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Glycoside Hydrolase Family 49

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Glycoside Hydrolase Family GH49
Clan GH-N
Mechanism inverting
Active site residues known
CAZy DB link

Substrate specificities

Glycoside hydrolases of family 49 cleave α-1,6-glucosidic linkages or α-1,4-glucosidic 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, and a dextranase from Talaromyces minioluteum (formerly known as Penicillium minioluteum), Dex49A, is currently the most characterized enzyme. Dextran 1,6-α-isomaltotriosidase (EC [1], isopullulanase (EC [2], endo-acting sulfated-arabinan hydrolase (EC 3.2.1-) [3], and 4-O-α-D-isomaltooligosaccharylmaltooligosaccharide 1,4-α-isomaltooligosaccharohydrolase (EC 3.2.1.-) [4] have also been described.

Kinetics and Mechanism

Family GH49 α-glycosidases are inverting enzymes, as first shown by NMR on a dextranase Dex49A from Talaromyces minioluteum [5] .

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 [5, 6], and all three of the Asp mutants of a GH49 enzyme, isopullulanase, lost their activities [7]. The general acid was first identified in Dex49A from Talaromyces minioluteum as Asp395 following the three-dimensional structure determination. To date, it is unclear whether either (or both) of the Asp residues (Asp376 and Asp396 in Dex49A) acts as a general base in the reaction of GH49 and GH28 enzymes [5, 8, 9].

Three-dimensional structures

Two structures of GH49 enzymes are available so far [5, 6], 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 [5, 6]. 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 PL1 enzyme, pectate lyase C [10].

Family Firsts

First gene cloning
Dextranase from Arthrobacter sp. CB-8 [11].
First sterochemistry determination
Dextranase (Dex49A) from Talaromyces minioluteum [5].
First general acid residue identification
Dextranase (Dex49A) from Talaromyces minioluteum [5].
First 3-D structure
Dextranase (Dex49A) from Talaromyces minioluteum by X-ray crystallography (PDB ID 1ogm) [5].


  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 | PubMed ID:10540747 [Mizuno1999]
  2. Sakano Y, Masuda N, and Kobayashi T. (1971). Hydrolysis of Pullulan by a Novel Enzyme from Aspergillus niger, Agric Biol Chem 1971;35(6):971-973.

  3. Helbert W, Poulet L, Drouillard S, Mathieu S, Loiodice M, Couturier M, Lombard V, Terrapon N, Turchetto J, Vincentelli R, and Henrissat B. (2019). Discovery of novel carbohydrate-active enzymes through the rational exploration of the protein sequences space. Proc Natl Acad Sci U S A. 2019;116(13):6063-6068. DOI:10.1073/pnas.1815791116 | PubMed ID:30850540 [Helbert2019]
  4. Kitagawa N, Watanabe H, Mori T, Aga H, Ushio S, and Yamamoto K. (2023). Cloning and sequence analysis of 4-O-α-d-isomaltooligosaccharylmaltooligosaccharide 1,4-α-isomaltooligosaccharohydrolase from Sarocladium kiliense U4520. Biosci Biotechnol Biochem. 2023;87(3):330-337. DOI:10.1093/bbb/zbac211 | PubMed ID:36592961 [Kitagawa2023]
  5. 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 | PubMed ID:12962629 [Larsson2003]
  6. 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 | PubMed ID:18155243 [Mizuno2008]
  7. 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 | PubMed ID:15560783 [Akeboshi2004]
  8. 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 | PubMed ID:10521427 [vanSanten1999]
  9. 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 | PubMed ID:12022868 [Shimizu2002]
  10. 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 | PubMed ID:8502994 [Yoder1993]
  11. 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 | PubMed ID:1859672 [Okushima1991]

All Medline abstracts: PubMed