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

REVIEW ARTICLE Plant z-amylase inhibitors and their interaction with insect a-amylases Structure, function and potential for crop protection Octavio L.. Parque Rural, Final W5, Asa Nor

REVIEW ARTICLE

Plant z-amylase inhibitors and their interaction with insect a-amylases Structure, function and potential for crop protection

Octavio L Franco’, Daniel J Rigden', Francislete R Melo’? and Maria F Grossi-de-Sá!

‘Centro Nacional de Recursos Genéticos e Biotecnologia, Cenargen|Embrapa, Brasilia-DF, Brazil; 7 Universidade Catélica de Brasilia, Brastlia-DF,, Brazil; 3Depio de Biologia Celular, Brasilia-DF, Brazil

Insect pests and pathogens (fungi, bacteria and viruses) are

responsible for severe crop losses Insects feed 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 through

their action on 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 improved through the use of transgenic technology

Current research in the area focuses particularly on weevils

as these are highly dependent on starch for their energy

supply Six different o-amylase inhibitor classes, lectin-like,

knottin-like, cereal-type, Kunitz-like, y-purothionin-like

and thaumatin-like could be used in pest control These

classes of inhibitors show remarkable structural variety

leading to different modes of inhibition and different

specificity profiles against diverse a-amylases Specificity of inhibition is an important issue as the introduced inhibitor must not adversely affect the plant’s own o-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 benefited from the recent deter- mination of many structures of o-amylases, inhibitors and complexes These structures highlight the remarkable variety in structural modes of o-amylase inhibition The continuing discovery of new classes of a-amylase inhibitor ensures that exciting discoveries remain to be made In this review, we summarize existing knowledge of insect œ-am- ylases, plant o-amylase inhibitors and their interaction Positive results recently obtained for transgenic plants and future prospects in the area are reviewed

Keywords: o-amylase inhibitors; knottin-like; lectin-like;

thaumatin-like; Kunitz; cereal-type; bean weevils; bifunc-

tional inhibitors

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

Correspondence to O L Franco, Centro Nacional de Recursos

Genéticos e Biotecnologia, Cenargen/Embrapa, S.A.I.N Parque

Rural, Final W5, Asa Norte, Biotecnologia, Laboratory 05, CEP:

70770-900, Brasilia-DF, Brazil, Fax: + 55 61 340 3624,

Tel.: + 55 61 448 4705, E-mail: ocfranco@cenargen.embrapa.br

Abbreviations: AAI, Amaranthus o-amylase inhibitor; «-AI1 and

œ-Al2, a-amylase inhibitors | and 2 from the common bean; AMY1

and AMY2, a-amylases from barley seeds; BASI, barley «-amylase

subtilisin inhibitor; BLA, Bacillus licheniformis œamylase; CAI,

cowpea o-amylase inhibitor; CHFI, corn Hageman factor inhibitor;

HSA, human salivary o-amylase; LCAI, Lachrima jobi chitinase/

a-amylase inhibitor; PAI, pigeonpea œ-amylase inhibitor; PPA, por-

cine pancreatic a-amylase; RASI, rice «-amylase/subtilisin inhibitor;

RBI, ragi bifunctional inhibitor; Slal, Sla2 and SIa3, Sorghum

a-amylase inhibitors 1-3; TASI, triticale «-amylase/subtilisin inhibi-

tor; TMA, Tenebrio molitor a-amylase; WASI, wheat œ-amylase

subtilisin inhibitor; ZSA, Zabrotes subfasciatus a-amylase

(Received 28 August 2001, accepted 6 November 2001)

and a source of dietary protein in many countries These seeds are rich in protein, carbohydrate and lipid and therefore suffer extensive predation by bruchids (weevils) and other pests The larvae of the weevil burrow 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 reflected in the limited number of insects capable of feeding on a given plant This resistance is the result of a set of defence mechanisms acquired by plants during evolution [2] It is only recently that many secondary chemical compounds have been definitively associated with plant defence, for example through their synthesis in response to pest or pathogen

attack Plant defences are, however, incomplete, as bruchids

and other insects are able to infest seeds and different plant tissues despite the presence of plant defence compounds Two factors 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 with better nutritional value has led, concom- itantly, to crops that are more susceptible to predation Secondly, just as plants evolve defences, their predators evolve means to evade those defence mechanisms; this is the

phenomenon of host-parasite coevolution, as described by

Ehrlich & Raven [4] Among these means are detoxification

or excretion of the defence compound, or simple adaptation

of the predator so that the toxin no longer has any effect

The relationship between leguminous seed plants and 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 seeds

