INTRODUCTION

-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 five- or 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), Ca²⁺ 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, Arg¹⁸³-Gly¹⁸⁴-Tyr¹⁸⁵ at this loop in AMY1 is distinctive to plant -amylases; others have Lys-His-Z, where Z is hydrophobic (9, 25, 27, 33-35). Arg¹⁸³ in AMY1 aligns with Lys¹⁸² 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 +2 and in the crystal structure of AMY2-BASI (36) interacts with a glutamic acid side chain of BASI.² In TAA and PPA, NZ, the side chain nitrogen, of the corresponding Lys forms the same hydrogen bond with substrate (1, 6). The K209F TAA mutant was inactive, whereas K209R showed 50 and 200% activity toward starch and 4-nitrophenyl -D-maltoside, respectively (37). Similarly, Lys²¹⁰ of Sfamy is found to be close to the catalytic site (38, 39).

Using local random mutagenesis we have been able to engineer barley -amylase to have improved activity even toward amylose. This approach moreover provided a powerful method to both extract information on the tolerance of individual positions to substitutions and to define the role of the target residues. Thus replacements obtained in the sequence Arg¹⁸³-Gly¹⁸⁴-Tyr¹⁸⁵ of AMY1 allowed detailed structure/function relationship investigations and resulted in enzyme variants with significantly improved activity and altered substrate specificity. This is the first report on random mutagenesis applied to a specific substrate-binding - loop of the catalytic (/)₈-barrel of a member of glycosylase family 13.

EXPERIMENTAL PROCEDURES

Soluble starch and the 2-chloro-4-nitrophenyl -D-maltoheptaoside kit (Granutest 3) were from Merck; G₁-PNP and G₂-PNP were purchased from Sigma; G₃-PNP, G₄-PNP, G₅-PNP, and G₆-PNP were from Calbiochem; and G₇-PNP was from Boehringer Mannheim. Degree of polymerization (DP) 17 amylose EX-I was from Hayashibara Chemical Laboratories (Okayama), and insoluble Blue Starch was from Pharmacia Biotech Inc. Acarbose was a generous gift of Dr. E. Moller (Bayer AG, Wuppertal). Restriction enzymes were from Promega and were used according to the manufacturer's recommendation. AMY2 was purified from barley malt (cultivars Triumph or Menuet) (19, 40) and BASI from barley seeds (cultivar Piggy) (41). Rabbit antibodies against barley -amylase were raised using AMY2 as antigen (custom immunization by Dako A/S, Denmark).

Escherichia coli NM522 (42) was used as the host of pBAL7 C95A (43), carrying the mutations in the parent AMY1 insert. Saccharomyces cerevisiae DBY746 (, his3 1, leu2-3, leu2-111, ura3-52, trp1-289) was used for the production of parent and mutant AMY1. pUC18 (44) and pBAL7 C95A were from house collections.

Standard cloning techniques (45) were used throughout. Because two SacI and two SacII sites were present in the AMY1 cDNA (46), silent mutations were introduced into the SacI (at position 646-651) and SacII (at 471-476) sites of the AMY1 C95A gene (43) to make the outer SacI (at 376-381) and SacII (at 1388-1393) sites unique. This involved using polymerase chain reaction (PCR) (Perkin-Elmer Instrument DNA Thermal Cycler) with Vent DNA polymerase (New England BioLabs), two outside primers (SAC01BAM, 5-TTTTTGGATCCTTTTTGAGCTCAAGTCGCTCATCGGC-3, and SAC02ECO, 5-TTTTTGAATTCTTTTTCCGCGGCACCGTTCTTCTCCC-3), and two partially complementary inside primers (SAC05DEL, 5-GACCGGGTCCAGCGCGAACTCAAGGAGTGGCTC-3, and SAC06DEL, 5-GAGCCACTCCTTGAGTTCGCGCTGGACCCGGTC-3) (nucleotides in bold type were replaced for mutation). The two outside primers have BamHI and EcoRI recognition sites, respectively, at the 5 end. The PCR product was purified with QIAquick Spin PCR Purification kit (QIAGEN) and was digested with EcoRI and BamHI. The purified fragment was ligated into pUC18, which was used to transform E. coli by electroporation (Gene Pulsar, Bio-Rad). Transformants were screened on ampicillin containing LB (LB+Amp) plates (47) incubated at 37 °C overnight and subsequently propagated in 5 ml of LB+Amp medium at 37 °C overnight, and pUCBAL C95A S I was purified after centrifugation using a Midi Plasmid kit (QIAGEN). Introduction of the silent mutation as well as the absence of additional mutations were verified by sequencing on Applied Biosystems 373A DNA sequencer (Taq DyeDeoxy Terminator Cycle Sequencing kit; Perkin-Elmer). The SacII silent mutation was subsequently introduced in pUCBAL C95A S I by PCR using the outside primers SAC01BAM and SAC02ECO (see above), with a different set of inside primers (SAC03DEL, 5-GCCGACTACAAGGATAGCAGAGGCATCTACTGCATCTTC-3, and SAC04DEL, 5-GAAGATGCAGTAGATGCCTCTGCTATCCTTGTAGTCGGC-3). The PCR product was subcloned into pUC18, and the recombinant plasmid pUCBAL C95A S I, II was purified as described above.

