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Pronase (type XXV serine protease from Streptomyces griseus) efficiently depolymerizes chitosan, a linear β→1,4-linked polysaccharide of 2-amino-deoxyglucose and 2-amino-2- N-acetylamino- d-glucose, to low-molecular weight chitosans (LMWC), chito-oligomers (degree of polymerization, 2–6) and monomer. The maximum depolymerization occurred at pH 3.5 and 37 °C, and the reaction obeyed Michaelis–Menten kinetics with a Km of 5.21 mg·mL−1 and Vmax of 138.55 nmoles·min−1·mg−1. The molecular mass of the major product, LMWC, varied between 9.0 ± 0.5 kDa depending on the reaction time. Scanning electron microscopy of LMWC showed an approximately eightfold decrease in particle size and characterization by infrared spectroscopy, circular dichroism, X-ray diffractometry and 13C-NMR revealed them to possess a lower degree of acetylation, hydration and crystallinity compared to chitosan. Chitosanolysis by pronase is an alternative and inexpensive method to produce a variety of chitosan degradation products that have wide and varied biofunctionalities. [ABSTRACT FROM AUTHOR]

The aminopeptidase from Streptomyces griseus is a calcium-activated metalloenzyme, which contains 2 mol tightly bound zinc/mol protein. This aminopeptidase rapidly hydrolyzes peptide bonds formed by N-terminal hydrophobic amino acids, such as leucine, methionine and phenylalanine. We have determined the complete primary structure of the protein, which contains 284 amino acid residues, yielding a molecular mass of 29723 Da. A search in the Swiss-Prot database for sequence similarities revealed a low degree of identity (26-34%) to Saccharomyces cerevisiae aminopeptidase Y. Aeromonas proteolytica aminopeptidase, and a hypothetical 49.5-kDa protein from Bacillus subtilis, which is supposed to belong to the aminopeptidase Y family. In all these proteins, the residues that are known to be involved in zinc coordination are conserved. [ABSTRACT FROM AUTHOR]

Streptomyces griseus aminopeptidase is a calcium-activated zinc metalloenzyme characterized by a high enzymic reactivity, high thermal stability and low molecular mass [Spungin, A. and Blumberg, S. (1989) Eur. J. Biochem. 183, 471-477]. A study of the specificity of S. griseus aminopeptidase using amino acid 4-nitroanilide substrates shows that the leucine derivative is the best substrate. Derivatives of other hydrophobic amino acids, methionine and phenylalanine, are also excellent substrates for the enzyme. The 4-nitroanilides of alanine, valine, proline and lysine are good substrates whereas those of the small size glycine and the acidic amino acids are very poor. No hydrolysis of a terminal Xaa residue can be detected with Xaa-proline N-terminal sequences. Calcium ions bind to the enzyme and modulate its activity in a substrate-dependent manner. The catalytically essential zinc of S. griseus aminopeptidase is removed by dialysis against 1,10-phenanthroline and replaced by manganese or cobalt ions, resulting in enzyme derivatives of altered specificities. Thus, whereas the zinc enzyme hydrolyzes leucine 4-nitroanilide at a 10-fold faster rate than the manganese or cobalt enzymes, the cobalt enzyme hydrolyzes alanine 4-nitroanilide at a more than 20-fold faster rate than the zinc or manganese enzymes. [ABSTRACT FROM AUTHOR]

A heat-stable aminopeptidase with an N-terminal Ala-Pro-Asp-Ile-Pro-Leu sequence has been purified from Streptomyces griseus by heat treatment followed by gel-exclusion and anion-exchange chromatographic procedures. The enzyme is a monomeric zinc metalloenzyme showing an apparent molecular mass of 33 kDa by sodium dodecyl sulfate/polyacrylamide gel electrophoresis and 21 kDa by gel filtration on Superose 12. Calcium ions bind to the enzyme, pKCa 4.5, and activate it about sixfold when the substrate is leucine-4-nitroanilide (0.4 mM in 50 mM Tris/HCl pH 8.0, 25 °C). Binding of Ca2+ also contributes to the thermal stability of the protein. This aminopeptidase may be useful for two-stage assays of bacterial and mammalian metalloendopeptidases; it may also serve in studies of proteolytic enzyme activation by calcium ions. [ABSTRACT FROM AUTHOR]

