Tài liệu Báo cáo khoa học: Plant a-amylase inhibitors and their interaction with insect a-amylases ppt

REVIEW ARTICLE Plant a-amylase inhibitors and their interaction with insect a-amylases Structure, function and potential for crop protection Octa  vio L. Franco 1,2,3 , Daniel J. Rigden 1 , Francislete R. Melo 1,3 and Maria F. Grossi-de-Sa  1 1 Centro Nacional de Recursos Gene  ticos e Biotecnologia, Cenargen/Embrapa, Brasõ  lia-DF, Brazil; 2 Universidade Cato  lica de Brasõ  lia, Brasõ  lia-DF, Brazil; 3 Depto. de Biologia Celular, Brasõ  lia-DF, Brazil Insect pests a nd patho gens (fungi, bacteria and viruses) are responsible for severe crop losses. Insects f eed directly on the plant tissues, while the pathogens lead to damage or death of the plant. Plants have evolved a certain degree of resistance through the production of defence compounds, which may be aproteic, e.g. antibiotics, alkaloids, terpenes, cyanogenic glucosides or proteic, e.g. chitinases, b-1,3-glu- canases, lectins, arcelins, vicilins, systemins and enzyme inhibitors. The enzyme inhibitors impede digestion t hrough their action o n insect gut digestive a-amylases and pro- teinases, which play a key role in the digestion of plant starch and proteins. The natural defences of crop plants may be i mproved through the use o f transgenic t echnology. Current research in the area focuses particularly on weevils as these are highly dependent on starch for their energy supply. Six dierent a-amylase inhibitor classes, lectin-like, knottin-like, cereal-type, Kunitz-like, c-purothionin-like and thaumatin-like could be used in pest control. These classes of inhibitors show remarkable structural variety leading to dierent modes of inhibition and dierent speci®city pro®les against diverse a-amylases. Speci®city of inhibition is an important issue as the introduced inhibitor must not adversely aect t he plant's own a-amylases, nor the nutritional value of the crop. Of particular interest are some bifunctional inhibitors with additional favourable properties, such as proteinase inhibitory activity or chitin- ase activity. The area has bene®ted from the recent deter- mination of many structures of a-amylases, inhibitors and complexes. These structures highlight the remarkable variety in structural modes of a-amylase inhibition. The continuing discovery of new classes of a-amylase inhibitor ensures that e xciting discoveries remain to be made. In t his review, w e summarize existing knowledge of insect a-am- ylases, plant a-amylase inhibitors and their interaction. Positive results recently obtained for transgenic plants and future prospects in the area are reviewed. Keywords: a-amylase inhibitors; knottin-like; lectin-like; thaumatin-like; Kunitz; cereal-type; bean weevils; bifunc- tional in hibitors. Insect pests and pathogens such as fungi, bacteria and viruses are together, responsible for severe crop losses. Worldwide, losses in agricultural production due to pest attack are around 37%, with small-scale farmers hardest hit [1]. Starchy leguminous seeds are an important staple food and a source of dietary p rotein in many countries. These seeds a re rich in protein, carbohydrate and lipid and therefore suffer extensive predation by bruchids (weevils) and other pests. The l arvae of the weevil b urrow into the seedpods and seeds and the insects usually continue to multiply during seed storage. The damage causes extensive losses, especially if the seeds are stored for long periods. In general, plants contain a certain degree of resistance against insect predation, which is re¯ected in the limited number o f insects capable of feeding on a given plant. This resistance is the result of a set of defenc e mechanisms acquired by plants during evolution [2]. It is only recently that many secondary chemical compounds have been de®nitively associate d with plant d efence, fo r e xample through their synthesis in response t o pest or pathogen attack. Plant defences are, however, incomplete, as bruchids and o ther insects are able to infest seeds and different plant tissues despite the presence of plant defence compounds. Two f actors seem to have contributed to this phenomenon. First, many plants suffer reductions in defence compounds during domestication [3]. Thus, the selection of better- tasting plants w ith better nutritional value has led, c oncom- itantly, to crops that are more susceptible to predation. Secondly, just as plants evolve defences, t heir predators evolve means to evade those defence mechanisms; t his is the Correspondence to O. L. Franco, Centro Nacional de Recursos Gene  ticos e Biotecnologia, Cenargen/Embrapa, S.A.I.N. Parque Rural, Final W5, Asa Norte, Biotecnologia, Laboratory 05, CEP: 70770±900, Brasõ  lia-DF, Brazil, Fax: + 5 5 6 1 340 3624, Tel.: + 55 61 448 4705, E-m ail: ocfranco@cenargen.embrapa.br Abbreviations: AAI, Amaranthus a-amylase inhibitor; a-AI1 and a-AI2, a-amylase inhibitors 1 and 2 from the common bean; AMY1 and AMY2, a-amylases from barley seeds; BASI, barley a-amylase subtilisin inhibitor; BLA, Bacillus licheniformis a-amylase; CAI, cowpea a-amylase inhibitor; CHFI, corn Hageman factor inhibitor; HSA, human salivary a-amylase; LCAI, Lachrima jobi chitinase/ a-amylase inhibitor; PAI, pigeonpea a-amylase inhibitor; PPA, por- cine pancreatic a-amylase; RASI, rice a-amylase/subtilisin inhibitor; RBI, ragi bifunctional inhibitor; SIa1, SIa2 and SIa3, Sorghum a-amylase inhibitors 1±3; TASI, triticale a-amylase/subtilisin inhibi- tor; TMA, Tenebrio molitor a-amylase; WASI, wheat a-amylase subtilisin inhibitor; ZSA, Zabrotes subfasciatus a-amylase. (Received 28 A ugust 2001, accepted 6 N ovember 2001) Eur. J. Biochem. 