Molecular cloning and nucleotide sequence of the genomic DNA for 1,2-alpha-D-mannosidase gene, msdC from Penicillium citrinum.

A gene encoding 1,2-alpha-D-mannosidase (EC 3.2.1.113) was cloned from Penicillium citrinum genomic DNA using the polymerase chain reaction (PCR). The coding region of the gene, msdC, occupied 1737 bp and was separated into four exons by three introns. The predicted protein consisted of 511 amino acid residues with M(r) 56,569. Penicillium enzyme had a hydrophobic signal peptide at the N-terminal region as did mammalian membrane-bound alpha-mannosidases, but in this case a proteolytic cleavage occurred at Lys-35-Ser-36 to remove the signal sequence during cell growth. Parts of amino acid sequences were similar to those of mammalian Golgi alpha-mannosidase IA and IB, but the sequence around the aspartic acid residue which interacted with 1-deoxymannojirimycin (Yoshida et al. (1994) Biochem. J. 303, 97-103) was unique in Penicillium enzyme.

Primary structure of aspergillopepsin I deduced from nucleotide sequence of the gene and aspartic acid-76 is an essential active site of the enzyme for trypsinogen activation.

The coding region of the aspergillopepsin I (EC 3.4.23.18) gene occupies 1340 base pairs of the genomic DNA and is separated into four exons by three introns. The predicted amino-acid sequence of aspergillopepsin I consists of 325 residues and is 32% and 27% homologous with those of human pepsin and calf chymosin. The cDNA of the gene prepared from mRNA has been cloned and expressed in yeast cells. To identify the residue of the substrate binding pocket in determining the specificity of aspergillopepsin I towards basic substrates, this residue was replaced with a serine residue by site-directed mutagenesis. The mutation is a single amino-acid change, Asp-76 converted to Ser-D76S, in the enzyme. The striking feature of this is that only the trypsinogen activating activity was destroyed. We therefore concluded that Asp-76 is the binding site towards basic substrates.

Molecular cloning and nucleotide sequence of the complementary DNA for penicillolysin gene, plnC, and 18 kDa metalloendopeptidase gene from Penicillium citrinum.

A full-length cDNA encoding the penicillolysin, an 18 kDa metalloendopeptidase from Penicillium citrinum, was cloned. Analysis of the 1284 base pair nucleotide sequence of the cDNA revealed a single open reading frame coding for 351 amino acid residues. The coding region of penicillolysin gene, plnC, occupies 1053 base pairs of the cDNA. The sequence consists of a putative 19-residue signal sequence, a 155-residue propeptide segment, and the 177-residues of penicillolysin with a molecular weight of 18,529. The deduced primary structure of penicillolysin is unique and the enzyme is a member of a new metalloendopeptidase family. Two histidine residues, His-128 and His-132, and glutamic acid residue, Glu-65 in penicillolysin were assumed to correspond to zinc ligands in the homologous thermolysin.

Molecular cloning and nucleotide sequence of the 1,2-alpha-D-mannosidase gene, msdS, from Aspergillus saitoi and expression of the gene in yeast cells.

A full-length cDNA encoding 1,2-alpha-D-mannosidase (EC 3.2.1.113) from Aspergillus saitoi was cloned. Analysis of the 1718 bp nucleotide sequence of the cDNA revealed a single open reading frame with 1539 nucleotides of 1,2-alpha-D-mannosidase gene, msdS. The predicted amino-acid sequence of 1,2-alpha-D-mannosidase consists of 513 residues with a molecular mass of 55,767 and is 70%, 26% and 35% identity with those of Penicillium citrinum 1,2-alpha-D-mannosidase, yeast alpha-mannosidase, and mouse alpha-mannosidase. The cDNA of the msdS gene has been cloned and expressed in yeast cells. To identify the activity of expression product methyl-2-O-alpha-mannopyranosyl-alpha-mannopyranoside (Man alpha 1-->2Man-OMe) was used as a substrate at pH 5.0.

Purification of an acid proteinase from Aspergillus saitoi and determination of peptide bond specificity.

