Cloning and expression in yeast of a cDNA clone encoding Aspergillus oryzae neutral protease II, a unique metalloprotease

Abstract

The neutral protease II (NpII) from Aspergillus oryzae is a zinc-containing metalloprotease with some unique properties. To elucidate its structure, we isolated a full-length cDNA clone for NpII. Sequence analysis reveals that NpII has a prepro region consisting of 175 amino acids preceding the mature region, which consists of 177 amino acids. As compared with other microbial metalloproteases, NpII is found to be unique in that it shares only a limited homology with them around two zinc ligand His residues and that the positions of the other zinc ligand (Glu) and the active site (His) cannot be established by homology. When a plasmid designed to express the prepro NpII cDNA was introduced into Saccharomyces cerevisiae and the transformant was cultured in YPD medium (2% glucose, 2% polypeptone, 1% yeast extract), it secreted a proNpII. However, in a culture of the same medium containing 0.2 mM ZnCl2, it secreted a mature NpII with a specific activity and N-terminus identical to those of native NpII. This observation suggests that either an autoproteolytic activity or a yeast protease effected the processing.

Amino Acid Sequences of Metalloendopeptidases Specific for Acyl-Lysine Bonds from Grifola frondosa and Pleurotus ostreatus Fruiting Bodies

“…7) with the BLAST program (17). They are Aspergillus oryzae neutral protease II (accession number 1078624) (18), Aspergillus flavs 24-kDa metalloproteinase precursor (deuterolysin, accession number 1170905), Aspergillus fumigata (listed as Sartorya fumigata in protein sequence data base) MEP20, (accession number 780794) (19), and penicillolysin (accession number 1078641) from Penicillium citrinum (20). From the sequence alignment, it is apparent that they have evolved from a common ancestor, although only mushroom enzymes have the unique specificity.…”

PCR cloning and heterologous expression of cDNA encoding a peptidyl-Lys metalloendopeptidase precursor of Grifola frondosa.

“…The amino acid sequence of GfMEP was aligned with other aspzincins, AmMEP (EMBL AJ238718), MEP from Aeromonas hydrophila (AhMEP) (Chang et al, 1997), penicillolysin (Matsumoto et al, 1994), MEPs from Aspergillus orysae (NPII) (Tatsumi et al, 1991), A. flavus (AflMEP) (Ramesh et al, 1995), and A. fumigatus (AfuMEP) (Ramesh et al, 1995) (Fig. 2A).…”

Cloning and sequencing of a gene encoding a novel extracellular neutral proteinase from Streptomyces sp. strain C5 and expression of the gene in Streptomyces lividans 1326

“…Nevertheless, the deduced amino acid sequence of this proteinase (NpII) shows little similarity with that of SnpA. Moreover, the substrate specificities of NpII are very different from those of SnpA (52), and NpII is markedly thermostable whereas SnpA loses activity rapidly at 80°C (52), indicating that NpII is very different from SnpA.…”

“…The sequence of proteinase NpII, an Aspergillus oryzae protein with an Mr of 19,000 that also contains a single zinc binding site, was recently described (Fig. 4); however, it lacks the second zinc ligand Glu residue and the active-site His residue (52). Nevertheless, the deduced amino acid sequence of this proteinase (NpII) shows little similarity with that of SnpA.…”

“…Recently, an unusual neutral proteinase of B. subtilis (Mpr) that also appeared to contain disulfide bridges was purified (46), and the small, highly thermostable, neutral proteinase (NpII) from A. oryzae has six Cys residues that are thought to form disulfide bridges to yield the remarkable thermostability of that enzyme (52).…”