Streptomyces griseus aminopeptidase is a zinc metalloenzyme containing 2 mol zinc/mol protein, similar to the homologous enzyme Aeromonas proteolytica aminopeptidase. In addition, a unique Ca2+-binding site has been identified in the Streptomyces enzyme, which is absent in the Aeromonas enzyme. Binding of Ca2+ enhances stability of the Streptomyces enzyme and modulates its activity and affinity towards substrates and inhibitors in a structure-dependent manner. Among the three hydrophobic 4-nitroanilides of alanine, valine and leucine, the latter displays the largest overall activation (increase in kcat/Km). Large enhancements in affinity (1/Ki) upon Ca2+ binding have been observed for inhibitors with flexible (leucine-like) residues at their N-termini and smaller enhancements for inhibitors with rigid (phenylalanine-like) residues. [ABSTRACT FROM AUTHOR]

The fusion glycoprotein (F0) was isolated from Newcastle disease virus (NDV) particles metabolically labelled with [2-3H]mannose; it was successively digested with protease and with endo-β-N-acetylglucosaminidase from Streptomyces griseus. In this manner, the majority of the oligosaccharides in NDV F0 could be liberated. After reduction with NaBH4, they were separated by high-performance liquid chromatography, and were subjected to structural analysis. Using micromethylation/capillary gas chromatography/mass fragmentography, α-mannosidase digestion, and acetolysis, it was found that the enzymatically released NDV F0 oligosaccharides are common oligomannosidic glycoprotein glycans of size classes (Man)8GlcNAc, (Man)7GlcNAc, (Man)6GlcNAc, (Man)9GlcNAc, and (Man)5GlcNAc (in order of prevalence). The major structural isomers present in the NDV F0 (Man)8GleNAc to (Man)5GlcNAc fractions were shown to lack mannose residues D2, D1 D2 or D2 D3, D1D2D3, and CD1D2D3, respectively, of (Man)9GlcNAc: [This symbol cannot be presented in ASCII format] [ABSTRACT FROM AUTHOR]

A number of peptides of the general formula Ac-Pro-Ala-X-Phe-NH2, where X = Gly, Ala, Leu, Phe or Pro, have been synthesized for the study of S2-P2 enzyme-substrate interactions in three serine proteases: α-chymotrypsin and Streptomyces griseus proteases 1 and 3. All three enzymes interacted favorably with those peptides with hydrophobic, non-aromatic, P2 amino acid residues, leucine being optimal. The increase in kcat/Km on going from the P2 glycine to the P2 leucine peptide was 30-fold in α-chymotrypsin and greater than 150-fold and 350-fold in the Streptomyces enzymes, being due to both increased affinities and higher acylation rates. Hydrophobic S2-P2 interactions, particularly in S. griseus proteases 1 and 3, appear to be the most important enzyme-substrate interactions apart from S1-P1. The kinetic data is discussed in relation to the tertiary structures of the active centers of the enzymes. [ABSTRACT FROM AUTHOR]

dTDP-L-dihydrostreptose:streptidine-6-phosphate dihydrostreptosyltransferase, an enzyme in- volved in the biosynthesis of streptomycin, has been purified from Streptomyces griseus to near homogeneity by a six-step procedure involving chromatography on streptidine-6-phosphate- Sepharose. By gel filtration the apparent Mr of the enzyme was found to be about 63000. The subunit Mr found on sodium dodecylsulfate gels is about 35000. The transferase is dependent on Mn2+ or Mg2+ ions. Co2+ is as effective as Mg2+. From the substrates tested only streptidine 6-phosphate was an acceptor for dihydrostreptose in the synthesis of O-α-L-dihydrostreptose(1→4)- streptidine 6-phosphate. No activity was found with streptidine, 2-deoxystreptamine and 4-deoxystreptamine. The activity of the transferase in the course of fermentation runs parallel to the activity of dTDP-dihydrostreptose synthase and reaches a maximum after around 50h of fermentation, just before appearance of streptomycin in the medium. [ABSTRACT FROM AUTHOR]

A conidium-producing variant of Streptomyces griseus, strain 45-H, produces a substance, factor C, which is capable of inducing conidium formation in the hyphae of a conidium-non-producing mutant, strain 52-1. Factor C can be determined quantitatively on the basis of this biological effect. The biologically active substance can be purified by ion-exchange chromatography on cellulose phosphate combined with affinity chromatography on DNA-agarose. The purified substance is concentrated at least 1700 times. The molecular weight of factor C, estimated by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate, is about 34500. On determining the amino acid composition of factor C 60% of the amino acids were found to be hydrophobic. [ABSTRACT FROM AUTHOR]