Plant defence compounds include antibiotics, 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 kinds of

a-amylase and proteinase inhibitors, present in seeds and

vegetative organs, act to regulate numbers of phytophagous

insects [9-11] o-Amylase inhibitors are attractive candidates

for the control of seed weevils as these insects are highly

dependent on starch as an energy source The use of

a-amylase inhibitors, through plant genetic engineering, for

weevil control will be the focus of this review The properties

of insect œ-amylases and available inhibitors will be

reviewed and issues affecting their specificity of interaction

addressed

INSECT o-AMYLASES

a-Amylases (a-1,4-glucan-4-glucanohydrolases, EC 3.2.1.1)

constitute a family of endo-amylases that catalyse the

hydrolysis of a-p-(1 — 4)-glucan linkages in starch compo-

nents, glycogen and other carbohydrates The enzyme plays

a key role in carbohydrate metabolism of microorganisms,

plants and animals Moreover, several insects, especially

those similar to the seed weevils 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 «-amylase inhibitors

from Phaseolus vulgaris seeds are detrimental to the

development of cowpea weevil Callosobruchus maculatus

and Azuki bean weevil Callosobruchus chinensis {12,13}

The carbohydrate digestion of bruchid weevils, such as

the Mexican bean weevil Zabrotes subfasciatus and the

cowpea weevil C maculatus, occurs mainly in the lumen of

the midgut High enzymatic activities against starch,

maltose, maltodextrins and galactosyl oligosaccharides were

found in the luminal fluid, 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, the

cell apex undergoes structural disorganization with the

disappearance of cell organelles Eventually, the apical

cytoplasm with the large amylase-containing membranous

structure is discharged into the midgut lumen After

extruding the apical cytoplasm, the cell apparently remains

functional, as cells are found to lack the cell apex, but have

all the other normal ultra structural features [15]

To validate insect o-amylases as targets for crop protec-

tion, it is important to research their variety and understand

how the expression of different forms is controlled Studies

in this area are at an early stage, although some important observations have been made The presence of different forms of o-amylases in the insect midgut lumen has been observed in C maculatus and Z subfasciatus [14,16] Pat- terns of o-amylase expression vary in Z subfasciatus fed on different diets, apparently in response to the presence of antimetabolic proteins such as o-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 the concentration

of œ-plucosidases and a-amylases when reared on different diets [14]

Although the sequences of several insect œ-amylases are known [17,18], the only three-dimensional insect z-amylase yet determined is that of the 7 molitor enzyme (TMA) This enzyme is well adapted to 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 | [20]) The protein folds into three distinct domains, named A, B and 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/+)s-barrel; an eight-stranded, parallel 6-barrel embraced

by a concentric circle of eight helical segments (seven

ahelices and one 3)o-helix) This domain contains the

Fig 1 Ribbon three-dimensional structure of Tenebrio molitor a-amy- lase (PDB code 1tmq) The domain A, B and C are coloured in green, red and orange, respectively The structure contains one calcium ion (yellow) and one chloride ion (cyan) The figure was made using MOLSCRIPT 2 [148] as were all other figures except Fig 2A.

catalytic site and the ligand binding residues [20,21]