269, 397±412 (2002) Ó FEBS 2002 phenomenon of host-parasite coevolution, as described by Ehrlich & Raven [4]. Among thes e means are detoxi®cation or excretion of the defence compound, or simple adaptation of the predator so that the toxin no longer has a ny effect. The relationship between leguminous s eed plants a nd seed weevils provides an excellent example of host-parasite coevolution. The seeds are rich in defence compounds so that the majority of possible predators cannot eat them, yet seed weevils thrive on the same see ds. Plant defence compounds include a ntibiotics, alkaloids, terpenes, cyanogenic glucosides and some proteins. Among these proteins are chitinase and b-1,3glucanase enzymes, lectins, arcelins, vicilins, systemins and enzyme inhibitors [5±8]. The enzyme inhibitors act on key insect gut digestive hydrolases, the a-amylases and proteinases. Several k inds of a-amylase and proteinase inhibitors, present in seeds and vegetative organs, act to regulate numbers of phytophagous insects [ 9±11]. a-Amylase inhibitors are a ttractive candidates for the control of seed w eevils as these insects are highly dependent on starch as an energy source. The u se of a-amylase inhibitors, t hrough plant genetic engineering, for weevil control w ill be the focus of this review. The properties of insect a-amylases and available inhibitors will be reviewed and issues affec ting their speci®city of interaction addressed. INSECT a -AMYLASES a-A mylases ( a-1,4-glucan-4-glucanohydrolases, E C 3.2.1.1) constitute a family of endo-amylases that catalyse the hydrolysis of a- D -(1 ® 4)-glucan linkages in starch c ompo- nents, glycogen and o ther carbohydrates. The enzyme plays a key role in carbohydrate metabolism of microorganisms, plants and animals. Moreover, several insects, especially those similar t o the seed wee vils that feed on starchy seeds during larval and/or adult stages, depend on their a-amylases for survival. Research on starch digestion as a target for control of starch-dependent insects was stimulated in recent years after results showed that a-amylase inhibitors from Ph aseolus v ulgaris seeds are detrimental to the development of cowpea weevil Callosobruchus maculatus and Azuki bean weevil Callosobruchus chinensis [12,13]. The c arbohydrate digestion of bruchid w eevils, such as the Mexican bean weevil Zabrotes subfasciatus and the cowpea w eevil C. maculatus, occurs mainly in the lumen of the midgut. High enzymatic a ctivities against starch, maltose, maltodextrins and galactosyl oligosaccharides were found in the luminal ¯uid, while only aminopeptidase activity was predominantly associated with gut membrane [14]. In the yellow mealworm Tenebrio molitor,the a-amylases are synthesized in anterior midgut cells and packed in the Golgi area into secretory vesicles that undergo fusion, as they migrate to the cell apex. At the same time, t he cell apex undergoes s tructural disorganization with t he disappearance of cell organelles. Eventually, t he apical cytoplasm with the large amylase-containing membranous structure is d ischarged into the midgut lumen. After extruding the apical c ytoplasm, the cell a pparently remains functional, as cells are f ound to lack the cell apex, but have all the other normal ultra structural features [15]. To validate insect a-amylasesastargetsforcropprotec- tion, it is important to research their variety and understand how the expression o f different forms i s controlled. Studies in this area are at an early stage, although s ome important observations have been made. The presence of different forms of a-amylases in th e insect midgut lumen h as been observed in C. maculatus and Z. subfasciatus [14,16]. Pat- terns of a-amylase expression vary in Z. subfasciatus fed o n different diets, apparently in response to the presence of antimetabolic proteins such as a-amylase inhibitors, rather than as a response to structural differences in the starch granules. Bean bruchids, such as the Mexican bean weevil larvae, also have the ability to modulate t he concentration of a-glucosidases and a-amylases when reared on different diets [14]. Although the sequences of several i nsect a-amylases are known [17,18], the only three-dimensional insect a-amylase yet d etermined is that of the T. molitor enzyme (TMA). This enzyme is well adapted t o the slightly acidic physiological environment of the larval midgut with a pH optimum of 5.8 for the cleavage of starch [19]. The structure of TMA comprises a single polypeptide chain of 471 amino-acid residues, one calcium ion, one chloride ion and 261 water molecules (Fig. 1 [20]). The protein folds i nto t hree distinc t domains, named A, B a nd C (Fig. 1). Domain A, the major structural unit of TMA is composed of two segments (residues 1±97 and 160±379; green in Fig. 1) and forms a (b/a) 8 -barrel; an eight-stranded, parallel b-barrel embraced by a c oncentric circle of e ight helical segments (seven a helices and one 3 10 -helix). This domain contains the Fig. 1. Ribbon thr ee-dimensional structure o f Tenebrio molitor a-amy- lase (PDB code 1tmq). ThedomainA,BandCarecolouredingreen, red and o r ange, r espec tively. Th e s tructure c ont ains on e c alcium ion (yellow) and one chloride ion (cyan). The ®gure was made using MOLSCRIPT 2 [148] as were all other ®gures ex cept Fig. 2A. 398 O. L. Franco et al. (Eur. J. Biochem. 269) Ó FEBS 2002 catalytic site and the ligand b inding residues [20,21]. Domain B is globular and is inserted into domain A. It is formed by several extended segments and a short a helix (residue 98±159; red i n Fig. 1). This domain forms a c avity against the b barrel of domain A in which the calcium ion is bound. This cation is of fundamental importance for the structural integrity o f a-amylases [20,22]. Finally, domain C is located exactly opposite t o domain B on the o ther side of domain A. The C domain comprises the C-terminal residues 380±471 (orange in Fig. 1) and forms a separated folding unit, exclusively made of b sheet. Eight of the 10 strands fold into a b sandwich structure with ÔGreek keyÕ topology. The conservation of the interface of A and C domains among a-amylases from different sources suggests an important role for e nzyme activity, stability and folding [ 20]. In the porcine pancreatic a-amylase (PPA), the interface between C and A domains contains a secondary starch-binding site, occupied by maltose in one crystal structure [23], but it remains to be seen if this is also the case for TMA. TMA, in co mmon with almost all determined a-amylase structures (the exception being that of calcium-deplete d Bacillus licheniformis a-amylase (BLA) [ 21], contains a calcium ion at a c onserved position (yellow in Fig. 1). The removal of the calcium ion in BLA causes local disorder around the Ca 2+ -binding s ite, resulting in an inactive enzyme [24]. The calcium-binding site of TMA is l ocated at the interface between domains A and B ( Fig. 1), near to the catalytic centre. The Ca 2+ ion is important for activity due its contact with His189. This histidine r esidue interacts with the fourth sugar o f the substrate, bound in the active site, forming a hinge between the catalytic-site and the Ca 2+ -binding site [20]. TMA crystal structures also contain a chloride anion (cyan in Fig. 