Aminopeptidase Ey (EC 3.4.11.20) from chicken (Gallus gallus domesticus) egg yolk is a homodimeric exopeptidase with a broad specificity for N-terminal amino acid residues at P1 position of the substrate. Aminopeptidase Ey is a 300-k metalloexopeptidase, containing 1.0 g atom of zinc per mole of a subunit with a relative molecular mass of 150 k. A full-length cDNA was cloned from chicken (female) liver cDNA library. Analysis of the 3196-base pairs (bp) nucleotide sequence of the cDNA revealed a single open reading frame coding for 967 amino acid residues. The coding region of aminopeptidase Ey gene, apdE, occupies 2901 bp of the cDNA. The predicted amino acid sequence of the enzyme is 66, 65, 64 and 63% identical with those of aminopeptidases N (EC 3.4.11.2) from human, pig, rabbit and rat, respectively. Aminopeptidase Ey contains the metallo-binding sequence motif, His-Glu-Xaa-His, found in zinc metallopeptidases. Zinc binding sites, His-386, His-390 and Glu-409, and catalytic site, Glu-387, were conserved in the homologous aminopeptidases N.
We previously reported purification and characterization of a 90k serine protease with pI 3.9 from Bacillus subtilis (natto) No. 16 [Kato et al. 1992 Biosci Biotechnol Biochem 56:1166]. The enzyme showed different and unique substrate specificity towards the oxidized B-chain of insulin from those of well-known bacterial serine proteases from Bacillus subtilisins. The structural gene, hspK, for the 90k serine protease was cloned and sequenced. The cloned DNA fragment contained a single open reading frame of 4302 bp coding a protein of 1433 amino acid residues. The deduced amino acid sequence of the 90k-protease indicated the presence of a typical signal sequence of the first 30 amino acids region and that there was a pro-sequence of 164 amino acid residues after the signal sequence. The mature region of the 90k-protease started from position 195 of amino acid residue, and the following peptide consisted of 1239 amino acid residues with a molecular weight of 133k. It might be a precursor protein of the 90k-protease, and the C-terminal region of 43k might be degraded to a mature protein from the precursor protein. The catalytic triad was thought to consist of Asp33, His81, and Ser259 from comparison of the amino acid sequence of the 90k-protease with those of the other bacterial serine proteases. The high-molecular-weight serine protease, the 90k-protease, may be an ancient form of bacterial serine proteases.
Cultured cells of rice (Oryza sativa cv Sasanishiki) produce two alpha-amylase isozymes, AMY-I and AMY-III. Using a bacterial expression system, eight chimeric genes constructed with various combination of AMY-I and AMY-III cDNA fragments were expressed, and each recombinant chimeric protein was characterized. Four of the eight recombinant enzymes having region c (one of the four regions having unconserved base sequences between AMY-I and AMY-III cDNAs) of AMY-I showed the same enzyme characteristics as that of native AMY-I, which had high temperature optimum at 50 degrees C. The other four chimeric proteins carrying region c of AMY-III showed the AMY-III type characteristics, which were a low temperature optimum at 25 degrees C and susceptibility to a higher maltooligosaccharide (G17) substrate. The unconserved region c is involved in the decision of the characteristic of AMY-I or AMY-III.
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