Random mutants of AMY1 at Arg¹⁸³-Gly¹⁸⁴-Tyr¹⁸⁵ were made in pUCBAL C95A S I, II by PCR using the outside primers described above and the inside primers: PL02, 5-CGTGGCGCCTTGACTTCGCTNNNNNNNNNTCGCCGGAGATGGCCAAGGT-3, plus PL05RV, 5-AGCGAAGTCAAGGCGCCACGCGTCGAAGCCGAGGT-3; random mutants at Gly¹⁸⁴ were made using the same outside primers and the inside primers: PL11, 5-CGTGGCGCCTTGACTTCGCTAGGNNNTACTCGCCGGAGATGGCCAAGGT-3, plus PL12RV, 5-CCTAGCGAAGTCAAGGCGCCACGCGTCGAAGCCGA-3. The PCR products encoding the random mutations were purified, subcloned into pUC18, and then used to transform E. coli. For the Arg¹⁸³-Gly¹⁸⁴-Tyr¹⁸⁵ and Gly¹⁸⁴ random mutations, 10% (100 µl) of the transformation mixture was screened as above. The transformant colonies were counted, each of seven clones was propagated in 5 ml of LB+Amp medium, and the plasmids were purified and sequenced. The size of the pool of random mutants was estimated from the total number of transformants and the mutation efficiency as determined by DNA sequencing of the isolated plasmids. Each of the remaining portions of the transformation mixtures (900 µl) was propagated in 1 liter of fresh LB+Amp medium at 37 °C overnight. The plasmids were purified (Maxi Plasmid kit; QIAGEN) and designated as pUCBAL C95A X(183-185) S I, II and pUCBAL C95A X(184) S I, II, respectively.

Site-directed mutagenesis of Arg¹⁸³ to Lys in pUCBAL C95A S I, II was done by PCR as above using two different inside primers (PL13, 5-CGTGGCGCCTTGACTTCGCTAAAGGCTACTCGCCGGAGATGGCCAAGGT-3, and PL05RV). The BamHI-EcoRI fragment was subcloned into pUC18 to yield pUCBAL C95A R183K S I, II, and the DNA sequence was confirmed.

pBAL7 C95A was thoroughly digested with SacI and SacII, and the 0.98-kilobase fragment was replaced by the corresponding mutated fragment of pUCBAL C95A S I, II. DNA sequencing confirmed that the two silent mutations but no additional mutation had occurred in the AMY1 C95A coding region. The 10-kilobase fragment of pBAL7 C95A S I, II was ligated with the 0.85-kilobase ApaI-SacII fragment of pUCBAL C95A X(183-185) S I, II, or pUCBAL C95A X(184) S I, II to introduce the two pools of random mutants. S. cerevisiae DBY746 cells were transformed by electroporation using the ligation solutions (48) and grown at 30 °C for 1 week on selection plates containing 1% soluble starch (Merck). The selection medium (SD/Leu) contains; 0.04% adenine sulfate, 1% succinic acid, 0.6% sodium hydroxide, 0.67% yeast nitrogen base (without amino acids, Difco), 2% glucose, 5% amino acid mixture (0.05% Trp, 0.05% His, 0.05% Arg, 0.05% Met, 0.07% Tyr, 0.07% Ile, 0.07% Lys, 0.12% Phe, 0.3% Val), 0.025% Thr, 0.005% Asp, and 0.0025% uracil. The mutations were identified by DNA sequencing between the ApaI and SacII sites using PCR products amplified with yeast transformant clones obtained by single colony isolation on SD/Leu plates.

The wild type ApaI-SacII fragment was replaced to give pUCBAL C95A R183K S I, II. The expression vector pBAL7 C95A R183K S I, II was constructed as above and used for yeast transformation. The transformants were grown on SD/Leu + 1% soluble starch plates at 30 °C for 2 days. Clear halos in the starch were visualized by exposure to I₂ vapor for 15 min.

