 |
20. Christiansen C, Abou Hachem M, Janecek S, Viksø-Nielsen A, Blennow A, and Svensson B. (2009). The carbohydrate-binding module family 20--diversity, structure, and function. FEBS J. 2009;276(18):5006-29. DOI:10.1111/j.1742-4658.2009.07221.x |