TY - JOUR

T1 - Binding of carbohydrates and protein inhibitors to the surfaceof a-amylases

AU - Bozonnet, Sophie

AU - Bønsager, Birgit C.

AU - Kramhøft, Birte

AU - Mori, Haruhide

AU - Hachem, Maher Abou

AU - Willemoës, Martin

AU - Jensen, Morten T.

AU - Fukuda, Kenji

AU - Nielsen, Peter K.

AU - Juge, Nathalie

AU - Aghajari, Nushin

AU - Tranier, Samuel

AU - Robert, Xavier

AU - Haser, Richard

AU - Svensson, Birte

N1 - Key words: barley a-amylase, subsite mutants, isozyme chimeras, secondary binding sites, degree of multiple attack, amylopectin, proteinaceous inhibitors.

PY - 2005

Y1 - 2005

N2 - This review on barley a-amylases 1 (AMY1) and 2 (AMY2) addresses rational mutations at distal subsitesto the catalytic site, polysaccharide hydrolysis, and interactions with proteinaceous inhibitors. Subsite mapping of barleya-amylases revealed 6 glycone and 4 aglycone substrate subsites. Moreover, two maltooligosaccharide surface binding-amylases revealed 6 glycone and 4 aglycone substrate subsites. Moreover, two maltooligosaccharide surface binding sites have been identified. Engineering of outer subsites -6 and +4 alters action patterns and relative specificities. Thus,compared to wild-type, Y105A AMY1 (subsite -6) shows 140%, 15%, and <1% and T212Y (subsite +4) 32%, 370%, and90% activity towards starch, maltodextrin, and maltoheptaoside, respectively. The enzyme kinetic properties and modeledmaltododecaose complexes suggest binding mode multiplicity. Following an initial hydrolytic cleavage of amylose, an averageof 1.9 bonds are cleaved per enzyme-substrate encounter, defining a degree of multiple attack (DMA) of 1.9. DMA increasedto 3.3 for Y105A and decreased to 1-1.7 for other subsite mutants. The fusion of a starch-binding domain to AMY1 raisedthe DMA to 3.0 and increased the amount of higher oligosaccharide products. Remarkably, the subsite mutants had unchangeddistribution of released oligosaccharides of DP 5-9, but the profiles differed for the shorter products. A recentlyidentified surface binding site, found exclusively in AMY1, involves the conserved Tyr380 which has no effect on the DMA,but proved critical for ß-cyclodextrin binding as shown by mutational and surface plasmon resonance analyses. Accordingly,AMY2 has lower affinity for ß-cyclodextrin. Hydrolysis of amylopectin proceeds via a fast and a slow reaction rate, withß-cyclodextrin inhibiting the fast one, implicating a distinct role for Tyr380 in activity on amylopectin. Barley seeds produce-cyclodextrin inhibiting the fast one, implicating a distinct role for Tyr380 in activity on amylopectin. Barley seeds produce different proteinaceous inhibitors acting specifically on insect, animal or plant a-amylases. Rational mutagenesis of barleya-amylase/subtilisin inhibitor (BASI) identified structural elements responsible for AMY2 inhibition and demonstrated the-amylase/subtilisin inhibitor (BASI) identified structural elements responsible for AMY2 inhibition and demonstrated the importance of ionic bonds for inhibitory activity.

AB - This review on barley a-amylases 1 (AMY1) and 2 (AMY2) addresses rational mutations at distal subsitesto the catalytic site, polysaccharide hydrolysis, and interactions with proteinaceous inhibitors. Subsite mapping of barleya-amylases revealed 6 glycone and 4 aglycone substrate subsites. Moreover, two maltooligosaccharide surface binding-amylases revealed 6 glycone and 4 aglycone substrate subsites. Moreover, two maltooligosaccharide surface binding sites have been identified. Engineering of outer subsites -6 and +4 alters action patterns and relative specificities. Thus,compared to wild-type, Y105A AMY1 (subsite -6) shows 140%, 15%, and <1% and T212Y (subsite +4) 32%, 370%, and90% activity towards starch, maltodextrin, and maltoheptaoside, respectively. The enzyme kinetic properties and modeledmaltododecaose complexes suggest binding mode multiplicity. Following an initial hydrolytic cleavage of amylose, an averageof 1.9 bonds are cleaved per enzyme-substrate encounter, defining a degree of multiple attack (DMA) of 1.9. DMA increasedto 3.3 for Y105A and decreased to 1-1.7 for other subsite mutants. The fusion of a starch-binding domain to AMY1 raisedthe DMA to 3.0 and increased the amount of higher oligosaccharide products. Remarkably, the subsite mutants had unchangeddistribution of released oligosaccharides of DP 5-9, but the profiles differed for the shorter products. A recentlyidentified surface binding site, found exclusively in AMY1, involves the conserved Tyr380 which has no effect on the DMA,but proved critical for ß-cyclodextrin binding as shown by mutational and surface plasmon resonance analyses. Accordingly,AMY2 has lower affinity for ß-cyclodextrin. Hydrolysis of amylopectin proceeds via a fast and a slow reaction rate, withß-cyclodextrin inhibiting the fast one, implicating a distinct role for Tyr380 in activity on amylopectin. Barley seeds produce-cyclodextrin inhibiting the fast one, implicating a distinct role for Tyr380 in activity on amylopectin. Barley seeds produce different proteinaceous inhibitors acting specifically on insect, animal or plant a-amylases. Rational mutagenesis of barleya-amylase/subtilisin inhibitor (BASI) identified structural elements responsible for AMY2 inhibition and demonstrated the-amylase/subtilisin inhibitor (BASI) identified structural elements responsible for AMY2 inhibition and demonstrated the importance of ionic bonds for inhibitory activity.

M3 - Journal article

VL - 60

SP - 27

EP - 36

JO - Biologia (Poland)

JF - Biologia (Poland)

SN - 0006-3088

IS - 16

ER -