ABSTRACT
The insulinotropic hormone glucagon-like peptide-1 is metabolised extremely rapidly by the ubiquitous enzyme dipeptidyl peptidase IV (DPP-IV). Therefore, human DPP-IV is a key regulator involved in the prevention and treatment of type 2 diabetes. To simplify the method of producing an inhibitory peptide against DPP-IV, we focused on rice bran (RB) as a source and subjected proteins from defatted RB to enzymatic proteolysis using 2 commercial enzymes. The RB peptides produced with Umamizyme G exhibited 10 times the inhibitory activity as those produced with Bioprase SP. The half-maximal inhibitory concentration (IC(50)) value of the RB peptides was 2.3 ± 0.1mg/ml. Leu-Pro and Ile-Pro were identified as the inhibitory peptides among the RB peptides produced with Umamizyme G. Ile-Pro was the strongest DPP-IV inhibitor among the 15 Xaa-Pro dipeptides and Pro-Ile tested. Ile-Pro competitively inhibited DPP-IV (K(i)=0.11 mM). Mass spectrometry indicated that the contents of Leu-Pro and Ile-Pro in the RB peptides were 2.91 ± 0.52 µg/mg.
Subject(s)
Dipeptidyl-Peptidase IV Inhibitors/chemistry , Oryza/chemistry , Peptides/chemistry , Plant Extracts/chemistry , Dipeptidyl Peptidase 4/chemistry , Dipeptidyl Peptidase 4/metabolism , Dipeptidyl-Peptidase IV Inhibitors/isolation & purification , Humans , Kinetics , Peptides/isolation & purification , Plant Extracts/isolation & purification , Seeds/chemistryABSTRACT
We introduce a highly potent fibrinolytic serine protease from Streptomyces omiyaensis (SOT), which belongs to the trypsin family. The fibrinolytic activity of SOT was examined using in vitro assays and was compared with those of known fibrinolytic enzymes such as plasmin, tissue-type plasminogen activator (t-PA), urokinase, and nattokinase. Compared to other enzymes, SOT showed remarkably higher hydrolytic activity toward mimic peptides of fibrin and plasminogen. The fibrinolytic activity of SOT is about 18-fold higher than that of plasmin, and is comparable to that of t-PA by fibrin plate assays. Furthermore, SOT had some plasminogen activator-like activity. Results show that SOT and nattokinase have very different fibrinolytic and fibrinogenolytic modes, engendering significant synergetic effects of SOT and nattokinase on fibrinolysis. These results suggest that SOT presents important possibilities for application in the therapy of thrombosis.
Subject(s)
Fibrinolytic Agents/metabolism , Serine Proteases/metabolism , Streptomyces/enzymology , Thrombolytic Therapy/methods , Fibrin/metabolism , Fibrinolysin/metabolism , Fibrinolysis , Fibrinolytic Agents/therapeutic use , Fluorescence Resonance Energy Transfer , Humans , Subtilisins/metabolism , Tissue Plasminogen Activator/metabolism , Urokinase-Type Plasminogen Activator/metabolismABSTRACT
The synthesis of diverse DL-configuration dipeptides in a one-pot reaction was demonstrated by using a function of the aminolysis reaction of a D-stereospecific amidohydrolase from Streptomyces sp., a clan SE, S12 family peptidase categorized as a peptidase with D-stereospecificity. The enzyme was able to use various aminoacyl derivatives, including L-aminoacyl derivatives, as acyl donors and acceptors. Investigations of the specificity of the peptide synthetic activity revealed that the enzyme preferentially used D-aminoacyl derivatives as acyl donors. In contrast, L-amino acids and their derivatives were preferentially used as acyl acceptors. Consequently, the synthesized dipeptides had a DL-configuration when D- and L-aminoacyl derivatives were mixed in a one-pot reaction. This report also describes that the enzyme produced cyclo(D-Pro-L-Arg), a specific inhibitor of family 18 chitinase, with a conversion rate for D-Pro benzyl ester and L-Arg methyl ester to cyclo(D-Pro-L-Arg) of greater than 65%. Furthermore, based on results of cyclo(D-Pro-L-Arg) synthesis, we propose a reaction mechanism for cyclo(D-Pro-L-Arg) production.
