Glycoside Hydrolase Family 49

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• Author: Takashi Tonozuka and Takatsugu Miyazaki
• Responsible Curator: Takashi Tonozuka

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Glycoside Hydrolase Family GH49
Clan	GH-N
Mechanism	inverting
Active site residues	known
CAZy DB link
https://www.cazy.org/GH49.html

Substrate specificities

Figure 1. Substrates of GH49 enzymes. All enzymes except dextran 1,6-α-isomaltotriosidase are endo-acting glycoside hydrolases. Sulfated arabinan contains sulfate groups at 2- and 3-OH (3S) of L-arabinopyranose residue and possesses side chains of galactose and xylose, which are not shown in this figure.

Glycoside hydrolases of GH49 cleave α-(1→6)-glucosidic linkages or α-(1→4)-glucosidic linkages of polysaccharides and oligosaccharides containing α-(1→6)-glucosidic linkages, such as dextran and pullulan. The major activities reported for this family of glycoside hydrolases are dextranase (EC 3.2.1.11), and a dextranase from Talaromyces minioluteum (formerly known as Penicillium minioluteum), Dex49A, is currently the most characterized enzyme. GH49 dextranases have been found in some bacteria and fungi. Dextran 1,6-α-isomaltotriosidase (EC 3.2.1.95) from Brevibacterium fuscum var. dextranlyticum is an exo-acting enzyme that hydrolyzes dextran from the non-reducing ends to produce isomaltotriose [1]. Isopullulanase (EC 3.2.1.57)from Aspergillus brasiliensis ATCC 9642 (formerly Aspergillus niger ATCC 9642) hydrolyzes α-(1→4)-linkages of pullulan to produce isopanose [2]. 4-O-α-D-Isomaltooligosaccharylmaltooligosaccharide 1,4-α-isomaltooligosaccharohydrolase (IMM-4IH, EC 3.2.1.-) from Sarocladium kiliense possesses more strict substrate specificity than isopullulanase and cannot hydrolyze pullulan [3]. As an exception, endo-acting sulfated-arabinan hydrolase (EC 3.2.1-) from Phocaeicola plebeius degrades sulfated arabinan produced by green algae Chaetomorpha sp. and Cladophora sp. [4].

Kinetics and Mechanism

Family GH49 α-glycosidases are inverting enzymes, as first shown by NMR on a dextranase Dex49A from Talaromyces minioluteum [5] . Optical rotation analysis of the hydrolysis of panose by isopullulanase supported the inverting mechanism [6].

Catalytic Residues

Three Asp residues (Asp376, Asp395, and Asp396 in Dex49A) are conserved in the catalytic center of clan GH-N members, GH49 and GH28 enzymes [5, 7], which is also the case for GH87 and GH110. All three of the Asp mutants of A. brasiliensis isopullulanase, lost their activities [6]. 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

Figure 2. Overall structures of GH49 enzymes. (Left to right) Aspergillus brasiliensis isopullulanase (IPU) in complex with isopanose (PDB ID 2Z8G), Talaromyces minioluteum dextranase (Dex49A) in complex with isomaltose (PDB ID 1OGM), and Arthrobacter oxydans dextranase (AoDex) (PDB ID 6NZS).

Figure 3. Active sites of GH49 enzymes. (A-C) Molecular surfaces of Dex49A (A), IPU (B), and AoDex (C) are shown in gray. Amino acid residues and ligands are shown as stick models: catalytic residues, slate blue; residues forming active site clefts: yellow (C-terminal domain) and pink (N-terminal domain); isomaltose and isopanose, green. In (B), the isomaltose molecule (dark green) bound in N448A variant of IPU (PDB ID 3WWG [10]) is superimposed. (D) The superimposition of the catalytic residues of the three GH49 enzymes. Water molecules interacting with two general base candidates (asterisk) are shown as red sphere.

Three structures of GH49 enzymes are available so far [5, 7, 11], 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. Although GH49, GH28, GH87, and GH110 families contain enzymes with distinct substrate specificities and exhibit low overall sequence homology, they share similar β-helix folds and the three catalytic Asp residues are completely conserved [5, 7, 12, 13]. 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 [14].

Family Firsts

First gene cloning

Dextranase from Arthrobacter sp. CB-8 [15].

First stereochemistry 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].

References

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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. https://doi.org/10.1271/bbb1961.35.971

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 |
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 |
14. 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 |