The specificity and mode of action of an acid proteinase (EC 3.4.23.6) from Aspergillus saitoi were investigated with oxidized B-chain of insulin, angiotensin II and bradykinin. Further purification of acid proteinase was performed with N,O-dibenzyloxycarbonyl-tyrosine hexamethylene-diamino-Sepharose 4B affinity chromatography and isoelectric focusing. The purified enzyme was free of any other proteolytic activity demonstrated in Asp. saitoi. Acid proteinase from Asp. saitoi hydrolyzed primarily two peptide bonds in the oxidized B-chain of insulin, the Leu(15)-Tyr(16) bond and the Phe(24)-Phe(25) bond. Additional cleavages of the bonds His(10)-Leu(11), Ala(14)-Leu(15) and Tyr(16)-Leu(17) were also noted. Primary splitting sites at Leu(15)-Tyr(16) and Phe(24-)-Phe(25) with acid proteinase from Asp. saitoi were identical with those reported in the work of cathepsin D (EC 3.4.23.5) from human erythrocyte. Hydrolysis of angiotensin II was observed at the Tyr(4)-Ile(5) bond. In conclusion, peptide bonds which have a hydrophobic amino acid such as phenylalanine, tyrosine, leucine and isoleucine in the P'1 position (as defined by Berger and Schechter, [29]) are preferentially cleaved by the trypsinogenactivating acid proteinase from Asp. saitoi.

Multiple forms of protyrosinase from Aspergillus oryzae and their mode of activation at pH 3.0.

This paper describes the isolation of three molecular forms (I-III) of protyrosinase and catalytically active tyrosinase ( monophenol ,dihydroxyphenylalanine: oxygen oxidoreductase, E.C. 1.14.18.1) from fresh mycelia of Aspergillus oryzae BIR 128 by (NH4)2SO4 fractionation, successive chromatographies and disc-gel electrophoresis. Experimental evidence is presented that purified protyrosinase is contaminated with firmly attached proteinases, and that this contamination may account for both the multiple molecular forms of protyrosinase /tyrosinase and the activation of the proenzyme at acidic pH (2.5-3.0). The activation process of protyrosinase seems to be the result of a cleavage of the polypeptide chain by aspartic proteinase from Aspergillus (EC 3.4.23.6) in the purified protyrosinase preparation. Protyrosinase and tyrosinase have different conformation, different stabilities and some different properties.

Specificities of extracellular and ribosomal serine proteinases from Bacillus natto, a food microorganism.

The specificities of extracellular and ribosomal serine proteinase from Bacillus natto, a food microorganism, were investigated. Both proteins have highly restricted and characteristic specificities. With the extracellular serine proteinase, initial cleavage site was observed at Leu15-Tyr16, secondary site at Ser9-His10 and additional cleavage sites at Gln4-His5 and His5-Leu6 in the oxidized insulin B-chain. Hydrolysis of proangiotensin with the extracellular serine proteinase was observed primarily at Phe8-His9 and secondary at Tyr4-Ile5. The extracellular serine proteinase has a Km of 0.08 mM and kcat of 3 S-1 for angiotensin hydrolysis. With the ribosomal proteinase, initial cleavage site of the oxidized insulin B-chain was observed at Leu15-Tyr16 and additional cleavage site at Phe24-Phe25. Hydrolysis of proangiotensin was observed at Tyr4-Ile5 bond with the ribosomal proteinase.

Action of crystalline acid carboxypeptidase from Penicillium janthinellum.

Acid carboxypeptidase (EC 3.4.12.-) crystallized from culture filtrate of Penicillium janthinellum has been investigated for its use in carboxy-terminal sequence determination of Z-Gly-Pro-Leu-Gly, Z-Gly-Pro-Leu-Gly-Pro, angiotensin I, native lysozyme, native ribonuclease T1, and reduced S-carboxy-methyl-lysozyme. The examination indicated that proline and glycine were liberated from Z-Gly-Pro-Leu-Gly-Pro. At high enzyme concentration, the enzyme catalyzed complete sequential release of amino acids from the carboxy-terminal leucine to the amino-terminal aspartic acid of angiotensin I. The enzyme released the carboxy-terminal leucine from native lysozyme, however, no release of the threonine from native ribonuclease T1 was observed after a prolonged period of incubation with the enzyme. The sequence of the first nine carboxy-terminal residues of denatured lysozyme, leucine, arginine, S-carboxymethyl-cysteine, glycine, arginine, isoleucine, tryptophane, alanine, and glutamine, could be deduced unequivocally from a time release plot of an incubation mixture with the enzyme.

Purification of an acidic alpha-D-mannosidase from Aspergillus saitoi and specific cleavage of 1,2-alpha-D-mannosidic linkage in yeast mannan.