Subject(s)
Amidohydrolases/metabolism , Dipeptides/chemistry , Dipeptides/metabolism , Streptomyces/enzymology , Streptomyces/metabolism , Mass Spectrometry , Models, Biological , Stereoisomerism , Substrate SpecificityABSTRACT
We recently identified residue 71 of two homologous serine proteases from Streptomyces omiyaensis (SOT) and Streptomyces griseus (SGT) as a crucial residue for differences in their topological specificities, i.e. recognition of a distinct three-dimensional structure. To study the role of this key residue in substrate recognition, we used surface plasmon resonance analysis to evaluate the affinities of inactive mutants, in which residues 71 of SOT and SGT were substituted respectively with Leu and Tyr, toward different types of collagens. We identified another amino acid residue involved in the interaction with collagens from analyses of inactive chimeras between SOT and SGT using an in vivo DNA shuffling system. Results showed that residue 72 contributes to collagen binding. By substituting Leu71 and Gln72 with Tyr and Arg, respectively, SGT mutant showed a change in topological specificity and high hydrolytic activity toward type IV collagen comparable to SOT. We demonstrated that the neighboring residues 71 and 72 in the N-terminal ß-barrel domain of the enzyme synergistically play an important role in substrate recognition.
Subject(s)
Streptomyces/enzymology , Trypsin/chemistry , Trypsin/metabolism , Amino Acid Sequence , Collagen/metabolism , Models, Molecular , Molecular Sequence Data , Mutagenesis, Site-Directed , Mutant Proteins/chemistry , Mutant Proteins/metabolism , Protein Binding , Protein Conformation , Sequence Analysis, Protein , Sequence Homology, Amino Acid , Streptomyces/genetics , Streptomyces/metabolism , Streptomyces griseus/enzymology , Streptomyces griseus/genetics , Streptomyces griseus/metabolism , Substrate Specificity , Trypsin/geneticsABSTRACT
Despite the widespread industrial applications of ß-mannanase, the relations between the enzymatic properties and metal ions remain poorly understood. To elucidate the effects of metal ions on ß-mannanase, thermal stability and hydrolysis activity were characterized. The stman and tfman genes encoding ß-mannanase (EC.3.2.1.78) from Streptomyces thermolilacinus NBRC14274 and Thermobifida fusca NBRC14071 were cloned and expressed in Escherichia coli. The thermal stability of each enzyme shifted to the 7-9°C high temperature in the presence of Ca(2+) compared with that in the absence of Ca(2+). These results show that the thermal stability of StMan and TfMan was enhanced by the presence of Ca(2+). StMan, but not TfMan, required Ca(2+) for the hydrolysis activity. To identify the Ca(2+) sensitive region of StMan, we prepared eight chimeric enzymes. Based on the results of the relationship between Ca(2+) and hydrolysis activity, the region of amino-acid residues 244-349 of StMan was responsible for a Ca(2+) sensitive site.
Subject(s)
Calcium/chemistry , Streptomyces/enzymology , beta-Mannosidase/chemistry , Amino Acid Sequence , Calcium/metabolism , Cloning, Molecular , Hydrolysis , Molecular Sequence Data , Temperature , beta-Mannosidase/genetics , beta-Mannosidase/metabolismABSTRACT
The reducing tetrasaccharide TMG-chitotriomycin (1) is an inhibitor of ß-N-acetylglucosaminidase (GlcNAcase), produced by the actinomycete Streptomyces anulatus NBRC13369. The inhibitor shows a unique inhibitory spectrum, that is, selectivity toward enzymes from chitin-containing organisms such as insects and fungi. Nevertheless, its structure-selectivity relationship remains to be clarified. In this study, we conducted a structure-guided search of analogues of 1 in order to obtain diverse N,N,N-trimethylglucosaminium (TMG)-containing chitooligosaccharides. In this approach, the specific fragmentation profile of 1 on ESI-MS/MS analysis was used for the selective detection of desired compounds. As a result, two new analogues, named TMG-chitomonomycin (3) and TMG-chitobiomycin (2), were obtained from a culture filtrate of 1-producing Streptomyces. Their enzyme-inhibiting activity revealed that the potency and selectivity depended on the degree of polymerization of the reducing end GlcNAc units. Furthermore, a computational modeling study inspired the inhibitory mechanism of TMG-related compounds as a mimic of the substrate in the Michaelis complex of the GH20 enzyme. This study is an example of the successful application of a MS/MS experiment for structure-guided isolation of natural products.
