Nucleotide sequence of the maltohexaose-producing amylase gene from an alkalophilic Bacillus sp. #707 and structural similarity to liquefying type alpha-amylases

The nucleotide sequence of the gene for maltohexaose-producing amylase from an alkalophilic Bacillus sp. #707 was determined. Starting at an ATG initiation codon, an open reading frame was composed of 1554 bp (518 amino acids). The NH2-terminal portion encoded a 33 amino acid-long signal peptide. The deduced amino acid sequence of the extracellular mature enzyme was more than 60% homologous to those of the liquefying type alpha-amylases but not to those of the saccharifying type alpha-amylases. The sequence of its signal peptide was completely different from those of other alpha-amylases.     

Hydrophobic cluster analysis of the primary sequences of alpha-amylases

The amino acid sequences of 18 alpha-amylases have been compared by hydrophobic cluster analysis. The method was first calibrated with two alpha-amylases (Aspergillus oryzae and pig pancreas) whose three-dimensional structures are known. It was then applied to the other alpha-amylases resulting in straightforward sequence alignments which could be used for structure prediction. It was found that all alpha-amylases which were investigated display the same basic super-secondary structure with a (beta alpha)8 barrel. Most of the secondary structure elements of the protein cores could be assigned to segments of the amino acid sequences. In addition, six sub-families could be identified, based upon specific similarities occurring in the variable regions of alpha-amylases.     

Alpha-amylase inhibitors from Ficus sp. seeds and their activities towards coleoptera insect pests

Alpha-amylase Inhibitors were isolated from Ficus sp. (Gameleira) seeds by acetone fractionation and Sephadex G-50. Two inhibitors (alpha-PPAI and alpha-ZSAI) were tested against alpha-amylases from coleopteran larvae. alpha-PPAI was active to alpha-amylases of Callosobruchus maculatus (52%) and Zabrotes subfasciatus (53%). alpha-ZSAI was strongly active to Z. subfasciatus (100%) of and Mimosestes mimosae (98%). The alpha-ZSAI is a glycoprotein of approximately 50 kDa with an IC50 value of 0.074 microg microl(-1).     

Purification and characterization of alpha-amylase from rat pancreatic acinar carcinoma. Comparison with pancreatic alpha-amylase.

alpha-Amylase was purified to apparent homogeneity from normal pancreas and a transplantable pancreatic acinar carcinoma of the rat by affinity chromatography on alpha-glucohydrolase inhibitor (alpha-GHI) bound to aminohexyl-Sepharose 4B. Recovery was 95-100% for both pancreas and tumour alpha-amylases. They were monomeric proteins, with Mr approx. 54000 on SDS/polyacrylamide-gel electrophoresis. Isoelectric focusing of both normal and tumour alpha-amylases resolved each into two major isoenzymes, with pI 8.3 and 8.7. Tumour-derived alpha-amylase contained two additional minor isoenzymes, with pI 7.6 and 6.95 respectively. All four tumour isoenzymes demonstrated amylolytic activity when isoelectric-focused gels were treated with starch and stained with iodine. Two-dimensional electrophoresis, on SDS/10-20%-polyacrylamide-gradient gels after isoelectric focusing, separated each major isoenzyme into doublets of similar Mr values. Pancreatic and tumour-derived alpha-amylases had similar Km and Ki (alpha-GHI) values, but the specific activity of the tumour alpha-amylase was approximately two-thirds that of the normal alpha-amylase. Although amino acid analysis and peptide mapping with the use of CNBr, N-chlorosuccinimide or Staphylococcus aureus V8 proteinase gave comparable profiles for the two alpha-amylases, tryptic-digest fingerprint patterns were different. Antibodies raised against the purified pancreatic alpha-amylase and tumour alpha-amylase respectively showed only one positive band on immunoblotting after gel electrophoresis of crude extracts of rat pancreas and carcinoma, at the same position as that of the purified enzyme. More than 95% of the alpha-amylase activity in the pancreas and in the tumour was absorbed by an excess amount of either antibody, indicating that normal and tumour alpha-amylases are immunologically identical. The presence of additional isoenzymes in the carcinoma, and dissimilarity of tryptic-digest patterns, may reflect an alteration in gene expression or in the post-translational modification of this protein in this heterogeneously differentiated transplantable pancreatic acinar carcinoma.     

Purification and properties of alpha-amylases in morphological variants of Bacillus subtilis

Extracellular alpha-amylases were isolated from the culture medium filtrates of Bacillus subtilis R-623 morphological variants R, P and S by means of biospecific chromatography on artificial sorbents and then purified to homogeneity. Some properties of purified alpha-amylases were being studied. The molecular weight of alpha-amylases from Bacillus subtilis variants R, P and S equals 57,000, 58,000 and 56,000, and the isoelectric points are at pH 5.4, 5.6 and 5.1, respectively. pH optimum for alpha-amylase from variants R and P is 4.5, and for that from variant S--5.0. alpha-Amylases from Bacillus subtilis R-623 morphological variants are thermostable enzymes. According to the properties studied, they correspond to Bacillus subtilis alpha-amylases that were isolated and described by other researchers.     

