Glycoside Hydrolase Family 65

This page has been approved by the Responsible Curator as essentially complete. CAZypedia is a living document, so further improvement of this page is still possible. If you would like to suggest an addition or correction, please contact the page's Responsible Curator directly by e-mail.

• Author: ^^^Hiroyuki Nakai^^^
• Responsible Curator: ^^^Hiroyuki Nakai^^^

---

Glycoside Hydrolase Family GH65
Clan	GH-L
Mechanism	inverting
Active site residues	known
CAZy DB link
https://www.cazy.org/GH65.html

Substrate specificities

Glycoside hydrolases belonging to GH65 act on substrates containing α-glucosidic linkages. GH65 contains mainly phosphorylases; maltose (Glc-α-1,4-Glc) phosphorylase (EC 2.4.1.8), trehalose (Glc-α1,α1-Glc) phosphorylase (EC 2.4.1.64), kojibiose (Glc-α-1,2-Glc) phosphorylase (EC 2.4.1.230), and trehalose 6-phosphate (Glc-α1,α1-Glc6P) phosphorylase (EC 2.4.1.-). Noticeably α,α-trehalases (EC 3.2.1.28), a hydrolase, are also GH65 members.

Kinetics and Mechanism

Phosphorolysis by GH65 enzymes proceeds with inversion of anomeric configuration, as first shown by Fitting and Doudoroff [1] using maltose phosphorylase from Neisseria meningitidis, i.e. maltose + Pi ↔ β-glucose 1-phosphate + glucose. The reaction mechanism for inverting GH65 phosphorylase has been proposed to be similar to an general acid/base-catalysed one-step displacement mechanism for inverting glycoside hydrolases [2], as proposed for inverting GH94 phosphorylase (previously classified into glycoside transferase family 36) [3], where direct nucleophilic attack by phosphate on the anomeric C-1 carbon is assisted by proton donation from the general acid catalyst to the glycosidic oxygen, instead of water molecule activated by general base catalyst in the inverting glycoside hydrolase reaction. The inverting phosphorolysis catalyzed by GH65 enzyme is reversible, which confers the phosphorylase with a capacity to effectively synthesize various α-glucosides from β-glucose 1-phosphate as donor and acceptor molecules. Noticeably β-glucosy fluoride can be used as donor in the synthetic reaction instead of the β-glucose 1-phosphate [4].

Catalytic Residues

The general acid catalyst was first predicted by superimposing the active site structure of maltose phosphorylase from Lactobacillus brevis [5] with a catalytic (α/α)₆ barrel domain of GH15 glucoamylase (EC 3.2.1.28) from Aspergillus awamori [6]. Considering the similarities of the active site structure, Glu487 of L. brevis maltose phosphorylase was estimated as the general acid residue. Additionally it had been proved by site-direct mutagenesis on Glu487 of Paenibacillus sp. maltose phosphorylase [7], which corresponds to the Glu487 of L. brevis maltose phosphorylase.

Three-dimensional structures

The three-dimensional structure of L. brevis maltose phosphorylase (PDB ID 1h54) was determined [5] and shows similarities with the (α/α)₆ barrel fold of GH15 glucoamylase (EC 3.2.1.28) [6], GH94 cellobiose phosphorylase (EC 2.4.1.20) [8], and GH94 chitobiose phosphorylase (EC 2.4.1.-) [3]. GH15 and GH65 together constitute glycoside hydrolase clan L [9].

Family Firsts

First stereochemistry determination

maltose phosphorylase (EC 2.4.1.8) from Neisseria meningitidis [1].

First sequence identification

maltose phosphorylase (EC 2.4.1.8) from Lactobacillus sanfranciscensis DSM 20451T [10]

trehalose phosphorylase (EC 2.4.1.64) from Thermoanaerobacter brockii ATCC 35047 [11]

kojibiose phosphorylase (EC 2.4.1.230) from Thermoanaerobacter brockii ATCC 35047 [12]

trehalose 6-phosphate phosphorylase (EC 2.4.1.-) from Lactococcus lactis ssp. lactis 19435 [13]

α,α-trehalase (EC 3.2.1.28) from Saccharomyces cerevisiae S288C [14].

First general acid residue identification

maltose phosphorylase (EC 2.4.1.8) from Lactobacillus brevis by X-ray structure analysis [5] and confirmed by mutagenesis for Paenibacillus sp. maltose phosphorylase (EC 2.4.1.8) [7].

First three-dimentional structure determination

maltose phosphorylase (EC 2.4.1.8) from Lactobacillus brevis [5].