Subject(s)
Acetylglucosaminidase/antagonists & inhibitors , Enzyme Inhibitors/chemistry , Glucosamine/analogs & derivatives , Oligomycins/chemistry , Acetylglucosaminidase/chemistry , Aspergillus oryzae/drug effects , Aspergillus oryzae/enzymology , Enzyme Inhibitors/pharmacology , Glucosamine/chemistry , Models, Molecular , Molecular Structure , Protein Structure, Tertiary , Spectrometry, Mass, Electrospray Ionization , Streptomyces coelicolor/drug effects , Streptomyces coelicolor/enzymology , Structure-Activity Relationship , Tandem Mass SpectrometryABSTRACT
A new S9 family aminopeptidase derived from the actinobacterial thermophile Acidothermus cellulolyticus was cloned and engineered into a transaminopeptidase by site-directed mutagenesis of catalytic Ser(491) into Cys. The engineered biocatalyst, designated aminolysin-A, can catalyze the formation of peptide bonds to give linear homo-oligopeptides, hetero-dipeptides, and cyclic dipeptides using cost-effective substrates in a one-pot reaction. Aminolysin-A can recognize several C-terminal-modified amino acids, including the l- and d-forms, as acyl donors as well as free amines, including amino acids and puromycin aminonucleoside, as acyl acceptors. The absence of amino acid esters prevents the formation of peptides; therefore, the reaction mechanism involves aminolysis and not a reverse reaction of hydrolysis. The aminolysin system will be a beneficial tool for the preparation of structurally diverse peptide mimetics by a simple approach.
Subject(s)
Actinomycetales/enzymology , Aminopeptidases/metabolism , Anti-Bacterial Agents/chemistry , Biocatalysis , Oligopeptides/chemistry , Puromycin/analogs & derivatives , Anti-Bacterial Agents/metabolism , Anti-Bacterial Agents/pharmacology , Microbial Viability/drug effects , Molecular Structure , Oligopeptides/metabolism , Phylogeny , Puromycin/metabolism , Puromycin/pharmacologyABSTRACT
X-prolyl dipeptidyl aminopeptidases (X-PDAPs) are useful in various food industries. In this study, we performed sequence-based screening to obtain a stable X-PDAP enzyme from thermophilic Streptomyces strains. We found three genes that encoded X-PDAP from Streptomyces thermoluteus subsp. fuscus NBRC 14270 (14270 X-PDAP), Streptomyces thermocyaneoviolaceus NBRC 14271 (14271 X-PDAP), and Streptomyces thermocoerulescens NBRC 14273, which were subsequently cloned and sequenced. The deduced amino acid sequences of these genes showed high similarity, with ~80% identity with each other. The isolated X-PDAPs and an X-PDAP from Streptomyces coelicolor were expressed in Streptomyces lividans using a hyperexpression vector: pTONA5a. Among these genes, only 14270 and 14271 X-PDAPs caused overexpression and extracellular production without artificial signal peptides. We also characterized the biochemical properties of purified 14271 X-PDAP. In addition, we found that, in peptide synthesis via an aminolysis reaction, this enzyme recognized D-amino acid derivatives as acyl acceptors, similar to L-amino acid derivatives.
