Domain B protruding at the third β strand of the α/β barrel in barley α‐amylase confers distinct isozyme‐specific properties

Abstract

a-Amylases belong to the alp-barrel protein family in which the active site is created by residues located at the C-terminus of the p strands and in the helix-connecting loops extending from these ends. In the a-amylase family, a small separate domain B protrudes at the C-terminus of the third p strand of the @/a),-barrel framework. The 80% identical barley a-amylase isozymes 1 and 2 (AMYl and AMY2, respectively) differ in substrate affinity and turnover rate, CaC1, stimulation of activity, sensitivity to the endogenous 21-kDa a-amylaselsubtilisin inhibitor, and stability at low pH. To identify regions that confer these isozyme-specific variations, AMY1 -AMY2 hybrid cDNAs were generated by in vivo homologous recombination in yeast. The hybrids AMYl-(l-9O)-AMY2-(90-403) and AMY1-(1-161)-AMY2-(161-403) characterized in this study contain the 90-residue and 161 -residue N-terminal sequences, respectively, of AMY 1 and complementary C-terminal regions of AMY2. AMYl-(1-90)-AMY2-(90-403) comprises the 60-amino-acid domain B of AMY2 and resembles this isozyme in sensitivity to a-amylaselsubtilisin inhibitor and its low affinity for the substrates p-nitrophenyl a-D-maltoheptaoside, amylose and the inhibitor acarbose. Only AMY1-( 1-161)-AMY2-(161-403) and AMYl, which both share domain B, are stable at low pH. However, AMY2 and both hybrid AMY species, but not AMY1, show maximum enzyme activity on insoluble blue starch at approximately 10 mM CaC1,. Domain B thus determines several functional and stability properties that distinguish the barley a-amylase isozymes.a-Amylases hydrolyse internal a-l,4-glucosidic linkages of starch and related dextrins and are widely occurring in microorganisms, higher plants and animals. In cereals, aamylases represent a major starch-degrading activity important in seed germination (MacGregor, 1987). a-Amylases and related amylolytic enzymes are @/a),-barrel proteins (Matsuura et al

Rice Phospholipase D Isoforms Show Differential Cellular Location and Gene Induction

“…In cereals, B-domains are essential for enzyme regulation and activity. For example, B-domains of barley a-amylases (AMY1 and AMY2) are instrumental in binding structural Ca 2+ , conferring stability at low pH, specifying substrate binding, and in binding of the barley a-amylase subtilisin inhibitor, BASI (Rodenburg et al 1994, Juge et al 1995. One interesting possibility is that this region is the site for binding b-trefoiltype inhibitors (e.g.…”

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

“…Amylose-Initial rates of hydrolysis of amylose DP17 (average degree of polymerization of 17; Hayashibara, Okayama, Japan) at 8 concentrations (0.04 -10.8 mg ϫ ml Ϫ1 ) were determined in the above buffer by adding enzyme (2-4 nM) at 37°C (35) and measuring reducing sugar by the copper-bicinchoninate procedure (61). A 540 was measured using maltose (2.5-20 g ϫ ml Ϫ1 ) as standard, and k cat and K m were calculated as above.…”

Amylose chain behavior in an interacting context II. Molecular modeling of a maltopentaose fragment in the barley ?-amylase catalytic site

“…AMY1 and AMY2 contain, respectively, 414 and 403 amino acid residues. 13,15,16 Despite their high degree of homology, AMY1 and AMY2 possess distinct physical and chemical properties, such as calcium ion affinity, [17][18][19] sulfhydryl and chelating reagents sensitivity, stability at acidic pH, 19,20 and high temperature, activity toward starch granules 21,22 and affinity for soluble substrates. [23][24][25] This could explain their different physiological roles.…”