An acidic alpha-D-mannosidase (alpha-D-mannoside mannohydrolase, EC 3.2.1.24) has been isolated from culture filtrate of Aspergillus saitoi. The extracellular alpha-mannosidase was homogeneous in polyacrylamide gel electrophoresis. The molecular weight of the enzyme was 51 000 and the isoelectric point pH 4.5. The purified enzyme has a pH optimum of 5.0, a Km of 0.45 mM with baker's yeast mannan and has no activity towards p-nitrophenyl-alpha-D-mannoside. The mode of action of the enzyme has been studied with baker's yeast mannan and saké yeast mannan. The enzyme cleaves specifically the 1,2-alpha-linked side chain, producing free mannose.

Initial sites of insulin cleavage and stereospecificity of carboxyl proteinases from Aspergillus sojae and Pycnoporus coccineus.

Initial cleavage sites of native insulin at a pH of about 3 and stereospecificity were investigated by fungal carboxyl proteinases (EC 3.4.23.6) from ASpergillus sojae, a species of fungi imperfecti, and Pycnoporus coccineus (formerly designated Trametes sanguinea), a wood deteriorating Basidiomycete, respectively. Fungal carboxyl proteinases were used as a model of vertebrate insulin degradation. A. sojae carboxyl proteinase I primarily hydrolyzed two peptide bonds located on the surface of native insulin monomer, the B16-B17 (Tyr-Leu) and B24-B25 (Phe-Phe) bonds, and secondarily the buried bonds, A15-A16 (Gln-Leu), B15-B16 (Leu-Tyr) and B14-B15 (ala-Leu), at pH 3.2 and 30 degree C. The initial cleavage sites of A. sojae carboxyl proteinases I towards native insulin were not identical with the initial cleavage sites towards the oxidized B chain of insulin. P. coccineus carboxyl proteinase Ia selectively hydrolyzed B14-B15 (Ala-Leu), B16-B17 (Tyr-Leu) and B24-B25 (Phe-Phe) bonds in the native insulin at pH 2.7. Based on these findings we suggest that the stereospecificity of the fungal carboxyl proteinases is similar to that of cathepsin D (EC 3.4.23.5), and that the synthesis and degradation of insulin may occur in microorganisms.

Involvement of cell wall beta-glucan in the action of HM-1 killer toxin.

HM-1 killer toxin secreted from Hansenula mrakii inhibits the growth of Saccharomyces cerevisiae cells by interfering with beta-1,3-glucan synthesis. We found that HM-1 killer toxin killed intact cells but not protoplasts. In addition, cells lacking the functional KRE6 allele (kre6 delta) became resistant to higher concentration of HM-1 killer toxin. As reported by Roemer and Bussey [(1991) Proc. Natl. Acad. Sci. 88 11295-11299], cells lacking functional KRE6 had a reduced level of the cell wall beta-1,6-glucan compared to that in cells harboring the normal KRE6. These results suggest that the cell wall beta-glucan is involved in the action of HM-1 killer toxin. Addition of HM-1 killer toxin with several kinds of oligosaccharides revealed that either beta-1,3- or beta-1,6-glucan blocked the cytocidal action of HM-1 killer toxin whereas alpha-1,4-glucan and chitin did not. Mannan also interfered with HM-1 killer toxin action, but this inhibitory effect was much weaker than that observed with beta-1,3- or beta-1,6-glucans. Thus, it appears that the cell wall beta-glucan interacts with HM-1 killer toxin, and that this toxin-beta-glucan commitment is required for the action of HM-1 killer toxin.

Characterization of the S1 subsite specificity of aspergillopepsin I by site-directed mutagenesis.

The structural determinants of S1 substrate specificity of aspergillopepsin I (API; EC 3.4.23.18), an aspartic proteinase from Aspergillus saitoi, were investigated by site-directed mutagenesis. Aspartic proteinases generally favor hydrophobic amino acids at P1 and P1'. However, API accommodates a Lys residue at P1, which leads to activation of trypsinogen. On the basis of amino acid sequence alignments of aspartic proteinases, Asp-76 and Ser-78 of API are conserved only in fungal enzymes with the ability to activate trypsinogen, and are located in the active-site flap. Site-directed mutants (D76N, D76E, D76S, D76T, S78A, and delta S78) were constructed, overexpressed in Escherichia coli cells and purified for comparative studies using natural and synthetic substrates. Substitution of Asp-76 to Ser or Thr and deletion of Ser-78, corresponding to the mammalian aspartic proteinases, caused drastic decreases in the activities towards substrates containing a basic amino acid residue at P1. In contrast, substrates with a hydrophobic residue at P1 were effectively hydrolyzed by each mutant enzyme. These results demonstrate that Asp-76 and Ser-78 residues on the active site flap play important roles in the recognition of a basic amino acid residue at the P1 position.

