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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 the member of the α-amylase clan GH-H , together with GH13 and GH70 . The family is monospecific with the 4-α-glucanotransferase (EC 126.96.36.199), that is known as disproportionating enzyme (D-enzyme) in plants  or amylomaltase in bacteria  and archaeons . Around 2,000 members  originates almost exclusively from Bacteria; and the family contains also a few tens of additional sequences from each Archaea and Eucarya (plants and green algae).
Amylomaltase catalyses the 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 .
Five tertiary structures have been determined and only slightly above 1% of the family members have already been biochemically characterized . 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. The eight-fold symmetry of the catalytic barrel is in the family GH77 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 it 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 is represented by otherwise extremely well-conserved and functional arginine in position i-2 with respect to the catalytic nucleophile that is replaced naturally 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 throughout the α-amylase family . Its substitution is therefore of a special interest because the GH77 protein from Borrelia burgdorferi does exhibit the real amylomaltase activity . Since, however, the lysine could eventually 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 real 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 and some of them not, indicating the group of borreliae may occupy an outstanding position in evolution of this 4-α-glucanotransferase family.
In plants especially (although also in some bacteria) a longer version of D-enzyme (DPE1) was identified and named as DPE2 [12, 13]. It usually contains a ~140 amino acid residues long insert within the catalytic GH77 TIM barrel and two copies of 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 .
Kinetics and Mechanism
Reaction products were 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 exact kinetics were determined only for a few, e.g., [5, 18, 19].
They 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). These are, e.g., the Asp293, Glu340 and Asp395 in the amylomaltase from Thermus aquaticus . The somewhat unusual conformations exhibited mainly by the supposed catalytic nucleophile (Asp293) has been explained by the high experimental pH of 9.0 used during the crystallization . This catalytic triad has, however, been confirmed by a later site-directed mutagenesis study. All the family GH77 4-α-glucanotransferases share the 4-7 conserved sequence regions [9, 11] characteristic for the entire α-amylase clan GH-H .
The five above-mentioned 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 .
- First stereochemistry determination
- First catalytic nucleophile identification
- The assumed catalytic nucleophile, Asp293, was identified as a catalytic residue in the amylomaltase from Thermus thermophilus by making the site-directed mutant enzymes (D293N and D293A) that exhibited greatly reduced disproportionation activity .
- First general acid/base residue identification
- The assumed catalytic proton donor, Glu340, was identified as a catalytic residue in the amylomaltase from Thermus thermophilus by making the site-directed mutant enzymes (E340Q and E340A) that exhibited greatly reduced disproportionation activity .
- 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) .
- 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 |
- Takaha T, Yanase M, Okada S, and Smith SM. (1993) Disproportionating enzyme (4-alpha-glucanotransferase; EC 188.8.131.52) of potato. Purification, molecular cloning, and potential role in starch metabolism. J Biol Chem. 268, 1391-6.
