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Glycoside Hydrolase Family 77
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|Glycoside Hydrolase Family GH77|
|Active site residues||known|
|CAZy DB link|
Glycoside hydrolase family GH77 is a member of the α-amylase clan GH-H , together with GH13 and GH70 . The family is monospecific with the 4-α-glucanotransferase (EC 184.108.40.206), that is known as disproportionating enzyme (D-enzyme) in plants  or amylomaltase in bacteria  and archaeons . Around 2,000 members  are known that originate mostly from Bacteria; the family also contains a few tens of additional sequences from each Archaea and Eucarya (plants and green algae). Only slightly above 1% of the family members have been biochemically characterized .
Amylomaltase catalyses glucan-chain transfer from one α-1,4-glucan to another α-1,4-glucan (or to 4-hydroxyl group of glucose) or within a single linear glucan molecule to produce a cyclic α-1,4-glucan with degree of polymerization starting from 17 [3, 4, 5]. Cyclodextrin glucanotransferase, a member of the α-amylase family GH13, also produces cyclic α-1,4-glucans, but with a small degree of polymerization (6-8), called cyclodextrins .
Kinetics and Mechanism
Reaction products have been analysed for several family GH77 enzymes by TLC (mainly) and HPLC, including the D-enzyme from potato  as well as amylomaltases from Clostridium butyricum , Thermus aquaticus , Aquifex aeolicus , Pyrobaculum aerophilum , Thermus thermophilus  and Borrelia burgdorferi , but the kinetics were determined only for a few, e.g., [5, 10, 11].
The family GH77 4-α-glucanotransferases fold into a (β/α)8-barrel with the catalytic machinery consisting of a strand β4-aspartic acid (catalytic nucleophile), β5-glutamic acid (proton donor) and β7-aspartic acid (transition-state stabilizer). For example these are Asp293, Glu340 and Asp395 in the amylomaltase from Thermus aquaticus . The somewhat unusual conformations exhibited mainly by the supposed catalytic nucleophile (Asp293) have been explained by the high experimental pH of 9.0 used during crystallization . The catalytic triad is supported by a later site-directed mutagenesis study . All the family GH77 4-α-glucanotransferases share the 4-7 conserved sequence regions [12, 15] characteristic for the α-amylase clan GH-H .
Five 3-D structures have been solved for the following family GH77 members: (i) the amylomaltases from Thermus aquaticus , Aquifex aeolicus (unpublished; PDB ID 1tz7), Thermus thermophilus  and Thermus brockianus ; and (ii) the D-enzyme from potato (unpublished; PDB ID 1x1n). The crystallization of the amylomaltase from Corynebacterium glutamicum has also been reported .
The main structural feature that discriminates the family GH77 amylomaltases from typical α-amylase family GH13 members is the lack of domain C  that succeeds the catalytic (β/α)8-barrel (TIM-barrel) in the family GH13 enzymes. The eight-fold symmetry of the catalytic barrel in the family GH77 is disrupted by several insertions between the barrel β-strands that form the so-called subdomains B1, B2 and B3 . Subdomain B1 consists of a highly twisted four-stranded antiparallel β-sheet with two α-helices and is also present in other enzymes from the α-amylase clan GH-H (known as domain B). Subdomain B2 has predominantly an α-helical structure and it is unique to amylomaltases. Subdomain B3 could have a role of domain C from the α-amylase family .
Interestingly, primary structures of amylomaltases from borreliae contain unique sequence features , i.e. natural mutations in functionally important positions from conserved sequence regions. The most important and remarkable one is represented by otherwise extremely well-conserved and functional arginine in position i-2 with respect to the catalytic nucleophile that is replaced by a lysine . It is worth mentioning that this arginine positioned two residues before the catalytic nucleophile in the conserved sequence region II was considered to belong to the four residues conserved invariantly (along with the catalytic triad) throughout the α-amylase family . Its substitution is therefore of a special interest because the GH77 protein from Borrelia burgdorferi exhibits amylomaltase activity . Since, however, the lysine could play the role of the original arginine, it is not possible to say unambiguously that the catalytic triad alone (aspartic acid, glutamic acid and aspartic acid at strands β4, β5 and β7, respectively, of the catalytic TIM-barrel) is enough for a GH-H protein to be a functional member of the α-amylase family . There are several additional putative amylomaltases from various borreliae available; some of them possess the Arg-to-Lys mutation, indicating the borreliae enzymes may occupy an outstanding position in evolution of this 4-α-glucanotransferase family GH77 .