1). The chloride m ay be capable of allosterically activating T MA [19] due to its proximity to a water molecule, which probably initiates substrate cleavage [25]. The nucleophilicity of this water molecule might be enhanced by the negative charge of the chloride anion. The enzymatic mechanism of a-amylases h as not yet been completely elucidated. It is likely that d ifferent a-amylases have a similar mechanism of action with catalytic residues conserved among all the enzymes [26,27]. Three acidic side chains in PPA (Asp197, Glu233 and Asp300) (Fig. 2B), corresponding to Asp185, Glu222 and Asp287 in TMA [20] are direc tly involved in catalysis [28]. The polysacchari de-binding groove of a-amylases can accommodate at least six sugar units, as observed crystallographically in PPA [28], and cleavage occurs between the third and the fourth pyranose residues. The reaction is believed to proceed by a double displacement mechanism [27]. a -AMYLASE INHIBITORS Nonproteinaceous inhibitors The class of non proteinaceous inhibitors contains diverse types of organic compounds s uch a s a carbose, isoacarbose, acarviosine-glucose, hibiscus acid and the cyclo dextrins (Fig. 2 A). T he two h ibiscus a cid forms, puri®ed from Roselle tea (Hibiscus sabdaria), the acarviosine-glucose, the isoacarbose and a-, b-andc-cyclodextrins are highly active against porcine and human pancreatic a-amylase (PPA and HPA) [29±32]. The inhibitory activity of these compounds against a-amylase s i s due in part to their cyclic structures, which resemble a-amylase substrates and there- fore bind to a-a mylase catalytic sites. In previous X-ray crystallography studies [33], t hree a-cyclodextrins molecules I-III bound to PPA. a-Cyclodextrin I and II bound near to the catalytic binding cleft, while a-cyclodextrin III biound at Fig. 2. The structures of nonproteinaceous a-amylases and acarbose green bound to the catalytic site of PPA. (A) Nonproteinace ous a-amylase inhibitors. (B ) T he structure of ac arbose g reen bound to t he catalytic site of PPA. Only enzym e r esid ues m akin g h ydrogen bonds (dashed line s) or hydrophobic contacts with the i nhibitor are shown. The t hree catalytic acidic r esidu es are labeled. Ó FEBS 2002 Plant a-amylase inhibitors (Eur. J. Biochem. 269) 399 an accessory site. The a-andb-cyclodextrin are not hydrolyzed to any signi®cant extent b y a-amylases, except by fungi a mylases [34,35]. In contrast, h uman saliva a-amylase (HSA) and PPA are capable of hydrolysing c-cyclodextrin [36]. The cyclodextrin mechanism of PPA inhibition is pH-, temperature- and substrate-dependent. When amylose is used as substrate, the inhibition is of the competitive type, bu t when m altopentose is u sed, the inhibition becomes noncompetitive [37]. PPA inhibition by acarbose, in contrast, is noncompetitive, irrespective of substrate [ 38]. The s tructure of PPA wit h acarbose, a pseudotetrasaccharide (Fig. 2B), bound to the active site has been determined [39]. Two linked identical acarbose fragments occupy the PPA active site (coloured green in Fig. 2B), hindering substrate hydrolysis. These acarbose fragments are bonded to residues from the a-amylase active site by a hydrogen-bonding network (Fig. 2B). No dis- placement o f a carbose-binding residues is r equired f or acarbose binding, compared to their positions in the empty enzyme structure, enhancing the effectiveness of t he inhibi- tion [39]. The valienamine ring (Fig. 2A) of acarbose is considered to be crucial in the inhibition mechanism of a-glucosidases, a-amylases and other amylolytic enzymes [40,41]. Its unsaturated structure and half-chair conforma- tion are r eminiscent of a planar oxocarbonium ion, proposed as either a transition state or an intermediate during the hydrolytic pathway of g lucosidases [42]. The properties of nonproteinaceous inhibitors make them interesting in the ®eld of medicine, both f or treatment [43] and in diagnostic procedures [44]. Nevertheless, the use of nonproteinaceous inhibitors for production of insect resis- tant transgenic plants is much more dif®cult. The produc- tion of acarbose o r organic acids i n plants is very complex and several metabolic pathways are involved. Hence, the presence of multiple expressed transgenes would be r equired in order to confer protection. In this area, the proteinaceous inhibitors, coded by a single gene, are more suitable. Proteinaceous inhibitors Proteinaceous a-amylase inhibitors are found in microor- ganisms, plants and animals [5,45±47]. In plants, proteina- ceous inhibitors are m ainly p resent in cereals such as wheat Triticum aestivum [46,48,49], barley Hordeum vulgareum [50], s orghum So rghum b icolor [51], rye Secale cereale [47,52] and rice Oryza sativa [53] but also in leguminosae such as pigeonpea Cajanus cajan [54], cowpea Vigna ungu iculata [55] and bean P. vulgaris [56,57]. These inhibitors have showed monomeric molecular m asses of 5 kDa [51], 9 kDa [55] and 13 kDa [49], homodimeric and heterodimeric masses of  26 kDa [49,57] and tetrameric masses of 50 kDa [58]. Different plant a-amylase inhibitors exhibit different spec- i®cities against a-amylases from diverse sources (Table 1). Determination of speci®city o f inhibition is the important ®rst step towards the discovery of an inhibitor that could be useful for generating insect-resistant transgenic plants. In some cases, the a-amylase inhibitors act only against mammalian a-amylases or, on the contrary, just against insect a-amylases. In the latter case, this provides a highly speci®c potential weapon in plant defence. a-AI2, AAI and some wheat inhibitors are among those naturally possessing favourable in hibition pro®les (Table 1). However, in gen- eral, a-amylase inhibitors inhibit several a-amylases from different sources. I n these cases, an improved understanding of the structural b ases for inhibition pro®les (as discussed later) may e nable the rational design of mutants with more desirable characteristics. A s proposed by Richardson [59], a-amylase inhibitors may b e conveniently classi®ed by their tertiary structure (Table 2) into six classes: lectin-like, knottin-like, cereal-type, Kunitz-like, c-purothionin-like and thaumatin-like. Lectin like a-amylase inhibitors a-AIs has been puri®ed and characterized from different accessions and varieties of the common bean P. vulgaris, including the white, red and black kidney beans [58,60± 62]. The best-characterized isoform, known as a-AI1, was cloned and identi®ed as an