Domain B is globular and is inserted into domain A It is

formed by several extended segments and a short « helix

(residue 98-159; red in Fig 1) This domain forms a cavity

against the B barrel of domain A in which the calcium ion is

bound This cation is of fundamental importance for the

structural integrity of a-amylases [20,22] Finally, domain C

is located exactly opposite to domain B on the other 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 Bsandwich structure with “Greek key’ topology The

conservation of the interface of A and C domains among

a-amylases from different sources suggests an important

role for enzyme activity, stability and folding [20] In the

porcine pancreatic o-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 common with almost all determined o-amylase

structures (the exception being that of calcitum-depleted

Bacillus licheniformis a-amylase (BLA) [21], contains a

calcium ion at a conserved position (yellow in Fig 1) The

removal of the calcium ion in BLA causes local disorder

around the Ca*~ -binding site, resulting in an inactive

enzyme [24] The calcium-binding site of TMA is located at

the interface between domains A and B (Fig 1), near to

the catalytic centre The Ca** ion is important for activity

due its contact with His189 This histidine residue interacts

with the fourth sugar of the substrate, bound in the active

site, forming a hinge between the catalytic-site and the

Ca * -binding site [20] TMA crystal structures also contain

a chloride anion (cyan in Fig 1) The chloride may be

capable of allosterically activating TMA [19] due to its

proximity to a water molecule, which probably initiates

A CH,OH

HO `© ng CH;OH

HOH

CHạ

HO

HO HO ISOACARBOSE

ủ

~Gth _~

“>

HO “OH CH,OH

oe CO;H

O;R

a 2 HIBISCUS ACID (R=H)

HO HO —~=OH

1 HIBISCUS ACID 6-METHYL ESTER (R=CH;)

substrate cleavage [25] The nucleophilicity of this water molecule might be enhanced by the negative charge of the chloride anion

The enzymatic mechanism of o-amylases has not yet been completely elucidated It is likely that different 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 directly involved in catalysis [28] The polysaccharide-binding groove of o-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 nonproteinaceous inhibitors contains diverse types of organic compounds such as acarbose, isoacarbose, acarviosine-glucose, hibiscus acid and the cyclodextrins (Fig 2A) The two hibiscus acid forms, purified from Roselle tea (Hibiscus sabdariffa), the acarviosine-glucose, the isoacarbose and a-, B- and y-cyclodextrins are highly active against porcine and human pancreatic o-amylase (PPA and HPA) [29-32] The inhibitory activity of these compounds against o-amylases is due in part to their cyclic structures, which resemble «-amylase substrates and there- fore bind to a-amylase catalytic sites In previous X-ray crystallography studies [33], three o-cyclodextrins molecules I-III bound to PPA a-Cyclodextrin I and II bound near to the catalytic binding cleft, while «-cyclodextrin HI biound at

Fig 2 The structures of nonproteinaceous ¢-amylases and acarbose green bound to the catalytic site of PPA (A) Nonproteinaceous o-amylase inhibitors (B) The structure of acarbose green bound to the catalytic site of PPA Only enzyme residues making hydrogen bonds (dashed lines) or hydrophobic contacts with the inhibitor are shown The three catalytic acidic residues are labeled.

an accessory site The o- and ÿ-cyclodextin are not

hydrolyzed to any significant extent by a-amylases, except

by fungi amylases [34,35] In contrast, human saliva

a-amylase (HSA) and PPA are capable of hydrolysing

y-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, but when maltopentose is used, the

inhibition becomes noncompetitive [37] PPA inhibition by

acarbose, in contrast, is noncompetitive, irrespective of

substrate [38] The structure of PPA with 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 of acarbose-binding residues is required for

acarbose binding, compared to their positions in the empty

enzyme structure, enhancing the effectiveness of the 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 reminiscent of a planar oxocarbonium ion,

proposed as either a transition state or an intermediate

during the hydrolytic pathway of glucosidases [42] The

properties of nonproteinaceous inhibitors make them

interesting in the field of medicine, both for treatment [43]

and in diagnostic procedures [44] Nevertheless, the use of

nonproteinaceous inhibitors for production of insect resis-

tant transgenic plants is much more difficult The produc-

tion of acarbose or organic acids in plants is very complex

and several metabolic pathways are involved Hence, the

presence of multiple expressed transgenes would be required

in order to confer protection In this area, the proteinaceous

inhibitors, coded by a single gene, are more suitable

Proteinaceous inhibitors

Proteinaceous o-amylase inhibitors are found in microor-

ganisms, plants and animals [5,45—-47] In plants, proteina-

ceous inhibitors are mainly present in cereals such as wheat

Triticum aestivum [46,48,49], barley Hordeum vulgareum

[50], sorghum Sorghum bicolor [51], rye Secale cereale [47,52]

and rice Oryza sativa [53] but also in leguminosae such as

pigeonpea Cajanus cajan [54], cowpea Vigna unguiculata [55]

and bean P vulgaris [56,57] These inhibitors have showed

monomeric molecular masses 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 «-amylase inhibitors exhibit different spec-

ificities against o-amylases from diverse sources (Table 1)