S. cerevisiae DBY746 harboring mutant or wild type pBAL7 C95A S I, II was propagated in 10 liters of SD/Leu medium at 25 or 30 °C for 2 days (MBR BioReactor, AG Switzerland). The recombinant AMY1 was purified by affinity chromatography on -cyclodextrin-Sepharose (49) and analyzed by isoelectric focusing (IEF), SDS-polyacrylamide gel electrophoresis (PAGE) (50), and Western blotting using PhastSystem (Pharmacia). For IEF (PhastGel, pI 4-6.5) 30 ng of protein was applied (15 ng of RGC-AMY1) and visualized by silver staining according to the manufacturer's recommendation. A starch/I₂-zymogram was prepared as earlier described (49). Native PAGE was carried out on PhastGel (10-15%) using the same amount of protein for zymogram development as in IEF. For SDS-PAGE (PhastGel, 10-15%) enzyme (6 µl, 10 µg/ml; RGC-AMY1, 5 µg/ml) was mixed with sample buffer (6 µl) and boiled for 5 min, marker dye (1 µl) was added, and 3 µl was applied to the gel; protein was detected by silver staining. Western blotting was performed with ProtoBlot Western blot AP system (Promega) using rabbit anti-AMY2 serum at 10³-fold dilution. The concentration of AMY1, parent and mutants, and BASI was determined by amino acid analysis (15, 49). The secretion level of recombinant AMY1 in fermentor cultures was estimated from the amount of purified enzyme.

HPLC Analysis of the Hydrolysis Products of 4-Nitrophenyl -D-Maltooligosaccharides

Enzyme and G₅-PNP, G₆-PNP, or G₇-PNP (120 µl) in 50 mM sodium acetate, pH 5.5, 12.5 mM CaCl₂ were incubated at 37 °C. Aliquots (40 µl) were removed at appropriate time intervals, and the reaction was stopped by addition of glacial acetic acid (20 µl). Substrate and products were separated by HPLC on a TSK-GEL oligo-PW column (Toso, 7.8 × 300 mm) and eluted with distilled water at room temperature at a flow rate of 0.7 ml/min (2150 HPLC pump, LKB) (7, 51). 4-Nitrophenyl compounds were monitored at 313 nm (SPD-6A, Shimadzu ultraviolet detector). A mixture containing 1% of each of G₁-PNP, G₃-PNP, G₅-PNP, G₆-PNP, and G₇-PNP in 50 mM acetate buffer, pH 5.5, was used for calibration. The relative activities were calculated using [DC_M/(t_M·[E]_M)]·[(t_W·[E]_W)/DC_W]·100, where DC_M and DC_W are degrees of cleavage of the substrate by mutant and wild type enzymes, t_M and t_W are the reaction times, and [E]_M and [E]_W are the concentrations of mutant and wild type enzymes, respectively. DC was calculated as [P_t/(S_t + P_t)]·100, where S_t and P_t represent residual substrate and total amount of products at reaction time t.

Hydrolysis of DP 17 amylose by mutant and wild type AMY1 (1-5 nM) at pH 5.5 in 20 mM sodium acetate, 5 mM CaCl₂, and 0.5 mg/ml bovine serum albumin at 37 °C was followed by reducing sugar analysis using copper bicinchoninate (52, 53). Boiled samples were aliquoted into microtiter plates in triplicates of 300 µl, and the absorbance at 540 nm was measured using a Ceres UV-900-HDi scanning autoreader (Bio-Tek Instruments, Inc). Maltose was used as a reference. Kinetic parameters were calculated by fitting the initial velocities at seven amylose concentrations in the range 0.06-10.0 mg/ml to the Michaelis-Menten equation using Erithacus-software GraFit, version 3.0. Standard deviations were calculated from the data obtained by triplicate experiments performed independently. The activity toward G₇-Cl-PNP was determined using the Granutest 3 -amylase assay kit; -amylase (15 nM) was added at nine concentrations of G₇-Cl-PNP (0.26-16 mM) in 20 mM MES, pH 6.8, 5 mM CaCl₂ (total volume, 100 µl) at 30 °C in microtiter plates. The progress of hydrolysis was followed at 405 nm, and kinetic parameters were calculated as above. -Amylase (30 nM) was preincubated with the inhibitor acarbose (0.5-50 µM) for 15 min at pH 5.5 (20 mM sodium acetate, 5 mM CaCl₂, 0.5 mg/ml bovine serum albumin) at 25 °C, the substrate DP 17 amylose (6.25 mg/ml) was added at the given [acarbose], and the activity was measured as above. K_i app was calculated as (A_x  A₀) × L_x/(A_max  A_x), where A_max and A_x are A₅₄₀ values at 0 and L_x µM acarbose, respectively.

The activity of mutants of AMY1 and wild type AMY1 and AMY2 (2 nM) toward insoluble Blue Starch (6.25 mg/ml) was measured at varying [CaCl₂] (0.0025-100 mM) at pH 5.5 (20 mM sodium acetate, 0.5 mg/ml bovine serum albumin) at 37 °C. Standard deviations were calculated from the data obtained in triplicate experiments performed independently.

AMY1 mutants and wild type AMY2 (30 nM) were preincubated with BASI (0.02-992 µM) at pH 8.0 (40 mM Tris, 5 mM CaCl₂, 0.5 mg/ml bovine serum albumin) for 15 min at 37 °C. Residual activity was determined by adding an aliquot (25 µl) of this mixture to an insoluble Blue Starch suspension (4 ml, 6.25 mg/ml) in the same buffer. After 15 min the reaction was stopped by the addition of 0.5 M NaOH (1 ml). The absorbance at 620 nm was measured after centrifugation, and the percentage of inhibition by BASI was calculated as described previously (15, 41).

