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

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 (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

Three structures of GH49 enzymes are available so far [5, 7, 10], 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, 11, 12]. 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 [13].

Family Firsts

First gene cloning

Dextranase from Arthrobacter sp. CB-8 [14].

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

4. 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 |