Determination of specificity of inhibition is the important

first step towards the discovery of an inhibitor that could be

useful for generating insect-resistant transgenic plants In

some cases, the o-amylase inhibitors act only against

mammalian o-amylases or, on the contrary, just against

insect a-amylases In the latter case, this provides a highly

specific potential weapon in plant defence o-AI2, AAI and

some wheat inhibitors are among those naturally possessing

favourable inhibition profiles (Table 1) However, in gen-

eral, a-amylase inhibitors inhibit several œ-amylases from

different sources In these cases, an improved understanding

of the structural bases for inhibition profiles (as discussed later) may enable the rational design of mutants with more desirable characteristics As proposed by Richardson [59], œ-amylase inhibitors may be conveniently classified by their tertiary structure (Table 2) into six classes: lectin-like, knottin-like, cereal-type, Kunitz-like, y-purothionin-like and thaumatin-like

Lectin like z-amylase inhibitors a-AlIs has been purified 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 identified as an o-amylase inhibitor homolo- gous to phytohemagglutinin (PHA) [63] A second variant

of a-AI, called a-AI2, is found in some wild accessions of

the common bean [56] These two allelic variants have different inhibition specificities a-AI1 inhibits PPA as well

as the o-amylases of the C maculatus and C chinensis, but it does not inhibit the œ-amylases of the Z subfas- ciatus (ZSA) In contrast, o-AI2 does not inhibit the first three amylases mentioned above but it does inhibit ZSA [56]

To reach their active mature form, comprising two noncovalently bound glycopeptide subunits, « and B, of 7.8 and 14kDa, respectively [57,64], bean o-AIs are post- translationally modified The proteolysis leading to the activation of o-AIl has been studied by mass spectrometry

A simple cleavage at the carboxyl side of Asn77, presum- ably by an Asn-specific 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 Bchain of o-AIl are clipped o-AI2 shows similar cleavages to a-AI1, but a somewhat different glycosylation pattern [57] Both variant inhibitors in their mature form have a heterotetrameric structure of two achains and two Bchains [58,66] and are highly glycosy- lated [57] a-AIs contain glycans attached to Asn63 and Asn67 [57] but these may not be necessary for inhibitory activity [67]

A third isoform, o-AIL (also known as o-AJI3), isolated from P vulgaris cv Rico 23 is a single-chain o-amylase inhibitor-like protein completely inactive towards all o-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 of this inhibitor group specifically inhibits fungal o-amylases [70] and additionally possesses hemagglutination activity, showing that these two activities are not mutually exclusive and that cleavage probably is not

a prerequisite for «-amylase inhibition

The formation of the inhibitor-enzyme complex for this class of a-amylase inhibitors is pH-, time- and concentra- tion-dependent [56,62] One heterotetramer of o-AI1 binds

to and inhibits two molecules of PPA with Kp = 10 '°M [58] To elucidate the inhibitory mechanism of 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 hairpin

Table 1 Activity of amylase inhibitors from different plant sources against mammalian and insect ¢-amylases Low activity represents less than 40% of the maximum activity

Inhibitors Source

Inhibitory activities

a-All

œ-AI2

Wheat

Extract

0.19

0.53

0.28

WRP25

WRP26

WRP27

1,2 and 3

BIII

AAI

CAI

PAI

Zeamatin

SlIal, SIa2

and SIz3

P vulgaris

P vulgaris

T aestivum

T aestivum

T aestivum

T aestivum

T aestivum

T aestivum

T aestivum

S cereale

S cereale

A hypochondriacus

V.unguiculata

C cajan

Z mays

S bicolor

PPA

None activity PPA and HSA

PPA and HSA

HSA and PPA (low)