RESULTS

In the present study we focused on active mutants of barley AMY1 secreted from the S. cerevisiae host strain. Parent enzyme C95A-AMY1 was used to avoid inactivating glutathionylation of the free thiol group of Cys⁹⁵ in AMY1 during yeast growth and processing (43). C95A-AMY1 is fully active toward Blue Starch (43) and will here be referred to as wild type or parent AMY1. A pool of 10⁴ different random mutants was created on pUC18 and designated as pUCBAL C95A X(183-185) S I, II. 120 yeast transformants harboring pBAL7C95A X(183-185) S I, II were selected and subjected to DNA sequencing, and -amylase activity was measured. Of these transformants, 83% had a mutant gene, but only 4% secreted active enzyme. DNA sequencing showed that Gly¹⁸⁴ as well as a polar residue at position 183 and a hydrophobic residue at position 185 were retained in active mutants (Table I). No transformants harboring mutant genes encoding a residue different from glycine at position 184 were found to secrete immunoreactive protein. Although they encoded a polar and a hydrophobic residue at positions 183 and 185, respectively, different residues: Lys (24 and 25), Asp (59), Trp (66), or Thr (72) replaced Gly¹⁸⁴ (Table I). Transformants producing Asn¹⁸³-Gly¹⁸⁴-Tyr¹⁸⁵ (NGY), KGY, and wild type (RGY) AMY1 gave large starch plate halos, whereas those secreting SGM- and TGL-AMY1 produced smaller halos, and RGC-AMY1 produced a very small halo. The halo size agreed well with the estimated amount of enzyme in fermentor cultures: wild type, 82 µg/l; KGY, 120 µg/l; NGY, 80 µg/l; SGM, 20 µg/l; TGL, 10 µg/l; and RGC mutants, < 10 µg/l.

Table I. Sequences at the fourth - loop of AMY1 mutants evaluated by analysis of enzymatic activity on starch plate and Western blotting Clone number DNA sequence (amino acid sequence) Activity on starch plate Western blotting Wild type AGG GGC TAC ++++ +  R183 G   Y 31 AGA GGT TGC + +  R   G   C 73 AAT GGG TAT ++++ +  N   G   Y 89 ACC GGG CTG ++ +  T   G   L 117 AGT GGT ATG +++ +  S   G   M R183K-1a AAA GGC TAC ++++ +  K   G   Y 6 TTT CAT CAT    b  F   H   H 22 ATT ATG ATT      I   M   I 24 CGC AAG TAT      R   K   Y 25 TAT AAA GCA      Y   K   A 32 CTT AGG AGT      L   R   S 58 TTG TCT AAT      L   S   N 59 TGG GAT CTG      W   D   L 66 CGG TGG GGT      R   W   G 72 GAT ACT GGG      D   T   G 77 GGA AAC CAA      G   N   Q 98 GTG GTG TTG      V   V   L 119 ATA CTC AGT      I   L   S a R183K-AMY1 was obtained by site-directed mutagenesis. Other mutants resulted from random mutagenesis. The full length of the AMY1 insert was sequenced for transformant clones listed. b 800-fold concentrated culture filtrates were used. Italic letters and bold letters are polar and hydrophobic residues, respectively, as classified according to Ref. 45.

After random mutation of only the codon for Gly¹⁸⁴, 150 yeast transformants were isolated. Following repeated colony isolation, enzyme secreting transformants (11) were identified by a positive starch plate assay, and the target DNA region was sequenced and found to encode Gly¹⁸⁴. Codons for Trp, Tyr, Phe, Val, Arg, Ser, or Cys¹⁸⁴ were identified in genes from transformants not secreting immunoreactive enzyme.

In the IEF zymogram only NGY-AMY1 resembled wild type with the expected modest decrease in pI (Fig. 1A, lanes 4 and 5). RGC-AMY1 exhibited much weaker bands (Fig. 1A, lane 1). TGL- and SGM-AMY1 lacked activity in the zymogram and gave faint diffuse bands by silver staining (Fig. 1, A and B, lanes 2 and 3). Clear activity and protein staining, however, were obtained for these mutants in native PAGE (Fig. 1, C and D). Essentially the same molecular size and antigenecity were found for the mutants and wild type AMY1 in SDS-PAGE and Western blotting (Fig. 1, E and F). KGY-AMY1 also showed activity in an IEF zymogram and had the same mobility in SDS-PAGE and native PAGE, and immunoreactivity as the wild type (data not shown).