Subject(s)
Bacterial Proteins/biosynthesis , Bacterial Proteins/chemistry , Dipeptidyl-Peptidases and Tripeptidyl-Peptidases/biosynthesis , Dipeptidyl-Peptidases and Tripeptidyl-Peptidases/chemistry , Streptomyces/enzymology , Amino Acid Sequence , Amino Acids/metabolism , DNA, Bacterial/metabolism , Hydrogen-Ion Concentration , Molecular Sequence Data , Protein Sorting Signals , Streptomyces/metabolism , TemperatureABSTRACT
L-Asparaginase (ASNase) has proved its use in medical and food industries. Sequence-based screening showed the thermophilic Streptomyces strain Streptomyces thermoluteus subsp. fuscus NBRC 14270 (14270 ASNase) to positive against predicted ASNase primary sequences. The 14270 ASNase gene and four L-asparaginase genes from Streptomyces coelicolor, Streptomyces avermitilis, and Streptomyces griseus (SGR ASNase) were expressed in Streptomyces lividans using a hyperexpression vector: pTONA5a. Among those genes, only 14270 ASNase and SGR ASNase were successful for overexpression and detected in culture supernatants without an artificial signal peptide. Comparison of the two Streptomyces enzymes described above demonstrated that 14270 ASNase was superior to SGR ASNase in terms of optimum temperature, thermal stability, and pH stability.
Subject(s)
Asparaginase/biosynthesis , Asparaginase/isolation & purification , Isoenzymes/biosynthesis , Isoenzymes/isolation & purification , Streptomyces lividans , Amino Acid Sequence , Asparaginase/genetics , Asparaginase/metabolism , Asparagine/metabolism , Cloning, Molecular , Genes, Bacterial , Isoenzymes/genetics , Isoenzymes/metabolism , Molecular Sequence Data , Plasmids/genetics , Plasmids/metabolism , Streptomyces coelicolor/enzymology , Streptomyces coelicolor/genetics , Streptomyces griseus/enzymology , Streptomyces griseus/genetics , Streptomyces lividans/enzymology , Streptomyces lividans/genetics , TemperatureABSTRACT
We specifically examined an exopeptidase, prolyl aminopeptidase (PAP), as a target for synthesis of proline-containing peptides. A PAP from Streptomyces thermoluteus subsp. fuscus NBRC14270 (PAP14270) was obtained using sequence-based screening. From PAP14270, 144Ser was replaced by Cys (scPAP14270) to give aminolysis activity. In contrast to wild-type PAP14270, scPAP14270 produced a polymer of proline benzyl ester and cyclo[Pro-Pro]. The product mass was confirmed using liquid chromatography-mass spectrometry (LC/MS). Several factors affecting the reaction, such as the pH, concentration of the substrate, and reaction time, were measured to determine their effects. Furthermore, a correlation was found between substrate specificity in proline peptide synthesis and the log D value of acyl acceptors in aminolysis catalyzed by scPAP14270. Results showed that dipeptide synthesis proceeded in a weakly acidic environment and that cyclization and polymerization occurred under alkaline conditions. Furthermore, results suggest that almost all amino acid esters whose log D value is greater than 0, except hydroxyproline benzyl ester (Hyp-OBzl), can be recognized as acyl acceptors. These findings support the use of PAPs as a tool for production of physiologically active proline peptides.
Subject(s)
Aminopeptidases/metabolism , Peptide Biosynthesis/physiology , Proline/metabolism , Streptomyces/enzymology , Aminopeptidases/genetics , Benzyl Compounds , Chromatography, Liquid , Cloning, Molecular , DNA Primers/genetics , Escherichia coli , Hydrogen-Ion Concentration , Mass Spectrometry , Mutagenesis , Polymerase Chain Reaction , Proline/analogs & derivatives , Proline/biosynthesis , Proline/physiology , Substrate Specificity , Time FactorsABSTRACT
Prolyl dipeptide synthesis by S9 aminopeptidase from Streptomyces thermocyaneoviolaceus (S9AP-St) has been demonstrated. In the synthesis, S9AP-St preferentially used l-Pro-OBzl as the acyl donor, yielding synthesized dipeptides having an l-Pro-Xaa structure. In addition, S9AP-St showed broad specificity toward the acyl acceptor. Furthermore, S9AP-St produced cyclo (l-Pro-l-His) with a conversion ratio of substrate to cyclo (l-Pro-l-His) higher than 40%.