PPA and HSA

None

None

None

HSA HSA and PPA None activity

None HSA and PPA None activity

HSA (low)

Callosobruchus maculatus Callosobruchus chinensis Diabrotica virgifera virgifera Hypothenemus hampei Tenebrio molitor Zabrotes subfasciatus Diabrotica virgifera virgifera Lygus hesperus

Lygus lineoralis Diabrotica virgifera virgifera Callosobruchus maculatus Zabrotes subfasciatus Acanthoscelides obtectus Tenebrio molitor Sitophilus oryzae Tribolium castaneum Tenebrio molitor Callosobruchus maculatus Zabrotes subfasciatus Acanthoscelides obtectus Tenebrio molitor Sitophilus oryzae Tribolium castaneum Tenebrio molitor Callosobruchus maculatus Zabrotes subfasciatus Tenebrio molitor Sitophilus oryzae Tribolium castaneum Callosobruchus maculatus Tenebrio molitor (low) Sitophilus oryzae Tenebrio molitor Zabrotes subfasciatus Acanthoscelides obtectus Tenebrio molitor Hypothenemus hampei Prostephanus truncatus Callosobruchus maculatus (low) Helicoverpa armigera (low) Tribolium castaneum Sitophilus zeamais Rhyzoperta dominica Locusta migratoria Periplaneta americana

[12,18,31,149]

[56,150] [49,151] [18,46,49,97]

[46,90,97]

[46,49]

[77.82.149.152]

[55]

[54]

[112,113]

[S1]

loops of o-AIl (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 strongly bonded to the inhibitor residues

Tyr186 and Tyr37 that occupies the catalytic pocket When

compared to results obtained in the PPA—c-AI1 complex,

the strong contacts in the catalytic clefts are highly

conserved and only slight modifications occur in the

extended protein-protein interaction [71,72] Bean amylase

inhibitors have been extensively used in transgenic plants due to their insecticidal properties

Kidney bean crude extracts containing lectin-like a-amylase inhibitors originally found in vivo, were used as starch blockers in the early 1980s, for the control of human noninsulin-dependent diabetes mellitus and obesity

[43,44,73,74] Those early attempts were unsuccessful due

to the undesirable presence of PHAs and _ proteinase inhibitors in the extract Later work with purified o-AI

in diabetic patients met with more success [73] More

Table

Residue numbers

class

lectins/glucanases Knottins

2.60.120.60° Lectin ND?

recently this class of inhibitors has been used for its insecticidal properties to protect seeds for insect predation [13,75,76]

Knottin-type «-amylase inhibitors The o-amylase inhibitor from Amaranthus hypocondriacus seeds (AAI) is the smallest proteinaceous inhibitor of œ-amylases yet described, with just 32 residues and three

disulfide bonds [77] The structure of its inhibitor, as

determined by NMR [78,79], contains a knottin fold; three antiparallel § strands and a characteristic disulfide topology

It revealed structural similarity to other proteins such as the proteinase inhibitor from Cucubirta maxima [80], charybdo- toxin and conotoxins [81]

AAI specifically inhibits insect «-amylases and is inactive against mammalian o-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 from 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 conserved residues involved

in substrate recognition and orientation, are connected to AAI via an intricate water-mediated hydrogen-bonding network [82] The TMA—AAIT 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 AAI bounded to TMA [82] demonstrate that both backbone and side conformations are only slightly adjusted on formation of the complex [79] The specific activity of AAI against insect o-amylases 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 five disulfide 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 associated with cereal flour [85,86] N-glycosylation is involved in the reactivity of the most reactive allergen, an inhibitor from rye [87]