The mutants and wild type AMY1 migrated as two bands in IEF and native PAGE. Electrospray ionization mass spectrometry of SGM-AMY1, KGY-AMY1, and wild type AMY1 (data not shown) showed a full-length and a truncated form lacking the C-terminal Arg/Ser⁴¹⁴ dipeptide. The truncation occurs by the known processing of recombinant AMY1 in S. cerevisiae by Kex1p, a carboxypeptidase specific for basic side chains (43, 49). Mass spectrometry also indicated Thr⁴¹⁰ in a significant fraction of recombinant AMY1 to carry two hexose residues, as identified earlier for recombinant wild type AMY1. Further glycosylation was not observed (data not shown). These modifications did not affect activity (43, 54, 55).

To investigate the role of Arg¹⁸³ in substrate interaction in detail, the product distribution in hydrolysis of G₅-, G₆-, and G₇-PNP was determined. Drastic changes were seen in the action pattern on G₆-PNP, in particular for NGY-, SGM-, and TGL-AMY1, whereas small changes occurred with G₅-PNP (Table II). The dominant productive binding mode for hydrolysis of G₆-PNP with wild type, KGY-AMY1, and RGC-AMY1 resulted in release of G-PNP in 32-50% yield, whereas NGY-, SGM-, and TGL-AMY1 released PNP in 48-84% yield. Although wild type (RGY) released some PNP (24%), KGY- and RGC-AMY1 formed no or very little PNP (Table II). A similar shift of the preferred bond to be cleaved in G₇-PNP was seen for NGY-AMY1 and, to a lesser degree, SGM-AMY1, as a significantly increased hydrolysis of the first bond to release PNP. Wild type, KGY-AMY1, TGL-AMY1, and RGC-AMY1, in contrast, hydrolyzed the second bond in G₇-PNP in 63-81% yield (Table II). For the shorter G₅-PNP, NGY-, SGM-, and TGL-AMY1 having small side chains at position 183 showed 3-26% of the wild type activity, whereas KGY-AMY1 retained 72% (Table III). In no case, however, did the action pattern shift on G₅-PNP, although SGM-AMY1 generated an unusually large amount of glucose (Table II). The results reflect the subsite map of AMY1 (19) with its low binding affinity at subsites +3 and 3 and the high affinity at subsite 6 that plays an important role in binding near the nonreducing end of longer maltooligosaccharides.

Table II. Action patterns on G5-PNP, G6-PNP, and G7-PNP cleaved by wild type and mutant AMY1 in Arg183-Gly184-Tyr185 at the fourth - loop Substrate concentrations were 5.2, 4.5, and 3.9 mM for G5-PNP, G6-PNP, and G7-PNP, respectively. AMY1 Cleavage frequency [E] Reaction time Degree of cleavage % µM min % G5-PNP      G-G-G-G-G-PNP   Wild type        8 16 32 43 0.10 30 39   KGY        4 10 26 42 18 0.83 6 47   NGY        7 14 41 38 0.04 960a 42   SGM       24 10 31 33 0.10 960a 35   TGL        5 17 29 42  7 0.20 60 41 G6-PNP   G-G-G-G-G-G-PNP   Wild type        6  6 26 38 24 0.16 7 29   KGY    15  4  5 27 50 0.17 5 37   NGY        2  2  7  6 84 0.06 20 41   SGM     7  7  5 17 15 48 0.10 240a 23   TGL     1  4  4 16 19 55 0.05 30 32   RGC    25  8  6 27 32  2 0.02 60 33 G7-PNP G-G-G-G-G-G-G-PNP   Wild type  5  2  1  1  6 81  4 0.03 10 55   KGY  1  5          78 17 0.03 3 52   NGY  4  1     1  4 30 58 0.03 10 50   SGM  7  2  2  2  7 51 28 0.10 20 48   TGL  6  3  2  2  8 63 17 0.02 30 50   RGC  8  2  2  2  8 77 0.01 80 54 a NGY-AMY1 and SGM-AMY1 are stable under the incubation conditions used.

Table III. Kinetic parameters for the hydrolysis of DP 17 amylose and maltooligosaccharide derivatives by wild type and mutant AMY1 Enzyme Amylose DP 17 G7-Cl-PNP Relative activitya Acarbose Ki, app kcat Km kcat/Km kcat Km kcat/Km G7-PNP G6-PNP G5-PNP s1 mg ml1 ml mg1 s1 s1 mM mM1 s1 % µM Wild (RGY) 464  ± 49 (100)b 2.20  ± 0.57 221  ± 81 (100) 75  (100) 9.8 7.7  (100) 100  (100) 100  (100) 100  (100) 17.1  ± 4.9 KGY 596  ± 125 (128) 4.17  ± 0.42 146  ± 47 (66) 120  (160) 16.1 7.5  (97) 316  (304) 168  (221) 72  (70) 15.4  ± 5.7 NGY 445  ± 30 (96) 3.18  ± 0.47 142  ± 21 (64) 37  (52) 4.0 9.3  (121) 91  (33) 132  (21) 8  (7) 2.9  ± 2.3 SGM 822  ± 147 (177) 2.37  ± 1.17 379  ± 124 (171) 60  (80) 3.4 17.6  (229) 13  (9) 4  (3) 3  (2) 3.9  ± 2.5 TGL 366  ± 143 (79) 1.89  ± 0.38 191  ± 38 (86) 26  (35) 3.9 6.7  (87) 45  (35) 82  (42) 26  (26) 1.9  ± 0.8 RGC NDc ND ND ND ND ND 37  (35) 106  (89) ND ND a The relative activities were calculated as described under "Experimental Procedures" using the data shown in Table II. The values in parenthesis present the relative activities (%) in the hydrolysis of the substrate at the penultimate glycosidic bond from the reducing end. b The values in parenthesis represent activities expressed as percentage of the values of wild type AMY1. c ND, not determined.