a-amylase inhibitor homolo- gous to phytohemagglutinin (PHA) [63]. A second variant of a-AI, called a-AI2, is f ound in some wild accessions of the common bean [56]. These two allelic variants have different inhibition speci®cities. a-AI1 inhibits PPA a s well as the a-amylases of the C. maculatus and C. chinensis, but it does not inhibit the a-amylases of the Z. subfas- ciatus (ZSA). In contrast, a-AI2 does no t inhibit the ®rst three amylases mentioned above but it does inhibit ZSA [56]. To reach their active mature form, comprising t wo noncovalently bound glycopeptide subunits, a and b,of7.8 and 14 kDa, respectively [57,64], bean a-AIs are post- translationally modi®ed. The proteolysis leading to the activation o f a-AI1 has been studied by mass spectrometr y. A simple cleavage at the car boxyl side of Asn77, presum- ably by an Asn-speci®c seed protease of previously demonstrated importance in legume lectin processing [65], is made and Asn79 is removed apparently by the action of a carboxypeptidase. Furthermore, 19 residues at the C-terminus of the b chain of a-AI1 are clipped. a-AI2 shows s imilar c leavages to a-AI1, but a s omewhat d ifferent glycosylation pattern [57]. Both variant inhibitors in their mature form have a h eterotetrameric structure of two a chains and two b chains [58,66] and are highly glycosy- lated [57]. a-AIs contain glycans attached to Asn63 and Asn67 [57] but these m ay not be necessary for inhibitory activity [67]. A third isoform, a-AIL (also known as a-AI3), isolated from P. vulgaris cv Rico 23 is a single-chain a-a mylase inhibitor-like protein completely inactive towards all a-am- ylases tested [68]. This protein, as with the insecticidal isoform Grp29 [69], may represent an evolutionary inter- mediate between phytohaemagglutinnins, arcelins and a-amylase inhibitors [68,157,158]. Interestingly, another noncleaved member o f this i nhibitor group speci ®cally inhibits fungal a-amylases [70] and additionally possesses hemagglutination a ctivity, showing that these two activities are not mutually exclusive and that cleavage probably is not a prerequisite for a-amylase inhibition. The formation of the inhibitor±enzyme complex for this class of a-amylase inhibitors is pH-, time- a nd concentra- tion-dependent [56,62]. One heterotetramer of a-AI1 binds to and inhibits two molecules of PPA with K D  10 )10 M [58]. To e lucidate the i nhibitory mechanism o f these inhibitors, the structures of the common bean a-AI1 in complex with PPA [71] and TMA [72] have been deter- mined. Structural analysis demonstrated that two h airpin 400 O. L. Franco et al. (Eur. J. Biochem. 269) Ó FEBS 2002 loops of a-AI1 (residues 29±46 and 171±189) were inserted into the TMA reactive site (Fig. 3A), blocking substrate binding and establishing a hydrogen bond network with the residues of the substrate-binding region. The catalytic residues are str ongly bonded to the inhibitor residues Tyr186 and Tyr37 that occupies the catalytic pocket. When compared to results obtained in the PPA±a-AI1 complex, the s trong contacts in the c atalytic clefts are highly conserved and only slight modi®cations occur in the extended protein±protein interaction [71,72]. Bean amylase inhibitors have been extensively used in transgenic p lants due to their insecticidal properties. Kidney bean crude extracts containing lectin-like a-amylase inhibitors originally found in vivo,wereusedas starch blockers in the early 1980 s, for the control of human noninsulin-dependent diabetes mellitus and obesity [43,44,73,74]. Those early attempts were unsuccessful due to the undes irable presence of P HAs and proteinase inhibitors in the extract. Later work with puri®ed a-AI in diabetic patients met with more success [73]. More Table 1. Activity of amylase inhibitors from dierent plant sources aga inst mammalian and insect a-amylases. Low activity represents less than 4 0% of the maximum activity. Inhibitors Source Inhibitory activities References Mammalian Insect a-AI1 P. vulgaris PPA Callosobruchus maculatus Callosobruchus chinensis Diabrotica virgifera virgifera Hypothenemus hampei Tenebrio molitor [12,18,31,149] a-AI2 P. vulgaris None activity Zabrotes subfasciatus [56,150] Wheat T. aestivum PPA and HSA Diabrotica virgifera virgifera [49,151] Extract Lygus hesperus Lygus lineoralis 0.19 T. aestivum PPA and HSA Diabrotica virgifera virgifera Callosobruchus maculatus Zabrotes subfasciatus Acanthoscelides obtectus Tenebrio molitor Sitophilus oryzae Tribolium castaneum [18,46,49,97] 0.53 T. aestivum HSA and PPA (low) Tenebrio molitor Callosobruchus maculatus Zabrotes subfasciatus Acanthoscelides obtectus [46,90,97] 0.28 T. aestivum PPA and HSA Tenebrio molitor [97] WRP25 T. aestivum None Sitophilus oryzae Tribolium castaneum Tenebrio molitor Callosobruchus maculatus Zabrotes subfasciatus [46,49] WRP26 T. aestivum None Tenebrio molitor Sitophilus oryzae Tribolium castaneum Callosobruchus maculatus [46,49] WRP27 T. aestivum None Tenebrio molitor (low) Sitophilus oryzae [49] 1,2 and 3 S. cereale HSA Tenebrio molitor [52] BIII S. cereale HSA and PPA Zabrotes subfasciatus Acanthoscelides obtectus [47] AAI A. hypochondriacus None activity Tenebrio molitor Hypothenemus hampei Prostephanus truncatus [77,82,149,152] CAI V.unguiculata None Callosobruchus maculatus (low) [55] PAI C. cajan HSA and PPA Helicoverpa armigera (low) [54] Zeamatin Z mays None activity Tribolium castaneum Sitophilus zeamais Rhyzoperta dominica [112,113] SIa1, SIa2 S. bicolor HSA (low) Locusta migratoria [51] and SIa3 Periplaneta americana Ó FEBS 2002 Plant a-amylase inhibitors (Eur. J. Biochem. 