Glycoside Hydrolase Family 55

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• Authors: ^^^Takuya Ishida^^^ and ^^^Kiyohiko Igarashi^^^
• Responsible Curator: ^^^Shinya Fushinobu^^^

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Glycoside Hydrolase Family 55
Clan	none
Mechanism	inverting
Active site residues	not known
CAZy DB link
https://www.cazy.org/GH55.html

Substrate specificities

Glycoside Hydrolase family 55 consists exclusively of β-1,3-glucanases, including both exo- and endo-enzymes. All biochemically characterized members of this family had been limited to fungal enzymes until the extensive work by Bianchetti and Takasuka et al. reporting crystallography of exo-β-1,3-glucanase SacteLam55A from Streptmyces sp. SirexAA-E (Uniprot G2NFJ9) together with characterization of many other bacterial enzymes [1].

The enzymes belonging to this family are generally called "laminarinases," because they hydrolyze laminarin from brown algae (β-1,3-glucan having single β-1,6-glucoside side chains: β-1,3/1,6-glucan). However, the physiological substrate for the enzymes might be fungal cell wall, whose major component is also β-1,3/1,6-glucan. The majority of the members in this family are exo-glucan-1,3-β-glucosidases (EC 3.2.1.58), which cleave the terminal β-1,3-glycosidic linkage at the non-reducing end of β-1,3-glucans or β-1,3/1,6-glucans. Many produce gentiobiose (β-D-glucopyranosyl-1,6-D-glucose) in addition to glucose during the degradation of β-1,3/1,6-glucan [2, 3]. Bgn13.1 from Hypocrea lixii (formerly known as Trichoderma harzianum) [4] and LamAI from Trichoderma viride [5]were characterised as endo-acting enzymes (EC 3.2.1.39).

Kinetics and Mechanism

Family 55 enzymes are inverting enzymes, as shown by ¹NMR analysis on ExgS from Aspergillus phoenicis (formerly known as Aspergillus saitoi) [6]. Release of α-glucose was subsequently confirmed by polarimetric analysis on family 55 enzymes from Acremonium persicinum[2]. These results are consistent with many classical reports on gentiobiose-producing exo-β-1,3-glucanases from fungi [7, 8], although the genes for these enzymes have not yet been described.

Catalytic Residues

Crystal structure of exo-β-1,3-glucanase Lam55A from Phanerochaete chrysospoirum K-3 (PcLam55A) complexed with gluconolactone (PDB ID 3eqo) suggests that Glu633 is the general acid. A candidate nucleophilic water was found near the C-1 atom of gluconolactone. Crystal structure of bacterial enzyme SacteLam55A complexed with laminarihexaose (PDB ID 4pf0) and kinetic analysis on its mutants revealed that corresponding glutamic acid (Glu502 in SacteLam55A) is functioning as general acid in bacterial enzymes.

General base identification for this family is less clear compared to general acid as well as several inverting GH families. In crystal structure of both PcLam55A and SacteLam55A, the candidate nucleophilic water have no direct interaction with acidic residue but with highly conserved glutamine residue which is h-bonded by conserved glutamic acid (Glu480 in SacteLam55A). The mutation on the corresponding glutamic acid (E480Q and E480A) of SacteLam55A is crucial for catalytic activity, based on which along with active site structure, proton relay system for activation of the water has been proposed [1].

In classical studies of a exo-β-1,3-glucanase from Sporotrichum dimorphosporum (formerly known as Basidiomycete QM-806), Jeffcoat and Kirkwood reported that chemical modification of histidine residues in the catalytic site of the enzyme caused irreversible loss of activity, suggesting a crucial role of a histidine residue [9].

Three-dimensional structures

The first solved 3-D structure was Lam55A from P. chrysosporium [10]. In this structure, two tandem β-helix domains are positioned side-by-side to form a rib cage-like structure. The active site is located between the two β-helix domains. A duplicated motif had been found in the primary sequence of EXG1 from Cochliobolus carbonum [11], predicting the presence of two structurally similar domains in this family.

SacteLam55A E502A structure complexed with laminarioligosaccharides revealed binding of scissile ligand and conformation of proposed catalytic residues. The structure also shows solvent exposed secondary binding site [1].

Family Firsts

First sterochemistry determination

Probably ExgS from A. saitoi by H-NMR analysis [6]. See kinetics and mechanism.

First gene cloning

BGN13.1 from T. harzianum (Uniprot P53626) [4] and EXG1 from C. carbonum (partial gene coning and gene knockout) (Uniprot P49426) [12]. First bacterial gene was cloned from Arthrobacter sp. NHB-10 (Uniprot A4PHQ5) [13].