Two-codon mutagenesis of alpha-amylase gene of Bacillus amyloliquefaciens

The oligonucleotide encoding Bam HI recognition site having the structure pCGGGATC had been inserted into the recognition sites MspI of the B. amyloliquefaciens alpha-amylase gene, which was cloned in pTG29B plasmid. The alpha-amylase gene had no BamHI sites before mutagenesis. The set of pNSBamHI plasmids with BamHI site at four different positions was obtained. It was shown that all the mutant alpha-amylases possess different specific activities. One of the mutant proteins possesses reduced thermostability. The mutant alpha-amylases can be used for further experiments on protein-engineering of liquefying-type alpha-amylases.     

Inhibitory specificity and insecticidal selectivity of alpha-amylase inhibitor from Phaseolus vulgaris

The primary structure and proteolytic processing of the alpha-amylase isoinhibitor alpha AI-1 from common bean (Phaseolus vulgaris cv. Magna) was determined by protein chemistry techniques. The inhibitory specificity of alphaAI-1 was screened with a panel of the digestive alpha-amylases from 30 species of insects, mites, gastropod, annelid worm, nematode and fungal phytopathogens with a focus on agricultural pests and important model species. This in vitro analysis showed a selective inhibition of alpha-amylases from three orders of insect (Coleoptera, Hymenoptera and Diptera) and an inhibition of alpha-amylases of the annelid worm. The inhibitory potential of alphaAI-1 against several alpha-amylases was found to be modulated by pH. To understand how alphaAI-1 discriminates among closely related alpha-amylases, the sequences of the alpha-amylases sensitive, respectively, insensitive to alphaAI-1 were compared, and the critical determinants were localized on the spatial alpha-amylase model. Based on the in vitro analysis of the inhibitory specificity of alphaAI-1, the in vivo activity of the ingested alphaAI-1 was demonstrated by suppression of the development of the insect larvae that expressed the sensitive digestive alpha-amylases. The first comprehensive mapping of alphaAI-1 specificity significantly broadens the spectrum of targets that can be regulated by alpha-amylase inhibitors of plant origin, and points to potential application of these protein insecticides in plant biotechnologies.     

Conserved residues of liquefying alpha-amylases are concentrated in the vicinity of active site

Three dimensional structure of three liquefying type Bacillus alpha-amylases were modeled based on sequence analyses and refined structure of Aspergillus oryzae enzyme. The models suggest that the overall folding motif of alpha-amylases is conserved. The active site, substrate binding and stabilizing calcium binding residues are conserved and concentrated in a cleft between two domains. They constitute the core of alpha-amylases to which other, less conserved regions are attached. The bacterial enzymes have a loop of about 45 residues near the active site and Ca2+ binding region. The loop may be important for the liquefying function of these enzymes.     

Properties of Thermosensitive Extracellular α-Amylases of Bacillus subtilis

Enzymological properties of four thermosensitive alpha-amylases (M3, M9, M18, and M20) brought by different mutation sites in alpha-amylase structural gene of Bacillus subtilis were compared with those of the parental alpha-amylase NA64. Two thermosensitive alpha-amylases (M9 and M20) were altered not only in their thermosensitivity but also in their immunological properties, catalytic properties, molecular weights determined by the gel filtration on a Bio-Gel P-100 column, and others. The other two thermosensitive alpha-amylases (M3 and M18) were altered only in their thermosensitivity.     

Structural basis for the inhibition of mammalian and insect alpha-amylases by plant protein inhibitors

Alpha-amylases are ubiquitous proteins which play an important role in the carbohydrate metabolism of microorganisms, animals and plants. Living organisms use protein inhibitors as a major tool to regulate the glycolytic activity of alpha-amylases. Most of the inhibitors for which three-dimensional (3-D) structures are available are directed against mammalian and insect alpha-amylases, interacting with the active sites in a substrate-like manner. In this review, we discuss the detailed inhibitory mechanism of these enzymes in light of the recent determination of the 3-D structures of pig pancreatic, human pancreatic, and yellow mealworm alpha-amylases in complex with plant protein inhibitors. In most cases, the mechanism of inhibition occurs through the direct blockage of the active center at several subsites of the enzyme. Inhibitors exhibiting "dual" activity against mammalian and insect alpha-amylases establish contacts of the same type in alternative ways.     