269) 401 recently this c lass of inhibitors has b een us ed for i ts insecticidal prope rties t o p rotect seeds for insect predation [13,75,76]. Knottin-type a-amylase inhibitors The a-amylase inhibitor from Amaranthus hypocondriacus seeds (AAI) is the smallest proteinaceous inhibitor of a-amylases yet described, with just 32 residues and three disul®de b onds [77]. The structure o f its inhibitor, as determined by NMR [78,79], contains a knottin fold; three antiparallel b strands and a characteristic disul®de to pology. It revealed structural similarity to other proteins such a s the proteinase inhibitor from Cucubirta maxima [80], charybdo- toxin and conotoxins [81]. AAI s peci®cally inhibits insect a-amylases and is inactive against mammalian a-amylases ([77]; Table 1). The struc- ture of its inhibitor in complex revealed that inhibition, as with the lectin-like inhibitors, is through blockage of the catalytic site ([82], Fig. 3B). The inhibitor binds in the active site crevice interacting with catalytic residues f rom the A and B domains of TMA (Fig. 1 [ 82]). The residue Asp287, one of the catalytic residues of its enzyme, forms a salt bridge directly with Arg7 of AAI. The other two enzymatic catalytic residues, as well other c onserved residues involved in substrate recognition and orientation, are connected to AAI via an intricate water-mediated hydrogen-bonding network [82]. The TMA±AAI complex is characterized by a high complementarity of the interaction surfaces (Fig. 3B). Structural comparisons of the inhibitor structure in solution [79] to the X-ray structure of A AI bounded to TMA [82] demonstrate that both backbone and side conformations are only slightly adjusted on formation of the complex [79]. The speci®c activity of AAI against insect a-a mylases makes it an attractive candidate for the development of insect- resistant transgenic plants. Cereal-type a-amylase inhibitors a-Amylase inhibitors of the cereal family are composed of 120±160 amino-acid residues forming ®ve disul®de bonds (Table 2) [46,83,84]. These inhibitors are also known as sensitizing agents in humans upon repeated exposure, causing allergy, dermatitis and baker's asthma assoc iated with cereal ¯our [85,86]. N-glycosylation is involved in the reactivity of the most reactive allergen, an inhibitor from rye [87]. The e xogenous wheat a-amylase inhibitor coded 0.19 [46,49] and the bifunctional inhibitor from Indian ®nger millet R BI [88,89], are the most studied inhibitors from this family. T he a-amylase inhibitor 0.19, named according to its gel electrophoretic mobility relative to b romophenol blue, inhibits a-amylase s f rom birds, Bacilli, insects and mammals (Table 1). Its inhibition of human salivary a-amylase is characterized by K i  0.29 n M [160]. It has 124 amino-acid residues and is homologous to RBI [ 90]. Mass spectrometry results [46] clearly demonstrated the presence of homodi- mers of 0.19 with smaller quantities of other multimers, in accord with sedimentation [49] and X-ray crystallography results [91]. Nevertheless, some cereal inhibitors act as monomers, such the wheat inhibitors 0.28, W RP25, WRP26 and W RP27 [46]. T he dimeric a-amylase inhibitor 0.19 was crystallized [92] and its structure d etermined. It con tains ®ve Table 2. Dierent structural c lasses of a-amylase inhibitors, based on a c lassi®cation by Richardson [59]. Structural class Source and references Residue numbers Disul®de bonds CATH b code and Family SCOP fold Names Legume lectin type Common beans [31,56] 240±250 5 2.60.120.60 c Lectin Concanavalin A-like lectins/glucanases a-A11 & a-A12 Knottin type Amaranth [78,79] 32 3 ND a Knottins AA1 Cereal type Wheat [46], barley [59] & Indian ®nger millet [153] 124±160 5 1.10.120.10 Cereal inhibitor Bifunctional inhibitor/ lipid-transfer protein/ seed storage 2S albumin 0.19, 0.53, 0.28 WRP25, WRP26, WRP27 & RBI Kunitz type Barley [102], wheat [99] & rice [100] 176±181 1±2 2.80.10.50.6 Proteinase inhibitor b-Trefoil BASI, WASI & RASI Thaumatin type Maize [112,113,156] 173±235 5±8 2.60.110.10 Sweet tasting protein Osmotin Thaumatin-like proteins Zeamatin c-Purothionin type Sorghum [51] 47±48 5 3.30.30.10 Antibacterial protein Knottins SIa1, SIa2&SIa3 a This inhibitor class was not classi®ed by CATH program. b Orengo et al. [154]. c 1, 2, and 3 from CATH code represents mainly a helix, mainly b sheets and mixed a helix and b sheets, respectively. 402 O. L. Franco et al. (Eur. J. Biochem. 269) Ó FEBS 2002 a-helices arranged in an up-and-down manner, satisfying favorable packing modes, with al l 10 cysteine residues forming disul®de bonds [91]. The bifunctional a-amylase/trypsin inhibitor (RBI) is another prototype of the cereal i nhibitor f amily. T his inhibitor is a stable monomer of 122 amino acids with ®ve disul®de bonds, which is resistant to urea, guanidine hydrochloride and thermal denaturation [93]. This bifunc- tional inhibitor p resents a three-dimensional structure very similar t o that of 0.19 inhibitor [91], with a globular fold with four a helices in a simple Ôup-and-downÕ topology and a small antiparallel b sheet ([94]; Fig. 3C). Like other inhibitors of this class, it can competitively inhibit a variety of a-amylases, including PPA and TMA. This latter enzyme is inhibited with a K i  15  2 n M [88,89,95]. As the structure of RBI±TMA complex reveals, the inhibitor binds to the enzyme active site, once again impeding substrate binding (Fig. 3 C). Two RBI segments are responsible for the interaction with TMA. Segment 1, comprising the N-terminal residues Ser1±Ala11 and the residues Pro52±Cys55, protrudes like an a rrow head i nto TMA's substrate-binding groove and directly targets the active site of the enzyme. The N-terminus forms the arrow tip, adopting a 3 10 -helical conformation in complex. The Ser1 residue makes several hydrogen bonds with the catalytic Asp185 and Glu222 from enzyme while Val2 and S er5 from i nhibitor i nteract with the third conserved acidic residue of the catalytic site, Asp287. The second binding segment comprises several r esidues, which form a collar around the upper part of the arrow head an d stabilize Fig. 3. The various known modes o f a-amylase inhibition. A s tandard c olou ring sc he me is used with enzyme d rawn i n m age nta and inhibitor helix, strand and coil drawn in red, cyan and yellow, respectively. The calcium ion common to all structures is drawn i n orange. The structures are (A) TMA bound to a-AI1 (PDB code 1vi w) (B) TMA-AAI (1clv) (C) TMA-RBI ( 1tmq) and (D) AMY2-BASI ( 1ava). In (D) the calcium ion bound at the enzyme±inhibitor i nterface (see text for details) is d rawn in green. Ó FEBS 2002 Plant a-amylase inhibitors (Eur. J. Biochem. 269) 403 the complex by further interactions with this enzyme [88]. Tests with a peptide containing the N-terminal 10 residues of RBI showed that only segment 1 is necessary for a-amylase inhibition. Synthetic peptides containing muta- ted N -terminal R BI seq uences demonstrated different inhibitory potentials [89]. Alam et al.