The activity after mutation was either increased or essentially retained for amylose, which is a substrate that spans the entire active site binding area. k_cat thus increased nearly 2-fold for SGM-AMY1; K_m slightly increased, except for TGL-AMY1 (Table III). k_cat/K_m after mutation varied as 64-171% of the wild type value. For the oligosaccharide G₇-Cl-PNP, k_cat was highest for KGY-AMY1 (160%) and lowest for TGL-AMY1 (35%). On this substrate NGY- and SGM-AMY1 gave intermediary k_cat values, and K_m increased or decreased by a factor of two dependent on the mutant (Table III). G₇-Cl-PNP contains a -glucosidic bond at the reducing end, which may interfere adversely with Lys¹⁸³, the equivalent of Lys¹⁸² in AMY2 that interacts with the sugar OH-3 at subsite +2 in the crystal structure of the acarbose complex (71). In comparison, the shorter Asn, Ser, or Thr¹⁸³ gave decreased K_m of around 4 µM, resulting in k_cat/K_m above the wild type value for NGY- and SGM-AMY1 (Table III). K_i app of acarbose inhibition of the hydrolysis of amylose was 2-4 µM for the NGY-, SGM-, and TGL-AMY1. Because K_i, app of the parent and KGY-AMY1 was around 16 µM (Table III), the pseudotetrasaccharide acarbose more effectively inhibited the Asn, Ser, or Thr¹⁸³ containing mutants.

The isozymes AMY1 and AMY2 show distinct variations in activity for insoluble Blue Starch as a function of the Ca²⁺ concentration (Fig. 2). Thus, whereas the activity of AMY1 changed little from 0.0025 to 10 mM CaCl₂, that of AMY2 increased to reach a maximum around 10 mM CaCl₂ (15). Both isozymes are inhibited at [CaCl₂] > 20 mM. Whereas SGM- and TGL-AMY1 were clearly less active than the parent enzyme at [Ca²⁺] < 0.01 mM (Fig. 2), resulting in a profile that resembled that of AMY2, the behavior of KGY-AMY1 (data not shown) very much resembled that of the AMY1 parent.

KGY- and NGY-AMY1 were tested for inhibition by up to 3 × 10⁴-fold molar excess of BASI. The natural target AMY2, under the conditions used, is inhibited even by stoichiometric amounts of BASI. However, although Lys¹⁸² of AMY2 forms a salt bridge with a glutamate residue in BASI,² replacement of the equivalent Arg¹⁸³ in AMY1 by Lys, or Asn¹⁸³ (in KGY- and NGY-AMY1), did not confer these AMY1 variants detectable sensitivity for BASI.

DISCUSSION

Very few and short sequences are well conserved among -amylases and related amylolytic enzymes (28, 29); only seven residues are invariant (25, 27). Based on crystal structures (1-7) and mutational analyses, these residues are implicated either in catalysis and transition state stabilization or in the structural integrity of the active site (25, 27, 53). The molecular model of a barley -amylase-inhibitor complex (71) confirmed that conserved regions are important in substrate specificity and action pattern. An alignment focused on the fourth - loop of the catalytic (/)₈-barrel revealed remarkable sequence variation corresponding to Arg¹⁸³-Gly¹⁸⁴-Tyr¹⁸⁵ in AMY1 (boxed in Fig. 3), the present target of random mutagenesis. Family members with different specificity thus contain (i) (Arg/Lys)-Gly-Ar as in plant -amylases, (ii) Lys-His-Z predominant in animal and microbial -amylases, or (iii) a completely different sequence where Z and Ar signify hydrophobic and aromatic residues, respectively (9, 34, 35, 46, 56). The carbonyl oxygen of the aligned residues, AMY2 Gly¹⁸³ (4, 9), TAA His²¹⁰ (63), and PPA His²⁰¹ (3), is a ligand to the Ca²⁺ conserved in -amylases, i.e. Ca²⁺ (Ca 500 in Ref. 4) in AMY2 (4). Lys¹⁸² of AMY2 binds to sugar OH-3 at subsite +2, and Lys²⁰⁰ of PPA shows the same interaction (6). Furthermore, NE2 of His²⁰¹ from the Lys-His-Z motif in PPA forms a hydrogen bond with sugar OH-2 in subsite +1, i.e. on the reducing end side of the cleavage (6). Site-directed mutagenesis of His²⁰¹ in the 95% identical human pancreatic -amylase revealed its multifunctional role in substrate affinity, pH activity dependence, Cl activation, and binding of a proteinaceous inhibitor (61, 62).