The exogenous wheat o-amylase inhibitor coded 0.19

[46,49] and the bifunctional inhibitor from Indian finger

millet RBI [88,89], are the most studied inhibitors from this family The o-amylase inhibitor 0.19, named according to its gel electrophoretic mobility relative to bromophenol blue,

inhibits o-amylases from birds, Bacilli, insects and mammals

(Table 1) Its inhibition of human salivary o-amylase is

characterized by K; = 0.29 nm [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, WRP25, WRP26 and WRP27 [46] The dimeric o-amylase inhibitor 0.19 was

crystallized [92] and its structure determined It contains five

Fig 3 The various known modes of g-amylase inhibition A standard colouring scheme is used with enzyme drawn in magenta and inhibitor helix, strand and coil drawn in red, cyan and yellow, respectively The calcium ion common to all structures is drawn in orange The structures are (A) TMA bound to a-AIl (PDB code |viw) (B) TMA-AAI (Iclv) (C) TMA-RBI (1tmq) and (D) AMY2-BASI (Lava) In (D) the calcium ion bound at the enzyme—inhibitor interface (see text for details) is drawn in green

a-helices arranged in an up-and-down manner, satisfying

favorable packing modes, with all 10 cysteine residues

forming disulfide bonds [91]

The bifunctional o-amylase/trypsin inhibitor (RBI) is

another prototype of the cereal inhibitor family This

inhibitor is a stable monomer of 122 amino acids with five

disulfide bonds, which is resistant to urea, guanidine

hydrochloride and thermal denaturation [93] This bifunc-

tional inhibitor presents a three-dimensional structure very

similar to that of 0.19 inhibitor [91], with a globular fold

with four «helices in a simple ‘up-and-down’ topology and

a small antiparallel Bsheet ((94]; Fig 3C) Like other

inhibitors of this class, it can competitively inhibit a variety

of œ-amylases, including PPA and TMA This latter

enzyme is inhibited with a Kj; = 15 + 2 nm [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 3C) Two RBI segments are responsible for the interaction with TMA Segment 1, comprising the N-terminal residues Serl—Alall and the residues Pro52—Cys55, protrudes like an arrow head into TMA’s substrate-binding groove and directly targets the active site of the enzyme The N-terminus forms the arrow tip, adopting a 3,9-helical conformation in complex The Serl residue makes several hydrogen bonds with the catalytic Asp185 and Glu222 from enzyme while Val2 and Ser5 from inhibitor interact with the third conserved acidic residue of the catalytic site, Asp287 The second binding segment comprises several residues, which form a collar around the upper part of the arrow head and stabilize

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 RBI sequences demonstrated different

inhibitory potentials [89] Alam e¢ al [89] also showed that

this inhibitor binds to soluble amylase substrate, reducing

the apparent affinity of the enzyme for the substrate

Inhibitor-substrate interactions could explain the differ-

ences in the type of inhibition observed for different

substrates with the same enzyme [37,89] Exploiting the

overall structural similarity 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 family [96] Their different sequences yield a

remarkable array of inhibition specificity profiles (Table |

[46,52]) In wheat, some o-amylases inhibitors genes may

be silent or expressed at a much lower level [96] It can

be envisaged that the lack of the pertinent predator in

the ecological niche could silent the respective inhibitor

gene [97]

Kunitz-like «-amylase inhibitors

The Kunitz-like o-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 o-amylase inhibitor from the Kunitz class

is the barley o-amylase/subtilisin inhibitor (BASD, a

bifunctional double-headed inhibitor with a fast tight

inhibitory reaction with cereal oamylase AMY2

(K; = 0.22 nm) and serine proteinases of the subtilisin

family [50,101] The structure of BASI [102] revealed two

disulfide bonds and a Btrefoil topology (Fig 3D) shared

with the homologous wheat o-amylase subtilisin inhibitor

(WASI [103]), the Erythrina caffra trypsin inhibitor [104]

and the ricin B chain [105]

In the cases of a-AI1, AAI and RBI, inhibition involves

the insertion of inhibitor loops 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 inhibition of barley «-amylase 2 (AMY2) by