[89]alsoshowedthat this inhibitor binds to soluble amylase substrate, reducing the apparent af®nity of the enzyme f or the s ubstrate. Inhibitor±substrate interactions could explain the differ- ences in the type o f inhibition observed for d ifferent substrates with the same enzyme [37,89]. E xploiting the overall structural s imilarity between RBI and 0.19, molec- ular models of 0.19±TMA and 0.19±HSA complexes have been constructed [46]. Multiple genes encode the members of the cereal-type inhibitor f amily [96]. Their different sequences yield a remarkable array of inhibition speci®city p ro®les (Table 1 [46,52]). In wheat, some a-amylases i nhibitors genes may be silent or expressed at a much lower level [96]. It can be envisaged that the lack of the pertin ent predator in the e cological niche could silent th e respective inhibitor gene [97]. Kunitz-like a-amylase inhibitors The Kunitz-like a-amylase inhibitors contain around 180 residues and four cystines (Table 2). They are present in cereals such as barley [98], wheat [99] and rice [100]. The best-characterized a-amylase inhibitor from the Kunitz class is the barley a-amylase/subtilisin inhibitor (BASI), a bifunctional double-headed inhibitor with a fast tight inhibitory reaction with cereal a-amylase AMY2 (K i  0.2 2 n M ) and serine prote inases of the subtilisin family [50 ,101]. The structure of BASI [102] r evealed two disul®de bonds and a b trefoil topology (Fig. 3D) shared with the homologous wheat a-amylase subtilisin inhibitor (WASI [103]), the Erythrina cara trypsin inhibitor [104] andthericinBchain[105]. In the cases of a-AI1, AAI and RBI, inhibition involves the i nsertion of inhibitor l oops into the a-amylase active site, thereby establishing a network of hydrogen bonds with catalytic and substrate-binding residues (Fig. 3A±C). The mechanism of i nhibition of barley a-amylase 2 ( AMY2) by BASI [102] is different, i n t hat the inhibitor does not interact directly with any catalytic acidic residues of the enzyme. Nevertheless, this inhibitor interacts strongly with both the A and B domains near the catalytic site, through the formation of 12 hydrogen bonds, two salt bridges and multiple van der Waal's contacts, and thereby prevents substrate access (Fig. 3D). A c avity at the enzyme±inhibitor interface contains a trapped calcium ion whose presence is suggested to electrostatically enhance the network of water molecules a t t he complex interface and thereby raises the stability of the complex. BASI is involved in regulating the degradation of seed carbohydrate, preventing the e ndogenous a-amyla se 2 from hydrolysing starch during premature sprouting [41]. Ad di- tionally, it p rotects t he seeds against exogenous proteinases and a-amylases produced by various pathogens a nd pests [106]. BASI i nhibits t he endogenou s enzyme w ith a stoichi- ometry of 1 : 1 [107], but, interestingly, is unable to inhibit barley a-amylase 1 (AMY1), which bears 74% sequence identity to AMY2 [101]. Thaumatin-like a-amylase inhibitors type The t haumatin-like inhibitors are proteins with molecular masses of  22 kDa with signi®cant sequence similarity to pathogenesis-related group 5 (PR-5) proteins and to thaum- atin, an intensively sweet protein from Thaumatococcus danielli fruit [108,109]. The best-ch aracterized inhibitor from t his class is zeam- atin, a bifunctional inhibitor from Zea mays that is homologous to the sweet protein t haumatin. Zeamatin has a total of 13 bstrands, 11 of which form a bsandwich at the core of protein ([110]; Fig. 4A). Several loops extend from this inhibitor core and are secured by one or more of the Fig. 4. The structural classes of a-amylase inhibitors whose modes of inhibition are not yet known (A) zeamatin (PDB code 1 dl5) and (B) SIa1 (1gpt). The coordinates of SIa1 have n o t be en deposited in t he PDB so that (B) shows the structure of the homolo gous c-thionin [124]. 404 O. L. Franco et al. (Eur. J. Biochem. 269) Ó FEBS 2002 eight disul®de bon ds. E lectrostatic modelling o f z eamatin reveals an electrostatically polarized surface, heavily popu- lated w ith Arg and L ys residues [110]. This maize inh ibitor is not sweet, despite its similarity to thaumatin, probably d ue to changes in a putative receptor-binding site [111]. Zeam- atin was a ble to inhibit porcine pancreatic trypsin and digestive a-amylases of the insects T. castaneum, Sitophilus zeamays and Rizopherta d ominica [112,113]. Other proteins from this class, such as the thaumatin-like p roteins R and S from barley seeds, did not show any inhibitory activity against trypsin or a-amylases [114] despite their highly similar N-terminal sequences. Zeamatin is mainly known for its antifungal activity, but this is not related to inhibition of hydrolyic enzymes as this protein does not inhibit f ungal a-amylases [112] and fungi do not contain trypsin. Zeamatin binds to b-1,3-glucans [115] and permeabilizes fungal-cells leading to cell d eath [116] but the a ntifungal mode of action of this protein is still a matter o f d ebate. For t hese properties, zeamatin could be used as a medical agent, acting on vaginal murine candidosis cells [117] or in transgenic plants, increasing their resistance against pests and pathogens. c-Purothionins-like a-amylase inhibitors The a-amylase inhibitors of this family have 47 or 48 residues, are sulfur-rich and form part of the c-thionin superfamily (Table 2). Members of this superfamily are involved in plant defence through a remarkable variety of mechanisms: m odi®cation o f m embrane p ermeability [118,119], i nhibition of protein synthesis [120] and protein- ase inhibition [121] Inhibition of insect a-amylases has bee n observed for three isoforms from Sorghum bicolor called SIa-1, S Ia-2 an d S Ia-3 [51]. These molecules strongly inhibited the digestive a-amylases of guts of locust and cockroach, poorly inhibited a-amylases from A. oryzae and human saliva and failed to inhibit the a-amylases from porcine pancreas, barley and Bacillus sp. [51]. T he structure of SIa-1 has been solved by NMR [122] revealing a a + b sandwich structure [123] with a nine-residue helix packed tightly a gainst the sheet (Fig. 4B). T he hel ix is h eld i n place by two disul®de bridges, which link sequential turns of the helix to residues 41 and 43 in the middle of strand b3, the so-called cysteine-stabilized helix (CSH) motif [122]. As expected from sequ ence comparison, the structure is similar to those of w heat c1-purothionin [ 124] and scorpion toxins [122]. BIFUNCTIONAL INHIBITORS As the a bove d iscussion highlights, bifunctional a-amylase/ proteinase inhibitors are relatively c ommon (Table 3). As inhibition of predatory insect digestive proteinases is another attractive route for plant protection, the co mbina- tion of a-amylase and proteinase inhibition is potentially