Subject(s)
Aminopeptidases/metabolism , Bacterial Proteins/metabolism , Dipeptides/metabolism , Streptomyces/enzymology , Aminopeptidases/genetics , Bacterial Proteins/genetics , Dipeptides/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Substrate SpecificityABSTRACT
Synthesis of beta-alanine (beta-Ala) containing dipeptide using S9 aminopeptidase from Streptomyces thermocyaneoviolaceus NBRC14271 (S9AP-St) was demonstrated with beta-Ala-benzyl ester (-OBzl) and various L-aminoacyl derivatives. For synthesis of beta-Ala-containing dipeptide, beta-Ala-OBzl was used preferentially as the acyl donor for S9AP-St, producing synthesized dipeptides having beta-Ala-Xaa structure. In contrast, engineering of S9AP-St into "transaminopeptidase" by substitution of catalytic Ser with Cys--designated as aminolysin-S--produced only dipeptides having Xaa-beta-Ala structure. Investigation of the specificity of S9AP-St toward acyl acceptors showed that S9AP has a broad substrate specificity toward various aminoacyl derivatives. Furthermore, S9AP-St produced carnosine methyl ester (-OMe) with a conversion ratio of beta-Ala-OBzl to carnosine-OMe that was greater than 30%.
Subject(s)
Aminopeptidases/metabolism , Peptides/metabolism , Streptomyces/enzymology , beta-Alanine/biosynthesis , Amino Acid Sequence , Aniline Compounds/metabolism , Carnosine/metabolism , Hydrogen-Ion Concentration , Hydrolysis , Mass Spectrometry , Molecular Sequence Data , Substrate Specificity , Time Factors , beta-Alanine/chemistryABSTRACT
Aminopeptidase from Streptomyces thermocyaneoviolaceus NBRC14271 was engineered into transaminopeptidase and used to catalyze an aminolysis reaction to give linear and cyclic dipeptides from cost-effective substrates such as the ester derivatives of amino acids.
Subject(s)
Aminopeptidases/metabolism , Dipeptides/chemical synthesis , Mutagenesis, Site-Directed , Peptides, Cyclic/chemical synthesis , Streptomyces/enzymology , Aminopeptidases/genetics , Dipeptides/metabolism , Peptides, Cyclic/metabolismABSTRACT
A new polyhydroxylated pyrrolizidine alkaloid designated as pochonicine (1) was isolated from a solid fermentation culture of the fungal strain Pochonia suchlasporia var. suchlasporia TAMA 87. The structure of 1 was determined using NMR and MS techniques as (1R*, 3S*, 5S*, 6S*, 7R*, 7a S*)-5-acetamidomethyl-3-hydroxymethyl-1,6,7-trihydroxypyrrolizidine. Pochonicine (1) showed potent inhibition against beta-N-acetylglucosaminidases (GlcNAcases) of various organisms including insects, fungi, mammals, and a plant but no inhibition against beta-glucosidase of almond, alpha-glucosidase of yeast, or chitinase of Bacillus sp. The GlcNAcase inhibitory activity of pochonicine (1) was comparable to nagstatin, a potent GlcNAcase inhibitor of natural origin.
Subject(s)
Acetylglucosaminidase/antagonists & inhibitors , Enzyme Inhibitors/pharmacology , Fungi/chemistry , Pyrrolizidine Alkaloids/pharmacology , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/isolation & purification , Fermentation , Gas Chromatography-Mass Spectrometry , Magnetic Resonance Spectroscopy , Pyrrolizidine Alkaloids/isolation & purificationABSTRACT
Studies of substrate recognition by serine proteases have focused on specificities at the primary S1-Sn sites, but topological specificities (i.e. recognition at distinct three-dimensional structural motifs) have not been established. This is the first report to identify the key amino acid residue conferring topological specificity. A serine protease from Streptomyces omiyaensis (SOT), which is a trypsin-like enzyme, was chosen as a model enzyme to clarify the recognition mechanism of structural protein substrates in serine proteases. We have found previously that the topological specificities of SOT and S. griseus trypsin (SGT) for high molecular mass substrates differ greatly, even though the enzymes have similar primary structures. In this study, we constructed chimeras between SOT and SGT using an in vivo DNA shuffling system and several mutants to identify the key residues involved in topological specificities. By comparing the substrate specificities of chimeras and mutants, we found that residue 71 of SOT, which is separate from the catalytic triad, contributes to the topological specificity. Using site-directed mutagenesis, residue 71 of SOT was also found to be crucial for catalytic efficiency and enzyme conformation.