BASI [102] is different, in that 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 cavity at the enzyme—nhibitor

interface contains a trapped calcium ion whose presence is

suggested to electrostatically enhance the network of water

molecules at the complex interface and thereby raises the

stability of the complex

BASI is involved in regulating the degradation of seed

carbohydrate, preventing the endogenous o-amylase 2 from

hydrolysing starch during premature sprouting [41] Addi-

tionally, it protects the seeds against exogenous proteinases

and a-amylases produced by various pathogens and pests

[106] BASI inhibits the endogenous enzyme with a stoichi-

ometry of | : 1 [107], but, interestingly, is unable to inhibit

barley o-amylase 1 (AMY1), which bears 74% sequence

identity to AMY2 [101]

Thaumatin-like «-amylase inhibitors type The thaumatin-like inhibitors are proteins with molecular masses of +22 kDa with significant 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-characterized inhibitor from this class is zeam- atin, a bifunctional inhibitor from Zea mays that is homologous to the sweet protein thaumatin Zeamatin has

a total of 13 Bstrands, 11 of which form a B sandwich 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 du5) and (B) SIa1 (1gpt) The coordinates of SIal have not been deposited in the PDB so that (B) shows the structure of the homologous y-thionin [124].

eight disulfide bonds Electrostatic modelling of zeamatin

reveals an electrostatically polarized surface, heavily popu-

lated with Arg and Lys residues [110] This maize inhibitor is

not sweet, despite its similarity to thaumatin, probably due

to changes in a putative receptor-binding site [111] Zeam-

atin was able to inhibit porcine pancreatic trypsin and

digestive o-amylases of the insects T castaneum, Sitophilus

zeamays and Rizopherta dominica [112,113] Other proteins

from this class, such as the thaumatin-like proteins R and S$

from barley seeds, did not show any inhibitory activity

against trypsin or œ-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 fungal

a-amylases [112] and fungi do not contain trypsin Zeamatin

binds to B-1,3-glucans [115] and permeabilizes fungal-cells

leading to cell death [116] but the antifungal mode of action

of this protein is still a matter of debate For these

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

y-Purothionins-like «-amylase inhibitors

The o-amylase inhibitors of this family have 47 or 48

residues, are sulfur-rich and form part of the y-thionin

superfamily (Table 2) Members of this superfamily are

involved in plant defence through a remarkable variety of

mechanisms: modification of membrane permeability

[118,119], inhibition of protein synthesis [120] and protein-

ase inhibition [121] Inhibition of insect o-amylases has been

observed for three isoforms from Sorghum bicolor called

SIoa-1, Slo-2 and SlIa-3 [51] These molecules strongly

inhibited the digestive a-amylases of guts of locust and

cockroach, poorly inhibited o-amylases from A oryzae and

human saliva and failed to inhibit the o-amylases from

porcine pancreas, barley and Bacillus sp [51] The structure

of SIo-1 has been solved by NMR [122] revealing aa + B

sandwich structure [123] with a nine-residue helix packed

tightly against the sheet (Fig 4B) The helix is held in place

by two disulfide 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 sequence comparison, the structure is similar

to those of wheat yl-purothionin [124] and scorpion toxins

[122]

BIFUNCTIONAL INHIBITORS

As the above discussion highlights, bifunctional z-amylase/ proteinase inhibitors are relatively common (Table 3) As inhibition of predatory insect digestive proteinases is another attractive route for plant protection, the combina- tion of a-amylase and proteinase inhibition is potentially very useful It is therefore important to know whether simultaneous inhibition of proteinase and œ-amylase 1s possible for these inhibitors

In the case of RBI, with two independent inhibition sites, formation of a stable o-amylase-RBI-trypsin complex has 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-specificity Modelling

of the TMA-RBI-trypsin complex (Fig 5; [88]), confirms

Fig 5 A model of a ternary complex formed by TMA (magenta), RBI (blue) and pancreatic bovine trypsin (green) Constructed as described in the text

Table 3 Activity of bifunctional inhibitors from different plant sources against mammalian and insect ¢-amylases Low activity represents approx- imately 40% of a total activity

a-Amylase inhibition Inhibitor Source Insect Fungal Plant Mammalian References Other activity

WASI Wheat ND ND + ND [99,103] Subtilisin 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

that no steric clashes prevent simultaneous inhibition of

both enzymes The homologous bifunctional corn Hag-

eman factor inhibitor (CHIF) inhibits mammalian trypsin,

Factor XIIa (Hageman factor) of the contact 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 with RBI, o-amylase

inhibition requires the N-terminal region [129]

