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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).

In order to study the functional roles of the individual domains the barley alpha-amylase 1 and alpha-amylase 2 were subjected to limited proteolysis. A single bond was cleaved, however, it was located in 1 of the loops of the alpha/beta barrel and not in the region between the barrel and the carboxyl-terminal domain. This form had about 40% of the activity of the intact enzyme and the isolated fragments corresponding to residues 1-294 and 295-403 were purified for the high pI isozyme and found to be inactive. In the case of porcine pancreatic alpha-amylase parallel studies indicated cleavage in a different loop from the barrel domain. Thus the structural integrity of the enzyme does not lead to cleavage between domains indicating that they may both be required for function.

In order to examine the functional role of specific amino acid sidechains a yeast expression system was established for complementary deoxyribonucleic acids (cDNA) encoding a member of either isozyme family although reasonable levels of protein was produced only in the case of the low pI isozyme. By change of expression plasmid a similar expression level has been obtained also for the other isozyme. 4 products obtained for the low pI isozyme in the initial system and large scale production has allowed the purification of these individual forms for chemical and functional characterisation. In this way carboxyl terminal processing was concluded to occur in the germinating plant seed and the yeast expression system was found to give rise to disulphide formation between free sulphur hydrogen groups in the recombinant proteins and glutathione. The carboxyl terminal processing was demonstrated also with isolated protoplasts of aleurone cells to be performed by 1 or serval carboxypeptidase(s).

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.

Funding Scheme

CSC - Cost-sharing contracts

Coordinator

CARLSBERG LABORATORY
Address
Gamle Carlsberg Vej 10 Dk 2500 Copenhagen,valby

Denmark