Crystal structure of a catalytic-site mutant α-amylase from Bacillus subtilis complexed with maltopentaose

Oligosaccharide Binding to Barley α-Amylase 1

“…In contrast, binding of natural substrates was only described in three complexes known to date. These are the ␣-amylase from porcine pancreas complexed with maltopentaose (14,15), the inactive catalytic mutant E208Q ␣-amylase from Bacillus subtilis in complex with maltopentaose (16), and the "maltogenic" ␣-amylase from Bacillus stearothermophilus in complex with a maltose unit that was derived from a maltotriose (17). ␣-Amylases belong to the glycoside hydrolase family 13 (afmb.cnrs-mrs.fr/CAZY), which together with GH70 and GH77 constitute GH clan H (18).…”

Tyrosine 105 and Threonine 212 at Outermost Substrate Binding Subsites –6 and +4 Control Substrate Specificity, Oligosaccharide Cleavage Patterns, and Multiple Binding Modes of Barley α-Amylase 1

“…Substrate interactions along the extended binding site have traditionally been described by subsite maps that indicate the number of consecutive glucosyl binding subsites (ranging from [5][6][7][8][9][10][11], the cleavage position, and the affinity of substrate glucosyl residues at individual subsites (2)(3)(4)(5)(6)(7)(8). The binding cleft is formed by ␤ 3 ␣ loops of the catalytic (␤/␣) 8 barrel domain (9 -14).…”

“…Substrate analogs are very rarely seen to fill the entire binding site in crystal structures, one example being a Bacillus licheniformis/Bacillus amyloliquefaciens ␣-amylase chimera accommodating at subsites Ϫ7 through ϩ3 a decasaccharide inhibitor derived by transglycosylation from the pseudotetrasaccharide acarbose (14). Related inhibitors cover the only five subsite long binding crevice in pancreatic ␣-amylase (8,9,20), and occupy part of the longer binding sites in microbial ␣-amylases (11,13,21) and in cyclodextrin glucosyltransferase (CGTase) (16,22,23). The structures validate modeled substrate complexes and subsite maps (8,12,24,25) by highlighting (i) aromatic stacking and hydrogen bonds between carbohydrate and protein (9,10,21,24,26,27), (ii) conformational features of the bound carbohydrate (8,21,28), (iii) conserved geometry of the catalytic site (10,14,15,21,22,29,30), and (iv) substrate binding motifs in ␤ 3 ␣ loops of the catalytic (␤/␣) 8 barrel (15).…”

“…stacking of aromatic side chains on carbohydrate rings and hydrogen bonding by the tyrosine ␥OH or tryptophan ⑀NH to carbohydrate OH groups (26,27,48,49). The protein engineering strategy used took advantage of: (i) the modeled AMY2/maltodecaose (25), (ii) insight in earlier AMY1 mutants of substrate binding residues (4, 30, 39 -41), and (iii) numerous crystal structures of ␣-amylases from Aspergillus oryzae (21), Aspergillus niger (50), B. licheniformis (51), Bacillus subtilis (11), Pseudoalteromonas haloplanktis (29), AMY2 (10), porcine (9), and human pancreas (8), and other GH-H members; e.g. CGTase (16,22,52), TVAII ␣-amylase (53), and amylomaltase (28).…”

Engineering Thermus Maltogenic Amylase with Improved Thermostability: Probing the Role of the Conserved Calcium Binding Site in Cyclodextrin-degrading Enzymes

“…10 12) A conserved calcium binding site (Ca1 site), which is located at the interface between the A and B domains, plays an important role in stabilizing these enzymes by maintaining the conformation of the active site 5) and protecting the enzymes from proteolysis. 12,13) Interestingly, another calcium binding site (Ca2 site) has been found in cyclodextrin (CD) related enzymes such as CD producing enzymes (cyclodextrin glucanotransferases) 11) and CD degrading enzymes (maltogenic amylase, neopullulanase, and cyclodextrinase), 14 16) but not in typical amylases.…”