References

1. FITTING C and DOUDOROFF M. (1952). Phosphorolysis of maltose by enzyme preparations from Neisseria meningitidis. J Biol Chem. 1952;199(1):153-63. | Google Books | Open Library
2. Nakai H, Baumann MJ, Petersen BO, Westphal Y, Schols H, Dilokpimol A, Hachem MA, Lahtinen SJ, Duus JØ, and Svensson B. (2009). The maltodextrin transport system and metabolism in Lactobacillus acidophilus NCFM and production of novel alpha-glucosides through reverse phosphorolysis by maltose phosphorylase. FEBS J. 2009;276(24):7353-65. DOI:10.1111/j.1742-4658.2009.07445.x |
3. Hidaka M, Honda Y, Kitaoka M, Nirasawa S, Hayashi K, Wakagi T, Shoun H, and Fushinobu S. (2004). Chitobiose phosphorylase from Vibrio proteolyticus, a member of glycosyl transferase family 36, has a clan GH-L-like (alpha/alpha)(6) barrel fold. Structure. 2004;12(6):937-47. DOI:10.1016/j.str.2004.03.027 |
4. Tsumuraya Y, Brewer CF, and Hehre EJ. (1990). Substrate-induced activation of maltose phosphorylase: interaction with the anomeric hydroxyl group of alpha-maltose and alpha-D-glucose controls the enzyme's glucosyltransferase activity. Arch Biochem Biophys. 1990;281(1):58-65. DOI:10.1016/0003-9861(90)90412-r |
5. Egloff MP, Uppenberg J, Haalck L, and van Tilbeurgh H. (2001). Crystal structure of maltose phosphorylase from Lactobacillus brevis: unexpected evolutionary relationship with glucoamylases. Structure. 2001;9(8):689-97. DOI:10.1016/s0969-2126(01)00626-8 |
6. Aleshin A, Golubev A, Firsov LM, and Honzatko RB. (1992). Crystal structure of glucoamylase from Aspergillus awamori var. X100 to 2.2-A resolution. J Biol Chem. 1992;267(27):19291-8. DOI:10.2210/pdb1gly/pdb |

7. Hidaka Y, Hatada Y, Akita M, Yoshida M, Nakamura N, Takada M, Nakakuki T, Ito S, and Horikoshi K. Maltose phosphorylase from a deep-sea Paenibacillus sp.: Enzymatic properties and nucleotide and amino-acid sequences. Enzyme and Microbial Technology, Volume 37, Issue 2, 1 July 2005, Pages 185-194. doi:10.1016/j.enzmictec.2005.02.010

8. Hidaka M, Kitaoka M, Hayashi K, Wakagi T, Shoun H, and Fushinobu S. (2006). Structural dissection of the reaction mechanism of cellobiose phosphorylase. Biochem J. 2006;398(1):37-43. DOI:10.1042/BJ20060274 |
9. 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 |
10. Ehrmann MA and Vogel RF. (1998). Maltose metabolism of Lactobacillus sanfranciscensis: cloning and heterologous expression of the key enzymes, maltose phosphorylase and phosphoglucomutase. FEMS Microbiol Lett. 1998;169(1):81-6. DOI:10.1111/j.1574-6968.1998.tb13302.x |
11. Maruta K, Mukai K, Yamashita H, Kubota M, Chaen H, Fukuda S, and Kurimoto M. (2002). Gene encoding a trehalose phosphorylase from Thermoanaerobacter brockii ATCC 35047. Biosci Biotechnol Biochem. 2002;66(9):1976-80. DOI:10.1271/bbb.66.1976 |
12. Yamamoto T, Maruta K, Mukai K, Yamashita H, Nishimoto T, Kubota M, Fukuda S, Kurimoto M, and Tsujisaka Y. (2004). Cloning and sequencing of kojibiose phosphorylase gene from Thermoanaerobacter brockii ATCC35047. J Biosci Bioeng. 2004;98(2):99-106. DOI:10.1016/S1389-1723(04)70249-2 |
13. Andersson U, Levander F, and Rådström P. (2001). Trehalose-6-phosphate phosphorylase is part of a novel metabolic pathway for trehalose utilization in Lactococcus lactis. J Biol Chem. 2001;276(46):42707-13. DOI:10.1074/jbc.M108279200 |
14. Destruelle M, Holzer H, and Klionsky DJ. (1995). Isolation and characterization of a novel yeast gene, ATH1, that is required for vacuolar acid trehalase activity. Yeast. 1995;11(11):1015-25. DOI:10.1002/yea.320111103 |