Glycoside Hydrolase Family 49

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• Author: ^^^Takashi Tonozuka^^^
• Responsible Curator: ^^^Takashi Tonozuka^^^

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Glycoside Hydrolase Family GHnn
Clan	GH-N
Mechanism	inverting
Active site residues	known
CAZy DB link
http://www.cazy.org/fam/GHnn.html

Substrate specificities

Glycoside hydrolases of family 49 cleave α-1,6-linkages or α-1,4-linkages of polysaccharides containing α-1,6-glucosidic linkages, dextran and pullulan. The major activities reported for this family of glycoside hydrolases are dextranase (EC 3.2.1.11), and a dextranase from Penicillium minioluteum, Dex49A, is currently the most characterised enzyme.　Dextran 1,6-α-isomaltotriosidase (EC 3.2.1.95) [1] and isopullulanase (EC 3.2.1.57) activities have also been described.

Kinetics and Mechanism

Family GH49 α-glycosidases are inverting enzymes, as first shown by NMR on a dextranase Dex49A from Penicillium minioluteum [2] .

Catalytic Residues

Three Asp residues (Asp376, Asp395, and Asp396 in Dex49A) are conserved in the catalytic centre of members of Clan GH-N, GH49 and GH28 enzymes [2, 3], and all three of the Asp mutants of a GH49 enzyme, isopullulanase, lost their activities [4]. The general acid was first identified in Dex49A from Penicillium minioluteum as Asp395 following the three-dimensional structure determination. To date, it is unclear whether either of the Asp residues (Asp376 and Asp396 in Dex49A) acts as a general base in the reaction of both GH49 and GH28 enzymes [2, 5, 6].

Three-dimensional structures

Two structures of GH49 enzymes are available so far [2, 3], and they display a two domain structure. The N-terminal domain is a β-sandwich and the C-terminal domain adopts a right-handed parallel β-helix. The similarity of the β-helix fold between GH49 and GH28 enzymes has been described, although almost none of the amino acid residues other than the three catalytic Asp residues is conserved between the two families [2, 3]. Each coil forming the cylindrical β-helix fold is composed of three β-sheets, which are named PB1, PB2, and PB3, following the original definition for a GH28 enzyme, pectate lyase C [7].

Family Firsts

First gene cloning

Dextranase from Arthrobacter sp. CB-8 [8].

First sterochemistry determination

Dextranase (Dex49A) from Penicillium minioluteum [2].

First general acid residue identification

Dextranase (Dex49A) from Penicillium minioluteum [2].

First 3-D structure

Dextranase (Dex49A) from Penicillium minioluteum by X-ray crystallography (PDB ID 1ogm) [2].

References

1. Mizuno T, Mori H, Ito H, Matsui H, Kimura A, and Chiba S. (1999). Molecular cloning of isomaltotrio-dextranase gene from Brevibacterium fuscum var. dextranlyticum strain 0407 and its expression in Escherichia coli. Biosci Biotechnol Biochem. 1999;63(9):1582-8. DOI:10.1271/bbb.63.1582 |
2. Larsson AM, Andersson R, Ståhlberg J, Kenne L, and Jones TA. (2003). Dextranase from Penicillium minioluteum: reaction course, crystal structure, and product complex. Structure. 2003;11(9):1111-21. DOI:10.1016/s0969-2126(03)00147-3 |
3. Mizuno M, Koide A, Yamamura A, Akeboshi H, Yoshida H, Kamitori S, Sakano Y, Nishikawa A, and Tonozuka T. (2008). Crystal structure of Aspergillus niger isopullulanase, a member of glycoside hydrolase family 49. J Mol Biol. 2008;376(1):210-20. DOI:10.1016/j.jmb.2007.11.098 |
4. Akeboshi H, Tonozuka T, Furukawa T, Ichikawa K, Aoki H, Shimonishi A, Nishikawa A, and Sakano Y. (2004). Insights into the reaction mechanism of glycosyl hydrolase family 49. Site-directed mutagenesis and substrate preference of isopullulanase. Eur J Biochem. 2004;271(22):4420-7. DOI:10.1111/j.1432-1033.2004.04378.x |
5. van Santen Y, Benen JA, Schröter KH, Kalk KH, Armand S, Visser J, and Dijkstra BW. (1999). 1.68-A crystal structure of endopolygalacturonase II from Aspergillus niger and identification of active site residues by site-directed mutagenesis. J Biol Chem. 1999;274(43):30474-80. DOI:10.1074/jbc.274.43.30474 |
6. Shimizu T, Nakatsu T, Miyairi K, Okuno T, and Kato H. (2002). Active-site architecture of endopolygalacturonase I from Stereum purpureum revealed by crystal structures in native and ligand-bound forms at atomic resolution. Biochemistry. 2002;41(21):6651-9. DOI:10.1021/bi025541a |
7. Yoder MD, Keen NT, and Jurnak F. (1993). New domain motif: the structure of pectate lyase C, a secreted plant virulence factor. Science. 1993;260(5113):1503-7. DOI:10.1126/science.8502994 |
8. Okushima M, Sugino D, Kouno Y, Nakano S, Miyahara J, Toda H, Kubo S, and Matsushiro A. (1991). Molecular cloning and nucleotide sequencing of the Arthrobacter dextranase gene and its expression in Escherichia coli and Streptococcus sanguis. Jpn J Genet. 1991;66(2):173-87. DOI:10.1266/jjg.66.173 |