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Content archived on 2024-04-15

STUDIES ON STRUCTURE AND FUNCTION OF BARLEY MALT ALPHA-AMYLASE

Objective


Alpha-amylases are starch degrading enzymes with important biotechnical applications. So far a structural model is available for a mammalian alpha-amylases and 2 microbial alpha-amylases. Dependent on the enzyme, different products are obtained. It is therefore relevant to determine a structure for a cereal alpha-amylase which characteristically produces larger oligodextrins. A multidisciplinary strategy was thus applied to improve the insight at a molecular level into structure/function relationship and structure/stability relationship of the dominant barley malt alpha-amylase 2 and its interaction with the endogenous inhibitor barley amylase/subtilisin inhibitor (BASI).

The 2 dominant forms of the major alpha-amylase isozyme from barley malt, the so called high-pI alpha-amylase or alpha-amylase 2, were purified to charge homogeneity to be used crystallisation attempts. In spite of their very similar primary structures alpha-2-2 (pI 5.93) but not alpha-2-1 (pI 5.88) formed crystals under the conditions previously established. These crystals greatly facilitated the structural analysis, since they could be prepared routinely in a highly reproducible manner by the hanging drop vapour diffusion technique or sitting drop vapour diffusion technique without formation of amorphous precipitate due to the absence of the much less soluble alpha-2-1 form. On the other hand the new crystal diffracted to only slighter better resolution than those obtained with the unfractionated preparations of alpha-amylase 2.

The search for isomorphous heavy atom derivatives was performed by soaking native crystals in solutions containing the relevant heavy atom salts. 3 derivatives were obtained at present with HgC12, Eu(NO3)3 and K2PtCl4, respectively. The 2 former gave crystals isomorphous to at least 3 angstrom resolution while the latter was isomorphous to about 4 angstroms. The phase problem was solved by the method of multiple isomorphous replacement using the 3 heavy atom deriva tives. A preliminary electron density map was calculated a 5 angstrom resolution. The most striking feature in this map is the huge solvent channels penetrating the crystal parallel to 1 of the crystal axes and enveloping a minimum cylindrical space of about 75 angstroms in diameter. The volume in the crystals occupied by solvent is about 74%, corresponding to 1 molecule per asymmetric unit. To improve the phases the so-called solvent flattening technique was especially useful in the present case due to high solvent content. This technique served to clear the structural features of the 3 angstrom electron density map. The complete structure is currently under construction and will be compared to the available tertiary structure models for the porcine pancreatic and Aspergillus sp alpha-amylases displaying less than 20% sequence identity to the barley enzyme. In addition the structural analysis of substrate analogue/inhibitor complexes have been initiated.

Alpha-amylases are starch degrading enzymes with important biotechnical applications. So far a structural model is available for a mammalian alpha-amylases and 2 microbial alpha-amylases. Dependent on the enzyme, different products are obtained. It is therefore relevant to determine a structure for a cereal alpha-amylase which characteristically produces larger oligodextrins. A multidisciplinary strategy was thus applied to improve the insight at a molecular level into structure/function relationship and structure/stability relationship of the dominant barley malt alpha-amylase 2 and its interaction with the endogenous inhibitor barley amylase/subtilisin inhibitor (BASI). The cloned enzyme has been engineered to have enhanced activity.

The high pI isozyme is specifically inhibited by the endogenous inhibitor BASI and chemical modification experiments using a differential labelling approach have revealed that 1 arginine residue in BASI is essential for activity. Furthermore, that target amylase protects altogether 4 arginines in the inhibitor against inactivating modification by phenylglyoxal. A yeast expression system has been established for the complementary deoxyribonucleic acid (cDNA) encoding BASI which will shortly be employed in site directed mutagenesis experiments at the 4 candidating arginines at present evaluated on the basis of the 3-dimensional structure available for the 92% homologous wheat inhibitor. The chemical identification of the protected groups is performed in parallel. Small crystals of BASI have been obtained. The threee-dimensional structure of the alpha-amylase 2-BASI has been solved.

The structure of the first alpha-amylase from a higher plant, the barley malt high pI isozyme form alpha-2-2 has been determined and a model constructed at 3 angstroms resolution. Techniques have been developed to synthesize nonhydrolyzable oligosaccharide analogues suitable for photoaffinity labelling of endo-alpha-glucanases. A complementary deoxyribonucleic acid yeast expression system for production of high yields of higher plant proteins has also been developed. It was discovered that carboxy terminal processing of the amylase took place in the aleurone cells catalyzed by 1 or more carboxypeptidases from malt and resulting in several forms of the protein.

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Centre National de la Recherche Scientifique (CNRS)
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31 chemin Joseph Aiguier
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