Kunitz-like inhibitors are also bifunctional, this time

possessing inhibitory activity against the subtilisin class pf

proteinases Among them, the BASI and WAST are the best

characterized, and both structures have been determined by

X-ray crystallography [102,103] It has been suggested that

WASI has two different sites because the activity against

a-amylases is retained after incubation with proteinases K

[130] A complex of WASF-proteinase K showed that a

loop containing the Gly66 and Ala67 is crucial to proteinase

inhibition [131] This class of bifunctional inhibitors inhibits

insect and endogenous plant o-amylases but not mamma-

lian o-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 filling and

therefore found in the seed prior to AMY2, which is

synthesized de novo during germination This inhibitor has

been proposed to control the activity of AMY2 in the case

of premature sprouting or to act in plant defence [159] The

inhibitor RASI could also help to regulate seed development

by inhibiting a development-specific œ-amylase [53] This

a-amylase inhibition specificity 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 o-amylase/

proteinase inhibitor [112,113] with a known crystal structure

[110] However, the observation of trypsin inhibition, albeit

weak, was only made recently [113] and it remains to be seen

whether or not zeamatin possesses independent sites for

proteinase and o-amylase inhibition

In addition to bifunctional o-amylase/proteinase inhibi-

tion, a single report has found chitinase activity present in

an insect o-amylase inhibitor isolated from Lachrima jobi

seeds [132] Chitinase activity is another recognized plant

defence [7] so that this double activity is 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, o-amylase inhibitors should have appro-

priate specificity profiles On the one hand, they should

ideally be effective against the 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 | and 3), already highlight some inhibitors as more

promising candidates than others For example, the cereal

bifunctional o-amylase/subtilisin inhibitors have strong

affinity for plant enzymes [107] deriving from their role in regulation of starch metabolism, and are therefore less favoured The known o-amylase inhibitors selective for insect enzymes and inactive against mammalian enzymes include WRP25, WRP26 and WRP27 from the cereal-type

class [46], the Amaranthus o-amylase inhibitor [77] and

zeamatin [113] As well as differences in affinity for broad groups of o-amylases, some remarkable examples of fine specificity exist Among several interesting specificity differ- ences in the cereal-type inhibitors is the inability of WRP26

to inhibit ZSA, while WRP25, 98% identical in sequence to

WRP26, is an effective inhibitor [46]

Given the remarkable structural and functional variety naturally found among o-amylase inhibitors (Tables 1-3), screening for inhibitors with desirable characteristics is a

viable option An attractive alternative, however, would be

the rational redesign of known inhibitors in order to confer upon them the required specificity profile 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 recent studies have addressed experimen- tally observed issues of amylase-inhibitor specificity, generally through sequence analysis and modelling, some- times supported by mutagenesis studies [46,62,82,133,134] Two independent studies [62,133] address the specificity

of o-AIl for PPA, not inhibiting ZSA and the opposite specificity of o-AI2 for ZSA over PPA [56] The reliance on simple counting of hydrogen bonds weakens the conclusions

of Le Berre-Anton et al [135] regarding the o-AI1/o-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 oAIl/oa-AI2 specificity and the authors concluded that specificity 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 examined through site-directed mutagenesis [134]

Mutations of AMY2 residues known to make electro-

static interactions at the interface with this inhibitor,

Arg128 and Asp142, were made, weakening the enzyme— inhibitor interaction and reducing the effect of charge screening on the interaction Remarkably, the introduc- tion of just two AMY2 residues, Argl28 and Prol29, into the AMYI environment was enough to enhance BASI sensitivity at least 100-fold As well as the electrostatic characteristics of the Arg, the conformational properties of the proline, which forms a cis peptide in AMY?2, are implicated in the AMYI/AMY?2 specificity The lysine, which replaces Prol29 in AMY 1 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 specificity profiles of a family of cereal o-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 œ-amylase, the complex of TMA with the more distantly

related RBI [88] was used as the basis for model