Alkaline-resistance model of subtilisin ALP I, a novel alkaline subtilisin.

The alkaline-resistance mechanism of the alkaline-stable enzymes is not yet known. To clarify the mechanism of alkaline-resistance of alkaline subtilisin, structural changes of two typical subtilisins, subtilisin ALP I (ALP I) and subtilisin Sendai (Sendai), were studied by means of physicochemical methods. Subtilisin NAT (NAT), which exhibits no alkaline resistance, was examined as a control. ALP I gradually lost its activity, accompanied by protein degradation, but, on the contrary, Sendai was stable under alkaline conditions. CD spectral measurements at neutral and alkaline pH indicated no apparent differences between ALP I and Sendai. A significant difference was observed on measurement of fluorescence emission spectra of the tryptophan residues of ALP I that were exposed on the enzyme surface. The fluorescence intensity of ALP I was greatly reduced under alkaline conditions; moreover, the reduction was reversed when alkaline-treated ALP I was neutralized. The fluorescence spectrum of Sendai remained unchanged. The enzymatic and optical activities of NAT were lost at high pH, indicating a lack of functional and structural stability in an alkaline environment. Judging from these results, the alkaline resistance is closely related to the surface structure of the enzyme molecule.

C-Terminal peptidyl-L-proline hydrolase activity of Aspergillus acid carboxypeptidase.

Aspergillus saitoi acid carboxypeptidase hydrolyzed C-terminal peptidyl-L-proline bonds and released the C-terminal proline from Z-Gly-Pro-Leu-Gly-Pro and Z-Gly-Pro at pH 3.3. Proline liberated by the enzymic reaction was measured by a sensitive colorimetric ninhydrin method in glacial acetic acid at 513 nm. A Km value of 1.0 mM and a kcat value of 0.09 s-1 for Z-Gly-Pro-Leu-Gly-Pro hydrolysis, and a Km value of 5.0 mM and a kcat value of 0.0045 s-1 for Z-Gly-Pro hydrolysis were calculated from Lineweaver-Burk plots.

A heat-labile serine proteinase from Penicillium citrinum.

A serine proteinase from Penicillium citrinum was purified. The M(r) and isoelectric point were determined as about 26,000 and 9.5, respectively. Activity was retained up to above 40 degrees at pH 7 for 30 min, but the enzyme was completely inactivated at 50 degrees. The first amino acids in the N-terminal region were ANVVQSNVPSWGLARISSKRPGTTSYTYDSTAGEGVVFYGVDTG. The specificity differs from that of other serine proteinases. Kinetic studies on fluorogenic substrates were determined.

Specificity and molecular properties of penicillolysin, a metalloproteinase from Penicillium citrinum.

The specificity and mode of action of penicillolysin, a metalloproteinase from Penicillium citrinum, were investigated with several bioactive-oligopeptides. The enzyme showed a high affinity toward the Pro-X (X = Gln, Lys, Leu or Arg) bonds of substance P, dynorphin A (1-13), neurotensin and chicken brain pentapeptide, and the R-R bonds in dynorphin A and neurotensin. Preferential cleavage of bonds by the enzyme with hydrophobic amino acid residues at the P1 position were observed on the peptides used. The specificity of penicillolysin differs from that of other metalloproteinases. The M(r) and pI were determined as 18,000 and 9.6, respectively. The first 50 amino acids in the N-terminal region were TKETCSNASRKSALEKALSNTVKLANAAATAARSGSASKFSEYEKTTSSS. CD spectra on the hollo- and apo-enzymes of penicillolysin were studied.

Molecular and enzymatic properties of an aspartic proteinase from Rhizopus hangchow.