Subject(s)
Streptomyces/enzymology , Trypsin/chemistry , Amino Acid Sequence , Amino Acid Substitution , Animals , Base Sequence , Catalytic Domain/genetics , Cattle , Collagen Type I/metabolism , Collagen Type IV/metabolism , DNA Primers/genetics , Genes, Bacterial , Humans , In Vitro Techniques , Models, Molecular , Molecular Sequence Data , Mutagenesis, Site-Directed , Protein Conformation , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Sequence Homology, Amino Acid , Species Specificity , Streptomyces/genetics , Streptomyces griseus/enzymology , Streptomyces griseus/genetics , Substrate Specificity , Trypsin/genetics , Trypsin/metabolism , Tyrosine/chemistryABSTRACT
Oligopeptidase B from Streptomyces griseus was cloned and characterized to clarify the substrate recognition mechanism and the role of a reactive cysteine residue in family S9 prolyl oligopeptidases (POPs). The cloned enzyme, SGR-OpdB, was annotated as a putative family S9 prolyl oligopeptidase based on its deduced amino acid sequence, in which a sole cysteine residue Cys(544) is present close to the catalytic Asp residue in the C-terminal region. The protein was identified as oligopeptidase B, a member of the subfamily S9a of the family S9 POPs, as judged by its substrate specificity and enzymatic characteristics. Its enzymatic activity was markedly enhanced by high NaCl concentration and the reducing reagents dithiothreitol (DTT) and reduced glutathione (GSH). It is particularly interesting that oxidized glutathione (GSSG) also enhanced SGR-OpdB activity. The SGR-OpdB C544A mutant was constructed and characterized to clarify the role of the putative reactive Cys residue, Cys(544). Surprisingly, the enzymatic activity of the Cys-free mutant was also markedly activated by the general thiol-reacting reagent DTT, GSH, and GSSG. To our knowledge, this is the first report of activity-enhancing effects of thiol-reacting reagents toward Cys-free enzymes. Results clarified the role of additives in inducing conformational change of SGR-OpdB into active peptidase.
Subject(s)
Serine Endopeptidases/metabolism , Sulfhydryl Reagents/pharmacology , Amino Acid Sequence , Enzyme Activation/drug effects , Hydrogen-Ion Concentration , Protein Conformation/drug effects , Serine Endopeptidases/drug effects , Serine Endopeptidases/genetics , Sodium Chloride/pharmacology , Streptomyces griseus/enzymology , Substrate SpecificityABSTRACT
Aminopeptidases from Streptomyces griseus (SGRAP) and S. coelicolor (SCOAP) were cloned and characterized to clarify their biochemical characteristics. Although both enzymes had been annotated as putative oligopeptidases of family S9 enzymes, they showed "aminopeptidase" activities, not "oligopeptidase" activities. Although their deduced amino acid sequences showed high similarity (69% overall sequence homology), they showed distinct substrate specificities and sensitivities to the reducing reagent dithiothreitol (DTT). The reaction pH and addition of DTT dramatically affected the substrate preference of SGRAP. Furthermore, SCOAP selectively hydrolyzed phenyalanine p-nitroanilide (Phe-pNA) in the presence or absence of DTT. The chimera protein between SGRAP and SCOAP was constructed to identify the region responsible for the properties described above. Furthermore, Cys(409) of SCOAP was identified as a functional residue responsible for activation by reducing reagent DTT.
Subject(s)
Aminopeptidases/metabolism , Peptide Hydrolases/metabolism , Streptomyces coelicolor/enzymology , Streptomyces griseus/enzymology , Amino Acid Sequence , Cloning, Molecular , Dithiothreitol/pharmacology , Hydrogen-Ion Concentration , Molecular Sequence Data , Peptide Hydrolases/genetics , Recombinant Fusion Proteins/metabolism , Sequence Alignment , Streptomyces coelicolor/genetics , Streptomyces griseus/genetics , Substrate Specificity/drug effectsABSTRACT
We constructed the Streptomyces hyperexpression vector pTONA5 based on pIJ702 vector; it includes a metalloendopeptidase (SSMP) promoter isolated from Streptomyces cinnamoneus TH-2 and a metalloendopeptidase terminator isolated from Streptomyces aureofaciens TH-3. The vector contains recognition sites for restriction enzymes NdeI and EcoRI/XbaI/HindIII between the promoter and terminator to facilitate heterologous gene cloning. The plasmids were transferred from Escherichia coli to streptomycetes via conjugation from oriT; the transformants were able to be selected using kanamycin and/or thiostrepton. The SSMP promoter functions constitutively in the presence of a rich inorganic phosphate source and glucose. We constructed expression plasmids including three Streptomyces aminopeptidases-leucine aminopeptidase, proline aminopeptidase (PAP), and aminopeptidase P (APP)-using the pTONA5 vector and Streptomyces lividans. Although they lack signal peptides for secretion, PAP and APP were secreted at high levels in the culture broth.