very useful. It is therefore important to know whether simultaneous inhibition of proteinase and a-amylase is possible for these inhibitors. In the c ase of RBI, w ith two ind ependent inhibition sites, formation of a stable a-amylase±RBI±trypsin complex h as been observed [95]. The N-terminal site is responsible for a-amylase inhibition, as previously discussed, while on the opposite side a canonical substrate-like trypsin inhibitor region is present [89,94,125,126]. In this inhibitor, the exposed trypsin-binding loop is located between two a-helices, contains the residues Gly32±Tyr37 and also contains Arg34 that confers trypsin-speci®city. Modelling of the TMA±RBI±trypsin complex (Fig. 5; [88]), con®rms Table 3. Activity o f bifunctional inhibitors from dierent plant sources against m ammalian and insect a-amylases. Low a ctivity represents a pprox- imately 40% of a total activity. Inhibitor Source a-Amylase inhibition References Other activity Insect Fungal Plant Mammalian BASI Barley + ND + ± [50,102] Subtilisin Inhibition RASI Rice + ND + ND [53,100] Subtilisin Inhibition WASI Wheat ND ND + ND [99,103] Subtilisin Inhibition CHIF Maize + ND ND ND [128] Trypsin Inhibition Zeamatin Maize + ± ND Low [112,113] Trypsin Inhibition RBI Finger millet + ND ND + [88] Trypsin Inhibition LCAI Jobi tear's seeds + ND ND ± [132] Chitinase Fig. 5. A model of a ternary complex fo rmed by TMA (mage nt a), RB I (blue) and pan creatic bovine trypsin (green ). Constructed as described in the text. Ó FEBS 2002 Plant a-amylase inhibitors (Eur. J. Biochem. 269) 405 that no steric clashes prevent simultaneous inhibition of both enzymes. The homologous bifunctional corn H ag- eman factor inhibitor (CHIF) inhibits mammalian trypsin, Factor XIIa (Hageman factor) of the co ntact pathway of coagulation as well a-amylases from several insects [127]. Its structure has been solved revealing a similar proteinase inhibitory site to that in RBI [128]. As w ith RBI, a-amylase inhibition requires the N-terminal region [129]. Kunitz-like inhibitors are also bifunctional, this time possessing inhibitory activity against the subtilisin c lass pf proteinases. Among them, the BASI and WASI are the best characterized, and both structures have been determined by X-ray crystallography [102,103]. It has been suggested that WASI has t wo different sites because t he activity against a-amylases is retained after incubation with proteinases K [130]. A complex of WASI±proteinase K showed that a loop containing the Gly66 and Ala67 is crucial to proteinase inhibition [131]. This class o f bifunctional inhibitors inhibits insect and endogenous plant a-amylases but not mamma- lian a-amylases [53,100,101]. They are also inactive against different classes of proteinases such as trypsins and chym- otrypsins [53]. BASI is deposited during grain ®lling and therefore found in the seed prior to AMY2, which is synthesized de novo during germination. This inhibitor has been proposed to control t he activity of AMY2 in the case of premature sprouting or t o act in plant defence [159]. The inhibitor R ASI could also help to regulate seed development by inhibiting a development-speci®c a-amylase [53]. This a-amylase inhibition speci®city is in agreement with a dual role in starch control of the storage tissues at plant developmental stages and as defensive agents in response to pest attack. Zeamatin represents a third bifunctional a-amylase/ proteinase inhibitor [112,113] with a known crystal structure [110]. H owever, t he observation of trypsin inhibition, albeit weak, was only made r ecently [ 113] and it remains to be seen whether or not zeamatin possesses independent sites for proteinase and a-amylase inhibition. In addition to bifunctional a-amylase/proteinase inhibi- tion, a single report has found chitinase activity present in an insect a-amylase inhibitor isolated from Lachrima jobi seeds [132]. Chitinase activity is another recognized plan t defence [7] so that this double activity i s of great potential biotechnological interest. However, further characterization of the inhibitor is clearly required. ISSUES OF a -AMYLASE INHIBITOR SPECIFICITY In order to be of practical use for the production of transgenic plants, a-amylase inhibitors should have appro- priate speci®city pro®les. On the one hand, they should ideally be eff ective against t he full range of potential predatory insects. However, they must not interfere with the action of endogenous a-amylases, which are of demonstrated importance in, for example, germination [41]. They should also lack activity against the mammalian enzymes, although this is in general a lesser issue as cooking would denature any inhibitors before ingestion. These simple considerations, in combination with biochemical data in the literature (Tables 1 and 3), already highlight some inhibitors as more promising candidates than others. For example, the cereal bifunctional a-amylase/subtilisin inhibitors have strong af®nity for plant enzymes [107] deriving from their role in regulation of starch metabolism, and are therefore less favoured. The known a-amylase inhibitors selective for insect enzymes and inactive against mammalian enzymes include WRP25, WRP26 a nd WRP27 from the cereal-type class [46], the Amaranthus a-amylase inhibitor [77] and zeamatin [113]. As well as differences in af®nity for broad groups of a-amylases, some remarkable examples of ®ne speci®city e xist. A mong several interesting speci®city d iffer- ences in the cereal-type inhibitors is the inability of WRP26 to inhibit Z SA, while WRP25, 98% i dentical in sequen ce to WRP26, is an effective inhibitor [46]. Given the remarkable structural and functional variety naturally found among a-amylase inhibitors (Tables 1±3), screening for inhibitors with desirable characteristics is a viable option. An attractive alternative, however, would be the r ational redesign of known i nhibitors in order t o confer upon them the required speci®city pro®le. Although conceivably more rapid than the screening approach, inhibitor redesign clearly requires a full understanding of amylase±inhibitor interaction structural bases. With the availability of an ever-increasing number of crystal struc- tures a number of r ecent studies h ave addressed e xperimen- tally observed issues of amylase-inhibitor speci®city, generally through sequence analysis and modelling, some- times supported by mutagenesis studies [46,62,82,133,134]. Two independent studies [62,133] address