1. Boric acid acts as a competitive inhibitor of the Streptomyces griseus protease-3-catalyzed hydrolysis of p-nitrophenylacetate, indicating that there is no significant interaction between inhibitor and the acyl-enzyme formed during the reaction. 2. Boric acid affects the rate of the transient burst of p-nitrophenol formation from p-nitrophenylacetate in a way suggesting that the inhibitor binds equally firmly to the free enzyme and the Michaelis complex formed between enzyme and substrate. The same conclusion can be drawn from the observation that boric acid acts as a non-competitive inhibitor of the enzymatic hydrolysis of glutaryl-L-phenylalanine-p-nitroanilide. 3. Apparent equilibrium constants for the binding of boric acid to the free enzyme and the Michaelis complex have been determined kinetically at different pH between 5 and 10. The data obtained provide evidence that the stability of the enzyme, inhibitor complex is dependent upon an ionizing group in the protein with apparent pKa of 6.6, and further show that there is no interaction between enzyme and the ionized form of the inhibitor. 4. The above results suggest that boric acid has no effect on the process of Michaelis-complex formation, but inhibits enzyme activity by binding covalently to the active-site seryl residue under formation of a tetrahedral adduct, which might represent a transition-state analogue for the enzymatic conversion of bound substrate into bound product. It is concluded that the nucleophilic reactivity of the active-site seryl residue is unaffected by the binding of p-nitrophenylacetate to the protein, and that Streptomyces griseus protease 3 may be considered as homologous with chymotrypsin as concerns the structure and function of the catalytic site. [ABSTRACT FROM AUTHOR]

The kinetics of the Streptomyces griseus protease-3-catalyzed hydrolysis of the non-specific ester substrate p-nitrophenylacetate and the specific peptide substrate glutaryl-L-phenylalaninep-nitroanilide are consistent with a three-step mechanism of Michaelis complex formation followed by enzyme acylation and deacylation. Both substrates pre-equilibrate rapidly with the free enzyme, the acylation (deacylation) step being rate-limiting in the enzymatic hydrolysis of glutarylL-phenylalanine-p-nitroanilide (p-nitrophenylacetate). Examination of the effect of pit on steady-state and transient-state kinetic parameters for the enzymatic hydrolysis of the two substrates provides evidence that the stability of the Michaelis complex is essentially unaffected by pH over the range investigated, whereas both acylation and deacylation of the enzyme is dependent upon all ionizing group in the protein with apparent pKa of 6.6. It is concluded that closely similar mechanisms of activation of the reactive seryl residue may be operative in Streptomyces griseus protease 3 and pancreatic serine proteases such as chymotrypsin and elastase. [ABSTRACT FROM AUTHOR]

1. When thymidine diphospho-D-[U-14C]glucose was incubated with a cell-free extract from a streptomycin-producing strain of Streptomyces griseus in the presence of NADPH, radioactive dTDP-dihydrosteptose was formed in addition to radioactive dTDP-4-keto-6-deoxyglucose and dTDP-rhamnose. Dihydrostreptose was identified by chromatographic, gas chromatographic and electrophoretic comparison with synthetic dihydrostreptose characterized by its nuclear magnetic resonance spectrum. 2. dTDP-dihydrostreptose is very labile and decomposes to dihydrostreptose monophosphate and further to free dihydrostreptose. 3. dTDP-dihydrostreptose and dTDP-rhamnose were also obtained when dTDP-4-keto 6-deoxyglucose was the substrate of the reaction. Dihydrostreptose synthesis could be inhibited completely by 1 mM p-chloromercuribenzoate, whereas rhamnose formation was not inhibited. 4. In the course of fermentation the activities of arginine: x-amidinotransferase, an enzyme involved in streptomycin biosynthesis, and of the enzymatic activity for dTDP-dihydrostreptose formation ran parallel and reached a maximum before onset of streptomycin formation. 5. A good correlation between the activity of "dTDP-dihydrostreptose synthase", arginine :x-amidinotransferase activity and streptomycin production was found in several strains of S. griseus. No dihydrostreptose formation could be detected in a hydroxystreptomycin-producing strain of S. griseocarneus. 6. Streptomycin but not dihydrostreptomycin could be detected in the fermentation medium of the S. griseus strain used for preparation of the cell-free extract. The role of dTDP-dihydrostreptose in streptomycin biosynthesis is at present unknown. [ABSTRACT FROM AUTHOR]