Enzyme-coding genes as molecular clocks: The molecular evolution of animal alpha-amylases

Summary We constructed a cDNA library for the beetle,Tribolium castaneum. This library was screened using a cloned amylase gene fromDrosophila melanogaster as a molecular probe. Beetle amylase cDNA clones were isolated from this bank, and the nucleotide sequence was obtained for a cDNA clone with a coding capacity for 228 amino acids. Both the nucleotide sequence and predicted amino acid sequence were compared to our recent results forD. melanogaster alpha-amylases, along with published sequences for other alpha-amylases. The results show that animal alpha-amylases are highly conserved over their entire length. A borader comparison, which includes plant and microbial alpha-amylase sequences, indicates that parts of the gene are conserved between prokaryotes, plants, and animals. We discuss the potential importance of this and other enzyme-coding genes for the construction of molecular phylogenies and for the study of the general question of molecular clocks in evolution.     

Cloning of a gene encoding raw-starch-digesting amylase from a Cytophaga sp. and its expression in Escherichia coli

A raw-starch-digesting amylase (RSDA) gene from a Cytophaga sp. was cloned and sequenced. The predicted protein product contained 519 amino acids and had high amino acid identity to alpha-amylases from three Bacillus species. Only one of the Bacillus alpha-amylases has raw-starch-digesting capability, however. The RSDA, expressed in Escherichia coli, had properties similar to those of the enzyme purified from the Cytophaga sp.     

Proteinaceous alpha-amylase inhibitors

Proteins that inhibit alpha-amylases have been isolated from plants and microorganisms. These inhibitors can have natural roles in the control of endogenous alpha-amylase activity or in defence against pathogens and pests; certain inhibitors are reported to be antinutritional factors. The alpha-amylase inhibitors belong to seven different protein structural families, most of which also contain evolutionary related proteins without inhibitory activity. Two families include bifunctional inhibitors acting both on alpha-amylases and proteases. High-resolution structures are available of target alpha-amylases in complex with inhibitors from five families. These structures indicate major diversity but also some similarity in the structural basis of alpha-amylase inhibition. Mutational analysis of the mechanism of inhibition was performed in a few cases and various protein engineering and biotechnological approaches have been outlined for exploitation of the inhibitory function.     

Molecular identification of four different alpha-amylase inhibitors from baru (Dipteryx alata) seeds with activity toward insect enzymes

The endophytic bruchid pest Callosobruchus maculatus causes severe damage to storage cowpea seeds, leading to economical losses. For this reason the use of alpha-amylase inhibitors to interfere with the pest digestion process has been an interesting alternative to control bruchids. With this aim, alpha-amylase inhibitors from baru seeds (Dipteryx alata) were isolated by affinity chromatographic procedures, causing enhanced inhibition of C. maculatus and Anthonomus grandis alpha-amylases. To attempt further purification, this fraction was applied onto a reversed-phase HPLC column, generating four peaks with remarkable inhibition toward C. maculatus alpha-amylases. SDS-PAGE and MALDI-ToF analysis identified major proteins of approximately 5.0, 11.0, 20.0 and 55 kDa that showed alpha-amylase inhibition. Results of in vivo bioassays using artificial seeds containing 1.0% (w/w) of baru crude extract revealed 40% cowpea weevil larvae mortality. These results provide evidence that several alpha-amylase inhibitors classes, with biotechnological potential, can be isolated from a single plant species.     

Specific inhibition of insect alpha-amylases: yellow meal worm alpha-amylase in complex with the amaranth alpha-amylase inhibitor at 2.0 A resolution.

BACKGROUND: alpha-Amylases constitute a family of enzymes that catalyze the hydrolysis of alpha-D-(1,4)-glucan linkages in starch and related polysaccharides. The Amaranth alpha-amylase inhibitor (AAI) specifically inhibits alpha-amylases from insects, but not from mammalian sources. AAI is the smallest proteinaceous alpha-amylase inhibitor described so far and has no known homologs in the sequence databases. Its mode of inhibition of alpha-amylases was unknown until now. RESULTS: The crystal structure of yellow meal worm alpha-amylase (TMA) in complex with AAI was determined at 2.0 A resolution. The overall fold of AAI, its three-stranded twisted beta sheet and the topology of its disulfide bonds identify it as a knottin-like protein. The inhibitor binds into the active-site groove of TMA, blocking the central four sugar-binding subsites. Residues from two AAI segments target the active-site residues of TMA. A comparison of the TMA-AAI complex with a modeled complex between porcine pancreatic alpha-amylase (PPA) and AAI identified six hydrogen bonds that can be formed only in the TMA-AAI complex. CONCLUSIONS: The binding of AAI to TMA presents a new inhibition mode for alpha-amylases. Due to its unique specificity towards insect alpha-amylases, AAI might represent a valuable tool for protecting crop plants from predatory insects. The close structural homology between AAI and 'knottins' opens new perspectives for the engineering of various novel activities onto the small scaffold of this group of proteins.     