An aspartic proteinase, rhizopuspepsin (EC 3.4.23.21), from Rhizopus hangchow was purified. The M(r) and isoelectric point were determined as ca 37,000 and 4.5, respectively. The first 19 amino acids in the N-terminal region were SGSGVVPMTDYEYDIEYYG. The contents of the alpha-helix, beta-structure and random coil were calculated to be ca 7.5, 88.9 and 2.7%, respectively. The enzyme can activate trypsinogen at pH 3.0. The activity was completely inactivated by pepstatin A. The specificity and mode of action of the enzyme were investigated with oxidized insulin B-chain at pH 3. The enzyme hydrolysed primarily two peptide bonds, the Leu15-Tyr16 bond and the Tyr16-Leu17 bond, while additional cleavage of the bonds, Ala14-Leu15 and Phe24-Phe25 was also noted.

A 1,2-alpha-D-mannosidase from a Bacillus sp.: purification, characterization, and mode of action.

A 1,2-alpha-D-mannosidase was purified to homogeneity from the culture supernatant of Bacillus sp. M-90, which was isolated from soil by enrichment culture on baker's yeast mannan. The purified enzyme had M(r) 380,000 Da, and was comprised of two apparently identical 190,000 Da subunits. It had a neutral optimum pH (7.0) and an isoelectric point of 3.6. The enzyme was highly specific for alpha 1,2-linked D-mannose oligosaccharides. An N-linked high-mannose type oligosaccharide, Man9GlcNAc2, was a good substrate, yielding Man5GlcNAc2, and the alpha 1,2-linked side chains of Saccharomyces cerevisiae mannan were also specifically hydrolyzed by the enzyme. p-Nitrophenyl alpha-D-mannopyranoside and 1,2-alpha-D-mannobiitol were not hydrolyzed at all. Calcium ion, 1-deoxyman-nojirimycin, and swainsonine had no effect on the enzyme, but the activity was completely inhibited by EDTA. The mode of action on alpha 1,2-linked mannotetraose indicated that the enzyme is an exo-1,2-alpha-D-mannanase.

Miltpain, new cysteine proteinase from the milt of chum salmon, Oncorhynchus keta.

A new cysteine proteinase, salmon miltpain, was isolated and purified from the milt of chum salmon (Oncorhynchus keta). Native molecular mass was estimated as 67,000 by gel filtration column chromatography (Shodex WS2003) and 22,300 by SDS-polyacrylamide gel electrophoresis. Isoelectoric point was determined to be 3.9 by isoelectric focusing. The first 15 amino acid residues in the N-terminal region were LPSFLY-AEMVGYNIL. The cysteine proteinase, which had a pH optimum of 6.0 for Z-Arg-Arg-MCA hydrolysis, required a thiol-reducing reagent for activation and was inhibited by E-64, iodacetamide, CA-074 Me, TLCK, TPCK and ZPCK. The cysteine proteinase exhibited unique substrate specificity toward paired basic residues such as Lys-Arg, Arg-Arg at the subsites of P2-P1 and had a K(m) of 16.3 microM and kcat of 20.3 s-1 with Z-Arg-Arg-MCA as substrate and a K(m) of 52.9 microM and kcat of 1.79 s-1 with Z-Phe-Arg-MCA. This proteinase was found to considerably hydrolyze basic proteins such as histone, salmine and clupaine but not milk casein.

Miltpain, a cysteine proteinase, from milt of Pacific cod (Gadus macrocephalus): purification and characterization.

Miltpain (EC.3.4.22.-) is a cysteine proteinase that preferentially hydrolyzes basic proteins, previously found in the milt of chum salmon. Here we report a similar cysteine proteinase in the milt of the marine Pacific cod. The enzyme was isolated and purified 6900-fold and with an estimated mass of 63 kDa by gel filtration chromatography and 72 kDa by SDS/PAGE. Cod miltpain has an optimum pH of 6.0 for Z-Arg-Arg-MCA hydrolysis, and Km of 11.5 microM and kcat of 19.0 s-1 with Z-Arg-Arg-MCA. It requires a thiol-inducing reagent for activation and is inhibited by E-64, iodoacetamide, CA-074, PCMB, NEM, TLCK, TPCK, ZPCK and o-phenanthroline. This proteinase strongly hydrolyzes basic proteins such as salmine, clupeine and histone, and exhibits unique substrate specificity toward paired basic residues such as Lys-Arg, Arg-Arg on the substrates of P2-P1. The isoelectric point is 5.2 by isoelectric focusing. N-Terminal sequencing gave a sequence of < EVPVEVVRXYVTSAPEK. The cysteine proteinase from Pacific cod very closely matches the previously reported miltpain from chum salmon.