Improved Activity and Modulated Action Pattern Obtained by Random Mutagenesis at the Fourth β-α Loop Involved in Substrate Binding to the Catalytic (β/α)8-Barrel Domain of Barley α-Amylase 1

Abstract

␣-Amylase (␣-1,4-D-glucan glucanohydrolase, EC 3.2.1.1) hydrolyzes internal ␣-1,4-glucosidic bonds in starch and related oligo-and polysaccharides. High resolution x-ray structures are known for Taka-amylase A (TAA) 1 from Aspergillus oryzae(1), acid ␣-amylase from Aspergillus niger (2), isozyme I of porcine pancreatic ␣-amylase (PPA; Ref.3), the AMY2-2 isoform from barley malt (4), and an inactive, protease-cleaved form of ␣-amylase from Bacillus licheniformis (5). These enzymes are organized in three domains, an N-terminal catalytic (␤/␣) 8 -barrel fold (domain A) having a long ␤-␣ loop (domain B) protruding at the third ␤-strand and a C-terminal either fiveor eight-stranded ␤-sheet (domain C). Oligosaccharide inhibitor complexes are determined of TAA (1), PPA (6), and AMY2-2 (71). The structure of Saccharomycopsis fibuligera ␣-amylase (Sfamy) was modelled on that of TAA (7). Barley ␣-amylases occur in two isozyme families (8), a low pI (AMY1) and a high pI (AMY2), of 80% sequence identity (9) but significantly different substrate affinity, turnover rate (10 -12), Ca 2ϩ dependence of activity (13-15), and stability (13, 15, 16). Only AMY2 is inhibited by the endogenous ␣-amylase/subtilisin inhibitor (BASI) present in barley seeds (17, 18). The subsite maps of binding affinities for substrate glucosyl residues are very similar for AMY1 and AMY2 and comprise ten subsites, six toward the nonreducing and four toward the reducing end relative to the cleavage point in linear maltooligosaccharides (19). They moreover possess, like subsite maps from Bacillus subtilis (20) and Bacillus amyloliquefaciens ␣-amylases (21), TAA (22), and Sfamy (23), a large negative affinity, indicating distortion of the glucose residue at the catalytic site, flanked by two subsites of large positive affinity.Sequence alignment and prediction of secondary structures indicated that different starch hydrolases and related enzymes contain a catalytic (␤/␣) 8 -barrel similar to the ␣-amylases (24,25). In a recent structure-based classification of glycosylases, these amylolytic enzymes belong to family 13 (26), currently including 18 EC classes, representing variations in substrate/ product specificity (27). Seven short conserved sequences characterize family 13 members, but only seven amino acid residues are invariant (28, 29). Although ␣-amylases strictly hydrolyze the ␣-1,4, related enzymes can act on either ␣-1,4 or ␣-1,6 glucosidic bonds or have dual bond-type specificity. A conserved region in the ␤-␣ loop extending at the C terminus of the fourth ␤-strand of the (␤/␣) 8 -fold is interpreted to play an important role in the specificity (25, 27, 30 -32). Remarkably, Arg 183 -Gly 184 -Tyr 185 at this loop in AMY1 is distinctive to plant ␣-amylases; others have Lys-His-Z, where Z is hydrophobic (9,25,27,(33)(34)(35). Arg 183 in AMY1 aligns with Lys 182 in AMY2 (9), which in the crystal structure of AMY2-acarbose (71) forms a hydrogen bond to OH-3 of the sugar ring at subsite ϩ2 and in the crystal structure of AMY2-BASI (36) interacts with a ...

Molecular Structure and Novel DNA Binding Sites Located in Loops of Flap Endonuclease-1 from Pyrococcus horikoshii

“…For the triple mutant K193A/R194A/ K195A, the K m value increased markedly and the k cat value decreased moderately. The negatively charged triple mutant K193E/R194E/K195E showed similar but more magnified effects on both parameters compared with the alanine triple mutant K193A/R194A/K195A, indicating ionic interactions of the positive cluster with DNA phosphate groups in an analogous manner to Arg 40 and Arg 42 on small loop 1. The values of K199A differed little from those of WT.…”

“…The K a value of R40G/R42G and R118A/ K119A dropped 6-and 4-fold, respectively, and the values for each single mutant decreased moderately compared with that of WT, whereas the K a value of the other mutants showed little difference to that of WT. The results indicated that both these sites, Arg 40 archaea (14, 18) for which differences in substrate binding were proposed. According to the two-modeled structures of the protein-substrate complex, the 5Ј end of the single-stranded flap strand threads through the hole formed by the L1 loop (corresponding to the large loop in our work).…”

“…Fig. 2, two pairs of basic amino acids (Arg 40 and Arg 42 , and Lys 51 and Arg 53 ) were observed typically on both edges of small loop 1 (39 -55) with highly conserved amino acids (Leu 47 , Gly 44 , and Gly 52 ) between the eukaryotes and archaea. Various mutations were introduced into this typical region to investigate the function of these residues.…”

“…4 clearly indicated that the former pair, Arg 40 and Arg 42 , bound equally to the substrate, and the latter pair, Lys 51 and Arg 53 , played a less significant role for the substrate binding and the catalytic reaction. In the molecular structure, Leu 47 was located in the middle of the two positive pairs on small loop 1 and showed tight hydrophobic interactions with the methyl and methylene groups of Ile 39 , Gln 41 in the same loop, and Ser 59 located near the loop.…”

“…To clarify differences between the endo-and exo-type binding, a kinetic analysis of 5Ј-exonuclease was performed using nick substrate. The kinetics of exo-activity were analyzed using single, double and triple mutants (Arg 40 The K m values of two double mutants, R40G/R42G and R118A/K119A were magnified by 6-and 17-fold, respectively, compared with the K m of WT, suggesting a large contribution by these two regions to the exo-type binding as shown in Fig. 5.…”