No abstract

As a member of the a-amylase superfamily of enzymes, amylomaltase catalyzes either the transglycosylation from one a-1,4 glucan to another or an intramolecular cyclization. The latter reaction is typical for cyclodextrin glucanotransferases. In contrast to these enzymes, amylomaltase catalyzes the formation of cyclic glucans with a degree of polymerization larger than 22. To characterize the factors that determine the size of the synthesized cycloamyloses, we have analyzed the X-ray structure of amylomaltase from Thermus aquaticus in complex with the inhibitor acarbose, a maltotetraose derivative, at 1.9 A Ê resolution. Two acarbose molecules are bound to the enzyme, one in the active site groove at subsite 23 to 11 and a second one < 14 A Ê away from the nonreducing end of the acarbose bound to the catalytic site. The inhibitor bound to the catalytic site occupies subsites 23 to 11. Unlike the situation in other enzymes of the a-amylase family, the inhibitor is not processed and the inhibitory cyclitol ring of acarbose, which mimicks the half chair conformation of the transition state, does not bind to catalytic subsite 21. The minimum ring size of cycloamyloses produced by this enzyme is proposed to be determined by the distance of the specific substrate binding sites at the active site and near Tyr54 and by the size of the 460s loop. The 250s loop might be involved in binding of the substrate at the reducing end of the scissile bond.Keywords: acarbose; a-amylase family; (b, a) 8 barrel; glucanotransferase; protein crystallography.Besides cellulose, starch is one of the most abundant carbohydrate polymers in nature. Starch consists of two compounds, the linear amylose with a-1,4 linked glucoses and the branched amylopectin with a-1,4 and a-1,6 connected repeating units. Due to its important role in energy storage and uptake many enzymes are known to act on starch. All starch hydrolases and related enzymes have been classified together with the other O-glycosyl hydrolases into a group of glycoside hydrolase families, which covers more than 80 families [1,2]. Amylomaltase belongs to the a-amylase family (family 13), which includes about 20 different enzymes. These proteins catalyze the transformation of a-1,4 and a-1,6 glucosidic linkages with retention of the anomeric center [3±6]. Well studied enzymes of this family are the a-amylases and the cyclodextrin glucanotransferases (CGTases). a-Amylases catalyze the hydrolysis of the a-1,4 glycosidic bonds of amylose, whereas CGTases catalyze a reversible transfer of a-1,4 glucans. By intramolecular cyclization these enzymes synthesize small cycloamyloses, termed cyclodextrins. The final cycloamylose products of CGTase activity consist mainly of 6±8 glucose units. However, CGTase initially produces large cycloamyloses, which are subsequently transformed into the smaller cyclodextrins [7].The doughnut-shaped cyclodextrins can form complexes with guest molecules in their central hydrophobic cavity and have found many applications in the food, pharmaceutical and cosmetic...

As a member of the alpha-amylase superfamily of enzymes, amylomaltase catalyzes either the transglycosylation from one alpha-1,4 glucan to another or an intramolecular cyclization. The latter reaction is typical for cyclodextrin glucanotransferases. In contrast to these enzymes, amylomaltase catalyzes the formation of cyclic glucans with a degree of polymerization larger than 22. To characterize the factors that determine the size of the synthesized cycloamyloses, we have analyzed the X-ray structure of amylomaltase from Thermus aquaticus in complex with the inhibitor acarbose, a maltotetraose derivative, at 1.9 A resolution. Two acarbose molecules are bound to the enzyme, one in the active site groove at subsite -3 to +1 and a second one approximately 14 A away from the nonreducing end of the acarbose bound to the catalytic site. The inhibitor bound to the catalytic site occupies subsites -3 to +1. Unlike the situation in other enzymes of the alpha-amylase family, the inhibitor is not processed and the inhibitory cyclitol ring of acarbose, which mimicks the half chair conformation of the transition state, does not bind to catalytic subsite -1. The minimum ring size of cycloamyloses produced by this enzyme is proposed to be determined by the distance of the specific substrate binding sites at the active site and near Tyr54 and by the size of the 460s loop. The 250s loop might be involved in binding of the substrate at the reducing end of the scissile bond.

The crystal structure of a decameric HNA/RNA (HNA = 2',3'-dideoxy-1',5'-anhydro-d-arabinohexitol nucleic acid) hybrid with the RNA sequence 5'-GGCAUUACGG-3' is the first crystal structure of a hybrid duplex between a naturally occurring nucleic acid and a strand, which is fully modified to contain a six-membered ring instead of ribose. The presence of four duplex helices in the asymmetric unit allows for a detailed discussion of hydration, which revealed a tighter spinelike backbone hydration for the HNA- than for the RNA-strands. The reinforced backbone hydration is suggested to contribute significantly to the exceptional stability of HNA-containing duplexes and might be one of the causes for the evolutionary preference for ribose-derived nucleic acids.