Amylase sequencing

Using molecular biology techniques, Conrad et al.61 produced hybrids of the a-amylases from B. amyloliquefaciens and B. licheniformis. Thirty-three hybrids were formed. They consisted of the entire a-amylase sequence with variable proportions from B. amyloliquefaciens a-amylase and B. licheniformis a-amylase. The hybrid enzymes fell into six groups that retained the extra-thermostability of the licheniformis enzyme. A specific hybrid sequence (residues 34-76) was correlated with the enzymes product specificity for forming and accumulating maltohexaose. Two of the hybrids were less thermostable than either of the parent types, while two others were enzymatically inactive. 

To understand the inhibition of a-amylase by peptide inhibitors it is crucial to first understand the native substrate-enzyme interaction. The active site and the reaction mechanism of a-amylases have been identified from several X-ray structures of human and pig pancreatic amylases in complex with carbohydrate-based inhibitors. The structural aspects of proteinaceous a-amylase inhibition have been reviewed by Payan. The sequence, architecture, and structure of a-amylases from mammals and insects are fairly homologous and mechanistic insights from mammalian enzymes can be used to elucidate inhibitor function with respect to insect enzymes. The architecture of a-amylases comprises three domains. Domain A contains the residues responsible for catalytic activity. It complexes a calcium ion, which is essential to maintain the active structure of the enzyme and the presence of a chloride ion close to the active site is required for activation. 

While the Uterature is rich in scientific information on glucosylases, recent interest has focused on the hypothesis that all these enzymes share a common catal3iic mechanism, despite differences in their product specificity (57). Indeed, it has been proposed that all glycosylases share the same basic chemical mechanism (58). Tlie a-amylases have been the focus of much of this attention, as the primary protein sequence (59), tertiary protein structure (54,55) and catalytic mechanism (57) have been recently delineated. 

The tropomyosins of mite and insect species show some sequence identity (63-65%) with snail tropomyosin and share similar epitopes (EFSA, 2006 Fig. 4.1). Still, tropomyosin appears to play a minor role in the crossreactivity of dust mites and snails (Asturias et ah, 2002 Guilloux et ah, 1998 Van Ree et ah, 1996a). Other non-tropomyosin allergens are likely to be involved including Der p 4 (amylase), Der p 5, Der p 7, and hemocyanin (Martins et ah, 2005 Mistrello et ah, 1992 Van Ree et ah, 1996). While snail is the main molluscan shellfish species involved in cross-reactions with dust mites, some patients allergic to dust mites and snails are also sensitized to mussels (DeMaat-Bleeker et ah, 1995 Van Ree et ah, 1996b). In their study of 70 patients sensitized to molluscan shellfish, Wu and Williams (2004) noted that 90% were also sensitized to dust mites. However, the clinical significance of this sensitization was not documented. 

The sequences of a-amylases vary widely but a conserved cluster of one glutamate and two aspartates is usually present (Table 12-1). 

Table V. Partial Amino Acid Sequence of a-Amylase from B. subtilis°...

Several amylases have been partially sequenced (42-45). For example, a-amylase from Bacillus subtilis, which is composed of two subunits of 24,000 molecular weight each, has an amino terminal sequence as shown in Table V (42). Perhaps fortuitously, the sequence of residues 8 through 12 resembles residues 14 through 18 in xylanase A, in that a polar residue is surrounded by four aromatic residues. 

Leah, R. Mundy, J. (1989). The bifunctional a-amylase/subtilisin inhibitor of barley Nucleotide sequence and patterns of seed-specific expression. Plant Molecular Biology 12, 673-82. 

Whittier, R.F., Dean, D.A. Rogers, J.C. (1987). Nucleotide sequence analysis of a-amylase and thiol protease genes that are hormonally regulated in barley aleurone cells. Nucleic Acids Research 15, 2515-35. 

Rocher et al. (1992) reported the presence of an 11-kDa protein from oat endosperm that displayed a great resemblance in sequence to that of the ragi bifunctional a-amylase inhibitor. 

Among the EST database of ragi sequences, there are two groups of bifunctional proteinase inhibitor trypsin a-amylase from seeds of ragi sequences. The upper clade was further subdivided (Fig. 6.10). Wang et al. (2008) concluded that there was great diversity in the sequence of different Bowman-Birk inhibitors in emmer wheat both within and between populations. 

Campos, F. A. P. and Richardson, M. (1983). The complete amino acid sequence of the bifunctional a-amylase/trypsin inhibitor from seeds of ragi (Indian finger millet, Eleusine coracana Gaertn.). FEBS Lett. 152, 300-304. 

Kabuto S, Ogawa T, Muramoto K, Oosthuizen V, Naude R J. 2000. The amino acid sequence of pancreatic a-amylase from the ostrich, Struthio camelus. Comp Biochem Physiol Part B 127 481-490. 

Feller, G., T. Lonhienne, C. Deroanne, C. Libioulle, J. Van Beeummen, and C. Gerday. 1992. Purification, characterization, and nucleotide sequence of the thermolabile a-amylase from the Antarctic psychrotroph Alteromonas haloplanctis A23. Journal of Biology Cbemisty 267 5217-5221. 

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