Alignment of the sequence in the fourth - loop extending from -strand 4 in the -amylase family.

Site-directed mutagenesis earlier indicated the roles in catalysis and binding of invariant carboxylic acid and histidine residues in barley AMY1 (53). The present goal is to alter substrate specificity and action pattern by random mutagenesis of Arg¹⁸³-Gly¹⁸⁴-Tyr¹⁸⁵ involved in substrate binding. All active mutant AMY1 found had conserved the motif: Gly¹⁸⁴, a polar residue at position 183, and a hydrophobic residue at position 185. Gly¹⁸⁴ occurs in all known plant -amylase sequences (27, 64). We searched for possible variants by subjecting the corresponding codon to random mutagenesis, but no active mutants were obtained. Attempts to construct G184A failed repeatedly (not described). In accordance with the crystal structure of AMY2 (4), glycine is suggested therefore to be required at that position in plant -amylases.

The amount of mutant protein produced was influenced by the side chains at position 183 or 185. Although KGY- and NGY-AMY1 were produced at the same level as wild type AMY1 (RGY), yields of SGM-, TGL-, and RGC-AMY1 were around 10% of that amount. The segment centered on Gly¹⁸⁴ may thus be critical for stability and/or proper folding. In the crystal structure of AMY2, Lys¹⁸² (equivalent to Arg¹⁸³ of AMY1) is located at a turn in the fourth - loop, in which the side chain of Phe¹⁸⁰ (Phe¹⁸¹ in AMY1) and the C proton of Gly¹⁸³ (Gly¹⁸⁴ in AMY1) come close (4) (Fig. 4). It is remarkable that the -amylases from higher plants, amylomaltases, and maltotetraohydrolases (27, 64) have a large residue (Phe or His) together with glycine matching Phe¹⁸¹ and Gly¹⁸⁴ in AMY1 (Fig. 4), whereas -amylases from other sources, maltohexaohydrolase, and cyclodextrin glucanotransferase contain a small residue (Ala, Ser, or Thr) and histidine corresponding to Phe¹⁸¹ and Gly¹⁸⁴, respectively. This complementarity in the different enzymes (Fig. 4) supports that AMY1 Gly¹⁸⁴ is structurally important.

Schematic model of the function of the sequence (Arg¹⁸³-Gly¹⁸⁴-Tyr¹⁸⁵) extending from the fourth -strand of AMY1.

AMY2 has three Ca²⁺, one of which is conserved in the different -amylase structures (2-4, 63). The carbonyl oxygen of Gly¹⁸³ is the only Ca²⁺ ligand from domain A (4). Replacement of both residues flanking AMY1 Gly¹⁸⁴ may affect the position of this carbonyl oxygen, because SGM- and TGL-AMY1 both lost activity at [Ca²⁺] < 10 µM and showed no zymogram activity after IEF. In fact, the behavior of AMY1-AMY2 isozyme hybrids suggested that some part of the AMY1 structure after residue 161 was responsible for the high Ca²⁺ affinity (65); this may be the Gly¹⁸⁴ region. The other Ca²⁺ ligands belong to the long third - segment (amino acids 89-152), called domain B, that is grafted onto the (/)₈-fold (4). Although the AMY2 Ca²⁺ ligands are all conserved in AMY1 (4, 9), AMY1 compared with AMY2 has higher affinity for Ca²⁺ (14). It remains to be seen if AMY1 contains three Ca²⁺, and it remians to be understood why the activity of the isozymes depends differently on Ca²⁺.

Arg¹⁸³ in AMY1 most probably binds to substrate. In AMY2 acarbose (an inhibitory pseudotetrasaccharide) (66), NZ of Lys¹⁸² thus hydrogen bonds with OH-3 of the third acarbose ring (71) and is considered a critical determinant of subsite 2. The shifted action pattern on the substrate G₆-PNP is compatible with a weakening of this interaction in NGY-, SGM-, and TGL-AMY1 releasing PNP, whereas wild type and mutants having (Arg/Lys)¹⁸³ release G₁-PNP as major product.