Study of the inhibition of four alpha amylases by acarbose and its 4IV-alpha-maltohexaosyl and 4IV-alpha-maltododecaosyl analogues

Acarbose analogues, 4IV-maltohexaosyl acarbose (G6-Aca) and 4IV-maltododecaosyl acarbose (G12-Aca), were prepared by the reaction of cyclomaltodextrin glucanyltransferase with cyclomaltohexaose and acarbose. The inhibition kinetics of acarbose and the two acarbose analogues were studied for four different alpha-amylases: Aspergillus oryzae, Bacillus amyloliquefaciens, human salivary, and porcine pancreatic alpha-amylases. The three inhibitors showed mixed, noncompetitive inhibition, for all four alpha-amylases. The acarbose inhibition constants, Ki, for the four alpha-amylases were 270, 13, 1.27, and 0.80 microM, respectively; the Ki values for G6-Aca were 33, 37, 14, and 7 nM, respectively; and the G12-Aca Ki constants were 59, 81, 18, and 11 nM, respectively. The G6-Aca and G12-Aca analogues are the most potent alpha-amylase inhibitors observed, with Ki values one to three orders of magnitude more potent than acarbose, which itself was one to three orders of magnitude more potent than other known alpha-amylase inhibitors.     

High pressure, thermal, and combined pressure-temperature stabilities of alpha-amylases from Bacillus species

Three different alpha-amylases from Bacillus subtilis, B. amyloliquefaciens, and B. licheniformis, were mutually compared with respect to thermal stability, pressure stability, and combined pressure-temperature stability. Measurements of residual enzyme activity and residual denaturation enthalpy showed that the alpha-amylase from B. licheniformis has by far the highest thermostability and that the two other alpha-amylases have thermostabilities of the same order of magnitude. FTIR spectroscopy showed that changes in the conformation of the alpha-amylases from B. amyloliquefaciens, B. subtilis, and B. licheniformis due to pressure occurred at about 6.5, 7.5, and 11 kbar, respectively. It seemed that, for the enzymes studied, thermal stability was correlated with pressure stability. As to the resistance under combined heat and high pressure, the alpha-amylase from B. licheniformis was much more stable than the alpha-amylases from B. amyloliquefaciens and B. subtilis, the latter two being about equally stable. It appears that under high pressure and/or temperature, B. licheniformis alpha-amylase is the most resistant among the three enzymes studied. (c) 1996 John Wiley & Sons, Inc.     

Plant alpha-amylase inhibitors and their interaction with insect alpha-amylases.

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, beta-1,3-glucanases, lectins, arcelins, vicilins, systemins and enzyme inhibitors. The enzyme inhibitors impede digestion through their action on insect gut digestive alpha-amylases and proteinases, 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 alpha-amylase inhibitor classes, lectin-like, knottin-like, cereal-type, Kunitz-like, gamma-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 alpha-amylases. Specificity of inhibition is an important issue as the introduced inhibitor must not adversely affect the plant's own alpha-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 chitinase activity. The area has benefited from the recent determination of many structures of alpha-amylases, inhibitors and complexes. These structures highlight the remarkable variety in structural modes of alpha-amylase inhibition. The continuing discovery of new classes of alpha-amylase inhibitor ensures that exciting discoveries remain to be made. In this review, we summarize existing knowledge of insect alpha-amylases, plant alpha-amylase inhibitors and their interaction. Positive results recently obtained for transgenic plants and future prospects in the area are reviewed.     

Where do animal alpha-amylases come from? An interkingdom trip

Alpha-amylases are widely found in eukaryotes and prokaryotes. Few amino acids are conserved among these organisms, but at an intra-kingdom level, conserved protein domains exist. In animals, numerous conserved stretches are considered as typical of animal alpha-amylases. Searching databases, we found no animal-type alpha-amylases outside the Bilateria. Instead, we found in the sponge Reniera sp. and in the sea anemone Nematostella vectensis, alpha-amylases whose most similar cognate was that of the amoeba Dictyostelium discoideum. We found that this "Dictyo-type" alpha-amylase was shared not only by these non-Bilaterian animals, but also by other Amoebozoa, Choanoflagellates, and Fungi. This suggested that the Dictyo-type alpha-amylase was present in the last common ancestor of Unikonts. The additional presence of the Dictyo-type in some Ciliates and Excavates, suggests that horizontal gene transfers may have occurred among Eukaryotes. We have also detected putative interkingdom transfers of amylase genes, which obscured the historical reconstitution. Several alternative scenarii are discussed.