First general acid residue identification

SacteLam55A from Streptmyces sp. SirexAA-E (Uniprot G2NFJ9) by crystal structure and kinetic analysis on mutants [1].

First general base residue identification

SacteLam55A from Streptmyces sp. SirexAA-E (Uniprot G2NFJ9) by crystal structure and kinetic analysis on mutants [1].

First 3-D structure

Lam55A from P. chrysosporium by X-ray crystallography [10].

References

1. Bianchetti CM, Takasuka TE, Deutsch S, Udell HS, Yik EJ, Bergeman LF, and Fox BG. (2015). Active site and laminarin binding in glycoside hydrolase family 55. J Biol Chem. 2015;290(19):11819-32. DOI:10.1074/jbc.M114.623579 |
2. Pitson SM, Seviour RJ, McDougall BM, Woodward JR, and Stone BA. (1995). Purification and characterization of three extracellular (1-->3)-beta-D-glucan glucohydrolases from the filamentous fungus Acremonium persicinum. Biochem J. 1995;308 ( Pt 3)(Pt 3):733-41. DOI:10.1042/bj3080733 |
3. Bara MT, Lima AL, and Ulhoa CJ. (2003). Purification and characterization of an exo-beta-1,3-glucanase produced by Trichoderma asperellum. FEMS Microbiol Lett. 2003;219(1):81-5. DOI:10.1016/S0378-1097(02)01191-6 |
4. de la Cruz J, Pintor-Toro JA, Benítez T, Llobell A, and Romero LC. (1995). A novel endo-beta-1,3-glucanase, BGN13.1, involved in the mycoparasitism of Trichoderma harzianum. J Bacteriol. 1995;177(23):6937-45. DOI:10.1128/jb.177.23.6937-6945.1995 |
5. Nobe R, Sakakibara Y, Fukuda N, Yoshida N, Ogawa K, and Suiko M. (2003). Purification and characterization of laminaran hydrolases from Trichoderma viride. Biosci Biotechnol Biochem. 2003;67(6):1349-57. DOI:10.1271/bbb.67.1349 |

6. Kasahara S, Nakajima T, Miyamoto C, Wada K, Furuichi Y, and Ichishima E. Characterization and mode of action of exo-1,3-β-D-glucanase from Aspergillus saitoi. J Ferment Bioeng 74 (4), 238-240 (1992).DOI:10.1016/0922-338X(92)90118-E

7. Nelson TE (1970). The hydrolytic mechanism of an exo-beta-(1--3)-D-glucanase. J Biol Chem. 1970;245(4):869-72. | Google Books | Open Library

8. Nagasaki N, Saito K, and Yarnamoto S. Purification and characterization of an exo-β-l,3-glucanase from a fungi imperfecti. Agric Biol Cbem 41, 493-502 (1977).JOI:JST.Journalarchive/bbb1961/41.493

9. Jeffcoat R and Kirkwood S. (1987). Implication of histidine at the active site of exo-beta-(1-3)-D-glucanase from Basidiomycete sp. QM 806. J Biol Chem. 1987;262(3):1088-91. | Google Books | Open Library
10. Ishida T, Fushinobu S, Kawai R, Kitaoka M, Igarashi K, and Samejima M. (2009). Crystal structure of glycoside hydrolase family 55 {beta}-1,3-glucanase from the basidiomycete Phanerochaete chrysosporium. J Biol Chem. 2009;284(15):10100-9. DOI:10.1074/jbc.M808122200 |
11. Nikolskaya AN, Pitkin JW, Schaeffer HJ, Ahn JH, and Walton JD. (1998). EXG1p, a novel exo-beta1,3-glucanase from the fungus Cochliobolus carbonum, contains a repeated motif present in other proteins that interact with polysaccharides. Biochim Biophys Acta. 1998;1425(3):632-6. DOI:10.1016/s0304-4165(98)00117-2 |
12. Schaeffer HJ, Leykam J, and Walton JD. (1994). Cloning and targeted gene disruption of EXG1, encoding exo-beta 1, 3-glucanase, in the phytopathogenic fungus Cochliobolus carbonum. Appl Environ Microbiol. 1994;60(2):594-8. DOI:10.1128/aem.60.2.594-598.1994 |
13. Okazaki K, Nishimura N, Matsuoka F, and Hayakawa S. (2007). Cloning and characterization of the gene encoding endo-beta-1,3-glucanase from Arthrobacter sp. NHB-10. Biosci Biotechnol Biochem. 2007;71(6):1568-71. DOI:10.1271/bbb.70030 |