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, 13].

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 [14]. In both cases, i.e. in GH15 glucoamylases and GH13 α-amylases, the CBM21 precedes the catalytic domain [15]. 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 [16]. Recently, a structural comparison identified an N-terminal CBM21-like domain in the barley GH13 limit dextrinase [12, 13], 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, 17] and amino acid sequence alignment [18]. The CBM21 sequence was revealed as related to that of the starch binding domain of Aspergillus niger glucoamylase [19], which has been assigned the family CBM20 [15, 20, 21].

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 [22].

References

1. Chou WI, Pai TW, Liu SH, Hsiung BK, and Chang MD. (2006). The family 21 carbohydrate-binding module of glucoamylase from Rhizopus oryzae consists of two sites playing distinct roles in ligand binding. Biochem J. 2006;396(3):469-77. DOI:10.1042/BJ20051982 |
2. Chu CH, Li KM, Lin SW, Chang MD, Jiang TY, and Sun YJ. (2014). Crystal structures of starch binding domain from Rhizopus oryzae glucoamylase in complex with isomaltooligosaccharide: insights into polysaccharide binding mechanism of CBM21 family. Proteins. 2014;82(6):1079-85. DOI:10.1002/prot.24446 |
3. Liu YN, Lai YT, Chou WI, Chang MD, and Lyu PC. (2007). Solution structure of family 21 carbohydrate-binding module from Rhizopus oryzae glucoamylase. Biochem J. 2007;403(1):21-30. DOI:10.1042/BJ20061312 |
4. Tung JY, Chang MD, Chou WI, Liu YY, Yeh YH, Chang FY, Lin SC, Qiu ZL, and Sun YJ. (2008). Crystal structures of the starch-binding domain from Rhizopus oryzae glucoamylase reveal a polysaccharide-binding path. Biochem J. 2008;416(1):27-36. DOI:10.1042/BJ20080580 |
5. Jiang TY, Ci YP, Chou WI, Lee YC, Sun YJ, Chou WY, Li KM, and Chang MD. (2012). Two unique ligand-binding clamps of Rhizopus oryzae starch binding domain for helical structure disruption of amylose. PLoS One. 2012;7(7):e41131. DOI:10.1371/journal.pone.0041131 |
6. Stephen P, Cheng KC, and Lyu PC. (2012). Crystal structure of circular permuted RoCBM21 (CP90): dimerisation and proximity of binding sites. PLoS One. 2012;7(11):e50488. DOI:10.1371/journal.pone.0050488 |

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.

8. Bui DM, Kunze I, Horstmann C, Schmidt T, Breunig KD, and Kunze G. (1996). Expression of the Arxula adeninivorans glucoamylase gene in Kluyveromyces lactis. Appl Microbiol Biotechnol. 1996;45(1-2):102-6. DOI:10.1007/s002530050655 |
9. Houghton-Larsen J and Pedersen PA. (2003). Cloning and characterisation of a glucoamylase gene (GlaM) from the dimorphic zygomycete Mucor circinelloides. Appl Microbiol Biotechnol. 2003;62(2-3):210-7. DOI:10.1007/s00253-003-1267-x |
10. Steyn AJ, Marmur J, and Pretorius IS. (1995). Cloning, sequence analysis and expression in yeasts of a cDNA containing a Lipomyces kononenkoae alpha-amylase-encoding gene. Gene. 1995;166(1):65-71. DOI:10.1016/0378-1119(95)00633-0 |
11. Kang HK, Lee JH, Kim D, Day DF, Robyt JF, Park KH, and Moon TW. (2004). Cloning and expression of Lipomyces starkeyi alpha-amylase in Escherichia coli and determination of some of its properties. FEMS Microbiol Lett. 2004;233(1):53-64. DOI:10.1016/j.femsle.2004.01.036 |

12. øller2012 pmid=22949184

14. Chen W, Xie T, Shao Y, and Chen F. (2012). Phylogenomic relationships between amylolytic enzymes from 85 strains of fungi. PLoS One. 2012;7(11):e49679. DOI:10.1371/journal.pone.0049679 |
15. Machovic M, Svensson B, MacGregor EA, and Janecek S. (2005). A new clan of CBM families based on bioinformatics of starch-binding domains from families CBM20 and CBM21. FEBS J. 2005;272(21):5497-513. DOI:10.1111/j.1742-4658.2005.04942.x |
16. Bork P, Dandekar T, Eisenhaber F, and Huynen M. (1998). Characterization of targeting domains by sequence analysis: glycogen-binding domains in protein phosphatases. J Mol Med (Berl). 1998;76(2):77-9. DOI:10.1007/s001090050194 |
17. Takahashi T, Tsuchida Y, and Irie M. (1982). Isolation of two inactive fragments of a Rhizopus sp. glucoamylase: relationship among three forms of the enzyme and the isolated fragments. J Biochem. 1982;92(5):1623-33. DOI:10.1093/oxfordjournals.jbchem.a134088 |

18. 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.

19. 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 |
20. 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 |
21. 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 |
22. Lin SC, Lin IP, Chou WI, Hsieh CA, Liu SH, Huang RY, Sheu CC, and Chang MD. (2009). CBM21 starch-binding domain: a new purification tag for recombinant protein engineering. Protein Expr Purif. 2009;65(2):261-6. DOI:10.1016/j.pep.2009.01.008 |