the speci®city of a-AI1 for PPA, not inhibiting ZSA and the opposite speci®city of a-AI2 for Z SA over PPA [56]. The reliance on simple counting of hydrogen b onds weakens the conclusions of Le Berre-Anton et al . [135] regarding the a-AI1/a-AI2 comparison but they show that the bulkier, single chain a-AIL is sterically impeded from binding either ZSA or PPA. In another study, analysis of other factors, including electrostatics and hydrophobic interactions, fails to lead to a simple explanation of a-AI1/a-AI2 speci®city and the authors concluded that s peci®city was conferred by multiple factors [133]. In studies aimed at explaining the ability of BASI to inhibit AMY2 but not AMY1, previous indications of the importance of electrostatic interactions [136] have been e xamined through site-directed mu tagenesis [ 134]. Mutations of AMY2 residues known to make electro- static interactions at the interface with this inhibitor, Arg128 and Asp142, were made, weakening the e nzyme± inhibitor interaction and reducing t he effect of charge screening o n the interaction. Remarkably, the introduc- tion of just two AMY2 residues, Arg128 and Pro129, into the A MY1 e nvironment w as enough to enhance BASI sensitivity at least 100-fold. As well as the electrostatic characteristics of the Arg, the conformational properties of t he pro line, which forms a cis peptide in AMY2, are implicated in the AMY1/AMY2 spe ci®city. The lysine, which replaces Pro129 in AMY1 is unlikely to form a cis peptide bond, with consequent changes in the conformation of neighbouring Arg128 and other residues at the interface. The varied inhibition speci®city pro®les of a family of cereal a-amylase inhibitors have been addressed by sequence analysis and model building [46]. In the absence of crystal structures for any of the analysed inhibitors in complex with a-amylase, the complex of T MA with the more distantly related RBI [88] was used as the basis for model construc- 406 O. L. Franco et al. (Eur. J. Biochem. 269) Ó FEBS 2002 [...]... cleavage of inhibitors by insect digestive proteinases [138] may complicate pest control by a-amylase inhibitors and could explain the low in vivo ef®ciency of some inhibitors against insect pests [16] The a-amylase diversity found in a single insect [14] indicates that unless an a-amylase inhibitor has reasonably broad speci®city, being capable of inhibiting all the a-amylases produced by the insect, ... TRANSGENIC PLANTS EXPRESSING a-AMYLASE INHIBITORS The transgenic plant approach provides an attractive alternative to the use of chemical pesticides and insecticides and could contribute to the production of crop varieties that are inherently tolerant/resistant to their major target insect pests Besides the bene®t on agricultural crop production, the use of genes that encode insecticidal proteins in Plant a-amylase. .. cloning of bruchid (Zabrotes subfasciatus) a-amylase cDNA and interactions of the expressed enzyme with bean amylase inhibitors Insect Biochem Mol Biol 27, 271±281 18 Titarenko, E & Chrispeels, M.J (2000) cDNA cloning, biochemical characterization and inhibition by plant inhibitors of the a-amylases of the Western corn rootworm, Diabrotica virgifera virgifera Insect Biochem Mol Biol 30, 979±990 19 Buonocore,... Speci®c inhibition of insect a-amylases: yellow meal worm a-amylase in complex with the Ê Amaranth a-amylase inhibitor at 2.0 A resolution Structure 7, 1079±1088 Buonocore, V., Petrucci, T & Silano, V (1977) Wheat protein inhibitors of a-amylase Phytochemistry 16, 811±820 Lyons, A., Richardson, M., Tatham, A.S & Shewry, P.R (1987) Characterization of homologous inhibitors of trypsin and a-amylase Biochim... speci®city and stability Plant Mol Biol 25, 141±157 MacGregor, E.A., Janececk, S & Svensson, B (2001) Relationship of sequence and structure to speci®city in the a-amylase family of enzymes Biochem Biophys Acta 1546, 1±20  Machius, M., Vertesy, L., Huber, R & Wiegand, G (1996) Carbohydrate and protein-based inhibitors of porcine pancreatic a-amylase: structure analysis and comparison of their binding... structural variety of the a-amylase inhibitors so far characterized is striking, encompassing proteins with mainly alpha, beta and mixed folds The continuing discovery of new a-amylase inhibitors suggests that the list of a-amylase inhibitors is far from complete As has been emphasized, consideration of a-amylase inhibitor speci®city is of prime importance The inhibition spectra of inhibitors so far characterized... evolution in plant chitinases: molecular targets of selection in plant- pathogen co-evolution Proc Natl Acad Sci USA 97, 5322±5327  8 Sales, M.P., Gerhardt, I.R., Grossi-de-Sa, M.F & Xavier-Filho, J (2000) Do legumes storage proteins play a role in defending seeds against bruchids? Plant Physiol 124, 515±522 9 Konarev, A.V (1996) Interaction of insect digestive enzymes with plant protein inhibitors and host-parasite... inhibitor and design of a routine procedure Clin Chem 23, 560±566 45 Silano, V (1987) a-Amylase inhibitors In Enzymes and Their Role in Cereal Technology (Kruger, J & Lineback, D., eds), pp 141±199 American Association of Cereal Chemists, St Paul, MD, USA 46 Franco, O.L., Rigden, D.J., Melo, F.R., Bloch Jr, C., Silva, C.P  & Grossi de Sa, M.F (2000) Activity of wheat a-amylase inhibitors towards bruchid a-amylases. .. Further characterization studies of the alpha-amylase protein inhibitor of gel eletrophoretic mobility 0 19 from the wheat kernel Biochim Biophys Acta 420, 288±297 49 Feng, G.H., Richardson, M., Chen, M.S., Kramer, K.J., Morgan, T.D & Reeck, G.R (1996) a-Amylase inhibitors from wheat: a sequences and patterns of inhibition of insect and human a-amylases Insect Biochem Mol Biol 26, 419±426 50 Abe, J.I.,... Salcedo, G (1994) Rye inhibitors of animal a-amylases shown di€erent speci®cities, aggregative properties and IgE-binding capacities than their homologues from wheat and barley Eur J Biochem 224, 525±531 53 Yamagata, H., Kunimatsu, K., Kamasaka, H., Kuramoto, T & Iwasaki, T (1998) Rice bifunctional a-amylase/ subtilisin inhibitor: characterization, localization, and changes in developing and germinating seeds . REVIEW ARTICLE Plant a-amylase inhibitors and their interaction with insect a-amylases Structure, function and potential for crop protection Octa  vio. Amaranthus a-amylase inhibitor; a-AI1 and a-AI2, a-amylase inhibitors 1 and 2 from the common bean; AMY1 and AMY2, a-amylases from barley seeds; BASI, barley a-amylase subtilisin