Primary structure of a precursor to the aspartic proteinase from Rhizomucor miehei shows that the enzyme is synthesized as a zymogen.
In order to characterize the zymogen of the milk-clotting enzyme from Rhizomucor miehei, we constructed a cDNA library on pBR327 in Escherichia coli. Aspartic proteinase-specific recombinants were isolated by colony hybridization to a specific oligonucleotide mixture, and the cDNA sequence corresponding to a precursor form of the enzyme was determined. The deduced amino acid sequence shows that this secreted fungal proteinase is synthesized as a precursor. The first 22 amino acid residues in this precursor constitute a typical signal peptide. The amino acid sequence of the following 47-amino-acid-long prosegment shows homology to the prosegments from both the extracellular and intracellular vertebrate aspartic proteinases, and to the prosegments from the yeast and Mucor pusillus aspartic proteinases as well. These observations suggest that all aspartic proteinases are synthesized with a prosegment and that this prosegment is essential for the correct folding of all the mature enzymes. The active Rhizomucor miehei enzyme consists of 361 amino acid residues with a total molecular weight of 38,701. Clusters of identities around the active site cleft support the assumption that these proteinases have a common folding of their peptide chains. The disulphide bridges were localized in the fungal enzyme, and 2 N-glycosylation sites were identified.
Boel
E
E
Laboratory of Genetics, Novo Research Institute, Copenhagen, Denmark.
Bech
A M
AM
Randrup
K
K
Draeger
B
B
Fiil
N P
NP
Foltmann
B
B
D000595:Amino Acid Sequence; D016282:Aspartic Acid Endopeptidases; D001483:Base Sequence; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics; D004271:DNA, Fungal / Q000235:genetics; D004220:Disulfides; D010450:Endopeptidases / Q000096:biosynthesis / Q000235:genetics*; D004792:Enzyme Precursors / Q000096:biosynthesis / Q000235:genetics*; D006031:Glycosylation; D008969:Molecular Sequence Data; D009090:Mucorales / Q000201:enzymology* / Q000235:genetics
D004271:DNA, Fungal; D004220:Disulfides; D004792:Enzyme Precursors; D004247:DNA; D010450:Endopeptidases; D016282:Aspartic Acid Endopeptidases; C022517:rennin-like enzyme (Aspergillus ochraceus)