The gene and deduced protein sequences of the zymogen of Aspergillus niger acid proteinase A.
Proteinase A obtained from the culture medium of Aspergillus niger var. macrosporus is a unique acid endopeptidase that is insensitive (or less sensitive) to specific inhibitors of ordinary acid or aspartic proteinases, such as pepstatin, diazoacetyl-DL-norleucine methyl ester, and 1,2-epoxy-3-(p-nitrophenoxy)-propane. In the preceding paper (Takahashi, K., Inoue, H., Sakai, K., Kohama, T., Kitahara, S., Takishima, K., Tanji, M., Athauda, S. B. P., Takahashi, T., Akanuma, H., Mamiya, G., Yamasaki, M. J. Biol. Chem. 266, 19480-19483), we reported the complete primary structure of the mature enzyme determined at the protein level. The enzyme has a unique two-chain structure with a 39-residue light (L) chain and a 173-residue heavy (H) chain linked noncovalently. As an extension of this study, we isolated genomic and cDNA clones encoding this proteinase and determined their nucleotide sequences. To isolate a genomic clone, the genomic DNA was selectively amplified by polymerase chain reaction using mixed oligonucleotide primers designed from the amino acid sequence of the H chain, and a specific probe thus generated was used for screening a lambda gt10 genomic library. A cDNA for the enzyme was also selectively amplified by polymerase chain reaction using primers synthesized based on the sequence of the genomic DNA. Sequencing of the cloned genomic DNA and cDNA revealed the nucleotide sequence of the structural gene for the enzyme of 846 base pairs without introns. It encodes the precursor form of proteinase A, including an NH2-terminal preprosequence of 59 residues, the L chain of 39 residues, an intervening sequence of 11 residues, and the H chain of 173 residues linked in that order. Thus, proteinase A is thought to be synthesized as a single peptide chain preproenzyme of 282 residues, which is processed to generate the mature two-chain form.
Inoue
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H
Department of Biophysics and Biochemistry, Faculty of Science, University of Tokyo, Japan.
Kimura
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T
Makabe
O
O
Takahashi
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K
D000595:Amino Acid Sequence; D016282:Aspartic Acid Endopeptidases / Q000235:genetics*; D001234:Aspergillus niger / Q000201:enzymology*; D001483:Base Sequence; D015139:Blotting, Southern; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics; D004792:Enzyme Precursors / Q000235:genetics*; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D012689:Sequence Homology, Nucleic Acid; D016385:TATA Box
D004792:Enzyme Precursors; D004247:DNA; C022325:aspartic proteinase A; D016282:Aspartic Acid Endopeptidases