In plants particularly a longer version of D-enzyme (DPE1) was identified and named DPE2 [21, 22]. It was recently described also in certain bacteria . It usually contains a ~140 amino acid residues long insert within the catalytic GH77 TIM barrel and two copies of a starch-binding domain of family CBM20 succeeded by a short coiled coil motif positioned N-terminally . Interestingly, removing the insert leads to inactivation of the DPE2 although the insert itself has nothing to do with the catalytic action of the enzyme .
- First stereochemistry determination
- There is no direct evidence for the stereochemistry of hydrolysis of the glucosidic bond in the reaction of any family GH77 4-α-glucanotransferase. HPLC analyses of reaction products reported for amylomaltases from, e.g., Aquifex aeolicus , Pyrobaculum aerophilum  and Thermus thermophilus  was used to propose a retaining mechanism assumed in analogy with the mechanism confirmed in α-amylase family GH13.
- First catalytic nucleophile identification
- Asp293, was identified as a catalytic nucleophile in the amylomaltase from Thermus thermophilus on the basis of mutant enzymes (D293N and D293A) that exhibited greatly reduced disproportionation activity .
- First general acid/base residue identification
- The catalytic proton donor, Glu340, was identified as a catalytic residue in the amylomaltase from Thermus thermophilus by demonstration of a greatly reduced disproportionation activity of the E340Q and E340A mutant enzymes .
- First 3-D structure
- The first 3-D structure of a GH77 member was that of the amylomaltase from Thermus aquaticus solved first as a free enzyme (PDB ID 1cwy)  and subsequently also as a complex with acarbose (PDB ID 1esw) .
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- 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. 37, D233-8. DOI:10.1093/nar/gkn663 |
- MacGregor EA, Janecek S, and Svensson B. (2001) Relationship of sequence and structure to specificity in the alpha-amylase family of enzymes. Biochim Biophys Acta. 1546, 1-20. DOI:10.1016/s0167-4838(00)00302-2 |
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- Lombard V, Golaconda Ramulu H, Drula E, Coutinho PM, and Henrissat B. (2014) The carbohydrate-active enzymes database (CAZy) in 2013. Nucleic Acids Res. 42, D490-5. DOI:10.1093/nar/gkt1178 |
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- Uitdehaag JC, Mosi R, Kalk KH, van der Veen BA, Dijkhuizen L, Withers SG, and Dijkstra BW. (1999) X-ray structures along the reaction pathway of cyclodextrin glycosyltransferase elucidate catalysis in the alpha-amylase family. Nat Struct Biol. 6, 432-6. DOI:10.1038/8235 |
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- Bhuiyan SH, Kitaoka M, and Hayashi K. A cycloamylose-forming hyperthermostable 4-α-glucanotransferase of Aquifex aeolicus expressed in Escherichia coli. Journal of Molecular Catalysis B: Enzymatic 2003; 22:45-53. (DOI: 10.1016/S1381-1177(03)00005-5)
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- Przylas I, Terada Y, Fujii K, Takaha T, Saenger W, and Sträter N. (2000) X-ray structure of acarbose bound to amylomaltase from Thermus aquaticus. Implications for the synthesis of large cyclic glucans. Eur J Biochem. 267, 6903-13. DOI:10.1046/j.1432-1033.2000.01790.x |
- Machovic M, and Janecek S. The invariant residues in the α-amylase family: just the catalytic triad. Biologia 2003; 58(6):1127-32. (PDF)
- Janecek S. How many conserved sequence regions are there in the α-amylase family? Biologia 2002; 57(Suppl. 11):29-41. (PDF)
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