KGY-AMY1 shows a 3-fold increase in activity and maintains wild type action pattern for hydrolysis of G₇-PNP but has only 72% activity on G₅-PNP. NGY-AMY1 shows 91 and 8% activity, respectively, on these substrates. The three-dimensional structures around Lys¹⁸² of AMY2 and Lys²⁰⁹ of TAA are similar (4). In case of Sfamy, which is highly homologous to TAA (7, 23, 37, 38, 67), K210R (38) had 20 and 200% (k_cat/K_m) of wild type activity toward maltopentaose and maltotriose due to decreased affinity at subsite +2 and increased affinity of subsite +1, respectively. The k_cat/K_m values of K210N Sfamy for G₅- and G₃-PNP were only 15 and 1% of K210R Sfamy due to reduced affinity at both subsites (38). This behavior is reminiscent of the present AMY1 mutants. The mutant action patterns are consistent with Arg¹⁸³ being an important component of subsites +1 and +2, and the strong affinity at subsite +1 required for efficient hydrolysis of short substrates, such as G₅-PNP.

The AMY1 mutants have efficient k_cat/K_m for amylose hydrolysis varying at 64-171% of the wild type value. Noticeably the relative activity of especially SGM-AMY1 increased with substrate length. According to the subsite theory (68, 69) k_cat/K_m = k_int · K_p, where k_int and K_p are the intrinsic rate constant for bond hydrolysis and the binding constant of substrate in productive mode, respectively; k_int = k_cat/K_m in hydrolysis of long substrates that cover the entire binding site. The strong dependence of SGM-AMY1 on substrate length might be explained by the mutation affecting both parameters resulting in increased k_int and decreased affinity at one or more subsites near the catalytic site. For hydrolysis of short substrates, K_p decreases are probably more important than k_int increases. This agrees with affinities at subsites +1 and +2 being reduced by the replacement of Arg¹⁸³ by Ser, hence k_cat/K_m is expected to decrease for the mutant compared with the wild type enzyme. Except for an elevated k_cat/K_m toward G₇-Cl-PNP containing a -anomeric bond between the nitrophenyl group and glucose at the reducing end, the relative activities against -anomeric oligosaccharide derivatives, G₇-PNP, G₆-PNP, and G₅-PNP, showed drastic decreases from 13 to 3%. For amylose, however, SGM-AMY1 has a high k_cat/K_m value, which simply reflects an increase in k_int.

The parent (C95A) AMY1 has K_m of 9.8 mM for G₇-Cl-PNP, whereas normal wild type AMY1, both from malt and a recombinant (11, 15) produced in Pichia pastoris, gave K_m around 1 mM (55). Similarly an approximate 5-fold increase in K_m to 2.2 mg/ml was obtained for parent AMY1 acting on the large substrate amylose, as compared with K_m = 0.4 mg/ml for AMY1 from malt and Pichia. This poorer affinity (Table III) may stem from the C95A mutation, which is located near His⁹³ known to be critical for transition state stabilization (53).

Finally, BASI specifically inhibits AMY2 (17, 18) in a 1:1 complex with K_d = 2 × 10¹⁰ M (41). The AMY2-BASI complex was crystallized (36), and in the molecular model Lys¹⁸² of AMY2 forms a salt bridge to a glutamate in BASI.² Whereas Lys¹⁸² probably contributes important stabilization of AMY2-BASI, the lack of inhibition of the mutant R183K even by a large excess of BASI showed that Lys¹⁸³ alone does not confer AMY1 sensitivity for BASI. This finding agrees with the fact that AMY1-AMY2 isozyme hybrids inhibited by BASI possess AMY2 sequence from residue 116 (15, 65).

In a schematic of the Arg¹⁸³-Gly¹⁸⁴-Tyr¹⁸⁵ region in the fourth - loop, based on the crystal structure of AMY2 (4) complexed with acarbose (71), Ca²⁺ stabilizes the contact between domains A and B (Fig. 4). Gly¹⁸⁴ (domain A) binds to this Ca²⁺ together with Asn⁹², Asp¹³⁹, Ala¹⁴², and Asp¹⁴⁹ (domain B). Gly¹⁸⁴ indeed may play an important conformational role involving the steric complementarity with the side chain of Phe¹⁸¹, which most probably is obstructed by introduction of a side chain at Gly¹⁸⁴. Because the activity for amylose was improved up to 171% for engineered AMY1, the enhanced k_cat of the - loop 4 mutants might reflect increased flexibility in this segment, which has the catalytic nucleophile Asp¹⁸⁰ located at the C terminus of -strand 4 (4). Finally, k_cat/K_m of SGM-AMY1 is nearly 2-fold that of wild type on amylose, which occupies all ten subsites in the substrate binding site, but activity on oligosaccharides is below 9% of wild type. In contrast, KGY-AMY1 is up to three times more active than wild type on G₆-PNP and G₇-PNP. The results suggest that the long side chain of Arg¹⁸³ contributes to binding at subsites +1 and +2 (Fig. 4) but also confers constraints on substrate processing.

The present results give very promising prospects for activity improvement and modulation of action pattern in plant -amylases by protein engineering in the distinct sequence (Lys/Arg)-Gly-Tyr at the active site - 4 segment. This is the first report on an engineered -amylase with enhanced activity toward the natural substrate. Ultimately -amylase variants might be useful in transgenic barley for enhancement of malt quality by improving the enzyme activity during germination and mashing (70).