Subject(s)
Genetic Vectors/genetics , Plasmids/genetics , Streptomyces/genetics , Aminopeptidases/metabolism , Electrophoresis, Polyacrylamide Gel , Glutathione Transferase/metabolism , Leucyl Aminopeptidase/metabolism , Promoter Regions, Genetic/genetics , Streptomyces/enzymology , Terminator Regions, GeneticABSTRACT
A novel beta-N-acetylglucosaminidase (GlcNAcase) inhibitor named TMG-chitotriomycin (1) was isolated from the culture filtrate of Streptomyces anulatus NBRC13369. The strain produced 1 only when colloidal chitin was used as the sole carbon source in the production medium. The structure of 1 was determined by spectral and constitutive sugar analyses of the corresponding alditol derivatives to be an equilibrated mixture of alpha-d-N,N,N-triMeGlcNH2-(1,4)-beta-d-GlcNAc-(1,4)-beta-d-GlcNAc-(1,4)-d-GlcNAc and its C-2 epimer of the reducing end residue. TMG-chitotriomycin (1) showed potent and selective inhibition of insect and fungal GlcNAcases with no inhibition of mammalian and plant GlcNAcases. In contrast, the known GlcNAcase inhibitor nagstatin potently inhibited all GlcNAcases. It should be emphasized that synthesized d-N,N,N-triMeGlcNH2, which is the component sugar of 1, showed no inhibition of the insect Spodoptera litura GlcNAcase. These results suggest that the (GlcNAc)3 unit positioned at the reducing end of 1 is essential for its enzyme inhibitory activity. The unique inhibitory spectrum of 1 will be useful to study chitinolytic systems and to develop selective fungicides or pesticides.
Subject(s)
Enzyme Inhibitors/metabolism , Enzyme Inhibitors/pharmacology , Penicillium/enzymology , Spodoptera/enzymology , Streptomyces/metabolism , Sugar Alcohols/pharmacology , beta-N-Acetyl-Galactosaminidase/antagonists & inhibitors , Animals , Bacillus/classification , Bacillus/enzymology , Carbohydrate Conformation , Carbohydrate Sequence , Chitinases/antagonists & inhibitors , Enzyme Activation/drug effects , Enzyme Inhibitors/chemistry , Insecta , Molecular Sequence Data , Sensitivity and Specificity , Species Specificity , Streptomyces/classification , Streptomyces griseus/enzymology , Structure-Activity Relationship , Sugar Alcohols/chemistryABSTRACT
From among 2000 soil isolates, we purified a secreted serine protease from Streptomyces omiyaensis (SOT), which has extremely high gelatinolytic activity. Using sequence analysis, the primary structure of SOT showed 77% identity with that of S. griseus trypsin (SGT). We constructed recombinants SOT and SGT using S. lividans. They indicated similar properties on optimum pH and temperature, thermostability, and substrate preference using fluorescence energy transfer combinatorial libraries. SOT greatly hydrolyzed both type I and type IV collagens, but SGT has poor ability to hydrolyze type IV collagen. Furthermore, SOT exhibits higher hydrolytic activities toward other protein substrate such as gelatin and casein than SGT. These results suggest that these two enzymes have different topological specificities in spite of their similar primary structures. We also constructed chimeras between SOT and SGT to investigate which domain is associated with differences in their substrate specificity. In comparison to substrate specificities of chimeras, we found that the N-terminal domain contributes to the determination of topological specificity.