The kinetics of the Streptomyces griseus protease-3-catalyzed hydrolysis of p-nitrophenylacetate have been studied by stopped-flow techniques with widely varied initial concentrations of enzyme and substrate. The results obtained are consistent with a three-step mechanism in which there is a rapid equilibration between enzyme and substrate to form a Michaelis complex with a dissociation constant in the order of 10 mM, followed by a moderately rapid formation of an acyl-enzyme and a rate-limiting deacylation of this intermediate. Estimated rate constants for the latter two reaction steps are 15 s-1 and 0.09 s-1, respectively. The presence of a nucleophile such as methanol results in a partitioning of the deacylation process, reflected by a linear dependence of the maximum steady-state reaction velocity on the concentration of methanol. This observation provides confirmatory evidence for a rate-limiting deacylation step in the catalytic mechanism. [ABSTRACT FROM AUTHOR]

1. The elastolytic activities of porcine and Streptomyces griseus elastase are inhibited by increased salt concentrations while the proteolytic and esterolytic activities are unaffected. This inhibition results from prevention of adsorption of the latter enzymes on insoluble elastin. Elastase and other basic proteins are adsorbed on elastin in low ionic strength, at pH range of 5-10, whereas acidic proteins are not. The adsorption is salt-dependent, non-specific and electrostatic in nature It results from the formation of salt bonds between the carboxyl groups of elastin and the positively charged groups of the basic proteins. 2. Basic poly(amino acids), that inhibit the elastolytic activity but not the proteolytic or esterolytic activity of porcine elastase by preventing its adsorption on elastin, are also themselves adsorbed on elastin similarly to the natural basic proteins. 3. It was concluded that the elastolytic activity of any basic proteinase depends on two basic properties ability to be adsorbed on elastin in pH range of 7-10, and side-chain specificity toward the non-polar amino acids that form the bulk of amino acid of elastin. [ABSTRACT FROM AUTHOR]

1. Three elastase-like enzymes, designated Enzyme I, II and III, were purified from a commercial preparation of Streptomyces griseus (pronase) by successive chromatography on DEAEcellulose at pH 9.5 and CM-cellulose at pH 5.5. 2. Electrophoresis on cellulose acetate at pH 6.9, on polyacrylamide gels at pH 4.5 and 9.5 and exclusion chromatography on Sephadex G-75 indicated that all three fractions were essentially homogenous. 3. The amino acid composition of the bee enzymes was determined and their molecular weights were estimated by gel-chromatographic methods. 4. All three enzymes possess strong esterolytic activity when assayed on a specific elastase substrate N-acetyl-L-alanyl-L-alanyl-L-alanine methyl ester (AcAla3OMe) and are active, to a smaller extent, on N-acetyl-L-tyrosine ethyl ester. Their activity on α-N-benzoyl-L-arginine ethyl ester was negligible. All the enzymes have strong proteolytic activity at slight alkaline pH and Enzyme II and II possess also an elastolytic activity on congo red elastin. 5. All three enzymes are inhibited by diisopropylfluorophosphate. Enzymes II and III were slowly inhibited by N-tosyl-L-phenylalanine chloromethane. Enzyme I was inhibited by the esterolysis products of AcAla3OMe, thus indicating that the same active site controls both esterolytic activities. [ABSTRACT FROM AUTHOR]

The binding of the aminoglycoside antibiotic dihydrostreptomycin to defined cell-wall teichoic acids and to lipoteichoic acid isolated from various gram-positive eubacteria was followed by equilibrium dialysis. Dihydrostreptomycin was used at a wide range of concentration under different conditions of ionic strength, concentration of teichoic acid, presence of cationic molecules like Mg2+, spermidine, other aminoglycoside antibiotics (gentamicin, neomycin, paromomycin). Interaction of dihydrostreptomycin with teichoic acid was found to be a cooperative binding process. The binding characteristics seem to be dependent on structural features of teichoic acid and are influenced by cationic molecules. Mg2+, spermidine and other aminoglycosides antibiotics inhibit the binding of dihydrostreptomycin to teichoic acid competitively. The binding of aminoglycosides to teichoic acids is considered as a model system for the interaction of aminoglycoside antibiotics with cellular polyanions. Conclusions of physiological significance are drawn.