Search

An expression and secretion system for scytalidopepsin B, an acid protease from Scytalidium lignicolum, was constructed in yeast. Saccharomyces cerevisiae AH22 was transformed with an yeast-E. coli shuttle vector, pAM82, in which an yeast invertase signal segment and the cDNA encoding the pro- and mature enzyme regions were inserted. The transformant was found to secret a pepstatin-insensitive acid protease, when cultured aerobically in a low phosphate (Pi) medium. Amino terminal amino acid sequencing analysis indicated that the recombinant acid protease was accurately processed and secreted as a mature form.

A chromosomal DNA of Scytalidium lignicolum was digested with Sau3AI. The digest was self-ligated and amplified by inverse PCR procedure using primers designed based on the nucleotide sequences of up- and down-stream regions of an intron present in the scytalidopepsin B gene. Analysis of the nucleotide sequence of PCR product (700 bp) showed that the enzyme is synthesized as a precursor protein consisting of the prepro- and mature enzyme regions. The deduced amino acid sequence was highly similar to those of aspergillopepsin A and recently reported endothiapepsins B and C, but quite different from those of pepstatin-insensitive bacterial acid proteases and the pepstatin-sensitive aspartic protease family.

The role of cysteine residues for structure and function of formaldehyde dehydrogenase from Pseudomonas putida was analysed by amino acid sequence comparison, homology-based structure modeling, site-directed mutagenesis, and chemical modification. Five out of seven cysteine residues found in the enzyme were concluded to coordinate with an active site zinc (Cys-46) and structural zinc atoms (Cys-97, -100, -103, and -111) from the sequence comparison with other Zn-containing medium-chain alcohol dehydrogenase homologues. The three-dimensional structure model based on the known structure of the horse liver E-type alcohol dehydrogenase (ADH) indicated that Cys-257 is located very far from the active site Zn and NAD+ binding region, suggesting that Cys-257 does not participate in the enzyme reaction. The structure also suggested that Cys-166 does not coordinate to active site Zn, but Asp-169 functions as a Zn-ligand, instead.

A chromosomal DNA fragment of Scytalidium lignicolum that encodes the mature enzyme region of acid protease B (Scytalidopepsin B), was cloned and its nucleotides sequenced. The fragment contained a 76-bp intron at the middle of the mature enzyme-coding region. The mature enzyme was composed of 206 amino acid residues with a molecular weight of 21,550. There were some discrepancies between the amino acid sequence deduced from these results and that previously established by protein sequencing.

Prolidase (iminodipeptidase, EC 3.4.13.9) was purified from an extract of Xanthomonas maltophilia, by ammonium sulfate fractionation and sequential chromatographies on DEAE-Toyopearl, Toyopearl HW65C, FPLC-Hiload Superdex 200 pg, and FPLC-Hitrap Q columns, which an activity recovery of 2.3%. The enzyme was the most active at pH 7.5 with Leu-Pro as substrate. It was stable between pH 6.0 and 8.5 for 60 min at 37 degrees C and retained half of activity after 60 min at 37 degrees C. The isoelectric point of the enzyme was 3.7. Its molecular weight was estimated to be 100,000 by gel filtration on FPLC-Hiload Superdex 200 and 51,000 by SDS-PAGE, suggesting that it is a dimer. It hydrolyzed dipeptides only if proline is located at the carboxyl terminal position. The enzyme was inhibited by PCMB and o-phenanthroline, and was activated by Mn2+.

Prolylcarboxypeptidase (Angiotensinase C, EC 3.4.16.2) was purified to homogeneity from cell free extracts of Xanthomonas maltophilia by ammonium sulfate fractionation and sequential chromatographies on DEAE-Toyopearl, Sephadex G-150, FPLC-Hiload Superdex 200 pg, and FPLC-Hitrap SP columns, with an activity recovery of 15%. The molecular weight of the enzyme was found to be 330,000 by gel filtration and 83,000 by SDS-PAGE, suggesting a tetrameric form for the native enzyme. It had an optimum pH of 8.5 and stability between pH 8.0 and 11.0. The isoelectric point of the enzyme was 6.6. The enzyme hydrolyzed Pro-X bonds when proline was in the penultimate position from the carboxyl terminal. The enzyme was strongly inhibited by diisopropylfluorophosphate (DFP), while phenylmethylsulfonyl fluoride (PMSF), p-chloromercuribenzoic acid (PCMB), iodoacetamide, and metal chelators had no effect.

The zinc-containing neutral endopeptidase (neutral protease: BANP) from Bacillus subtilis var. amylosacchariticus was inhibited by the proteinaceous metalloprotease inhibitor isolated from Streptomyces nigrescens (SMPI). The degree of inhibition was, however, significantly less than that for thermolysin (TLN). During incubation of BANP with SMPI, the inhibitor was proteolytically degraded and inactivated. Analysis of the digestion products suggested that a minor diversity in their substrate specificities between TLN and BANP affects the sensitivity to the proteinaceous metalloprotease inhibitor, SMPI.