- Terada Y, Fujii K, Takaha T, and Okada S. (1999) Thermus aquaticus ATCC 33923 amylomaltase gene cloning and expression and enzyme characterization: production of cycloamylose. Appl Environ Microbiol. 65, 910-5. DOI:10.1128/AEM.65.3.910-915.1999 |
- Kaper T, Talik B, Ettema TJ, Bos H, van der Maarel MJ, and Dijkhuizen L. (2005) Amylomaltase of Pyrobaculum aerophilum IM2 produces thermoreversible starch gels. Appl Environ Microbiol. 71, 5098-106. DOI:10.1128/AEM.71.9.5098-5106.2005 |
- 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 |
- Leemhuis H, Kelly RM, and Dijkhuizen L. (2010) Engineering of cyclodextrin glucanotransferases and the impact for biotechnological applications. Appl Microbiol Biotechnol. 85, 823-35. DOI:10.1007/s00253-009-2221-3 |
- Przylas I, Tomoo K, Terada Y, Takaha T, Fujii K, Saenger W, and Sträter N. (2000) Crystal structure of amylomaltase from thermus aquaticus, a glycosyltransferase catalysing the production of large cyclic glucans. J Mol Biol. 296, 873-86. DOI:10.1006/jmbi.1999.3503 |
- 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)
- Godány A, Vidová B, and Janecek S. (2008) The unique glycoside hydrolase family 77 amylomaltase from Borrelia burgdorferi with only catalytic triad conserved. FEMS Microbiol Lett. 284, 84-91. DOI:10.1111/j.1574-6968.2008.01191.x |
- Lloyd JR, Blennow A, Burhenne K, and Kossmann J. (2004) Repression of a novel isoform of disproportionating enzyme (stDPE2) in potato leads to inhibition of starch degradation in leaves but not tubers stored at low temperature. Plant Physiol. 134, 1347-54. DOI:10.1104/pp.103.038026 |
- Lu Y and Sharkey TD. (2004) The role of amylomaltase in maltose metabolism in the cytosol of photosynthetic cells. Planta. 218, 466-73. DOI:10.1007/s00425-003-1127-z |
- Steichen JM, Petty RV, and Sharkey TD. (2008) Domain characterization of a 4-alpha-glucanotransferase essential for maltose metabolism in photosynthetic leaves. J Biol Chem. 283, 20797-804. DOI:10.1074/jbc.M803051200 |
- Ruzanski C, Smirnova J, Rejzek M, Cockburn D, Pedersen HL, Pike M, Willats WG, Svensson B, Steup M, Ebenhöh O, Smith AM, and Field RA. (2013) A bacterial glucanotransferase can replace the complex maltose metabolism required for starch to sucrose conversion in leaves at night. J Biol Chem. 288, 28581-98. DOI:10.1074/jbc.M113.497867 |
- 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 |
- Goda SK, Eissa O, Akhtar M, and Minton NP. (1997) Molecular analysis of a Clostridium butyricum NCIMB 7423 gene encoding 4-alpha-glucanotransferase and characterization of the recombinant enzyme produced in Escherichia coli. Microbiology (Reading). 143 ( Pt 10), 3287-3294. DOI:10.1099/00221287-143-10-3287 |
- 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)
- Kaper T, Leemhuis H, Uitdehaag JC, van der Veen BA, Dijkstra BW, van der Maarel MJ, and Dijkhuizen L. (2007) Identification of acceptor substrate binding subsites +2 and +3 in the amylomaltase from Thermus thermophilus HB8. Biochemistry. 46, 5261-9. DOI:10.1021/bi602408j |
- 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 |
- Barends TR, Bultema JB, Kaper T, van der Maarel MJ, Dijkhuizen L, and Dijkstra BW. (2007) Three-way stabilization of the covalent intermediate in amylomaltase, an alpha-amylase-like transglycosylase. J Biol Chem. 282, 17242-9. DOI:10.1074/jbc.M701444200 |
- Jung JH, Jung TY, Seo DH, Yoon SM, Choi HC, Park BC, Park CS, and Woo EJ. (2011) Structural and functional analysis of substrate recognition by the 250s loop in amylomaltase from Thermus brockianus. Proteins. 79, 633-44. DOI:10.1002/prot.22911 |
- Srisimarat W, Murakami S, Pongsawasdi P, and Krusong K. (2013) Crystallization and preliminary X-ray crystallographic analysis of the amylomaltase from Corynebacterium glutamicum. Acta Crystallogr Sect F Struct Biol Cryst Commun. 69, 1004-6. DOI:10.1107/S1744309113020319 |
- Hoon-Hanks LL, Morton EA, Lybecker MC, Battisti JM, Samuels DS, and Drecktrah D. (2012) Borrelia burgdorferi malQ mutants utilize disaccharides and traverse the enzootic cycle. FEMS Immunol Med Microbiol. 66, 157-65. DOI:10.1111/j.1574-695X.2012.00996.x |