Purification and characterization of a 7Fe ferredoxin from Streptomyces griseus

A ferredoxin has been purified from Streptomyces griseus grown in soybean flour-containing medium. The homogeneous protein has a molecular weight near 14000 as determined by both PAGE and size exclusion chromatography. The iron and labile sulfide content is 6–7 atoms/mole protein. EPR spectroscopy of native S. griseus ferredoxin shows an isotropic signal at g=2.01 which is typical of [3Fe-4S]¹⁺ clusters and which quantitates to 0.9 spin/mole. Reduction of the ferredoxin by excess dithionite at pH 8.0 produces an EPR silent state with a small amount of a g=1.95 type signal. Photoreduction in the presence of deazaflavin generates a signal typical of [4Fe-4S]¹⁺ clusters at much higher yields (0.4–0.5 spin/mole) with major features at g-values of 2.06, 1.94, 1.90 and 1.88. This latter EPR signal is most similar to that seen for reduced 7Fe ferredoxins, which contain both a [3Fe-4S] and [4Fe-4S] cluster. In vitro reconstitution experiments demonstrate the ability of the S. grisues ferredoxin to couple electron transfer between spinach ferredoxin reductase and S. griseus cytochrome P-450_soy for NADPH-dependent substrate oxidation. This represents a possible physiological function for the S. griseus ferredoxin, which if true, would be the first functional role demonstrated for a 7Fe ferredoxin.     

Purification and characterization of a soybean flour-induced cytochrome P-450 from Streptomyces griseus.

A soybean flour-induced, soluble cytochrome P-450 (P-450soy) was purified 130-fold to homogeneity from Streptomyces griseus. Native cytochrome P-450soy is a single polypeptide, with a molecular weight of 47,500, in association with one ferriprotoporphyrin IX prosthetic group. Oxidized P-450soy exhibited visible absorption maxima at 394, 514, and 646 nm, characteristic of a high-spin cytochrome P-450. The CO-reduced difference spectrum of P-450soy had a Soret maximum at 448 nm. When reconstituted with spinach ferredoxin and spinach ferredoxin:NADP+ oxidoreductase, purified cytochrome P-450soy catalyzed the NADPH-dependent oxidation of the xenobiotic substrates precocene II and 7-ethoxycoumarin. In vitro proteolysis of cytochrome P-450soy generated a stable and catalytically active cytochrome P-450, designated P-450soy delta.     

Purification and characterization of Streptomyces griseus catechol O-methyltransferase

A soluble (100,000 x g supernatant) methyltransferase catalyzing the transfer of the methyl group of S-adenosyl-L-methionine to catechols was present in cell extracts of Streptomyces griseus. A simple, general, and rapid catechol-based assay method was devised for enzyme purification and characterization. The enzyme was purified 141-fold by precipitation with ammonium sulfate and successive chromatography over columns of DEAE-cellulose, DEAE-Sepharose, and Sephacryl S-200. The purified cytoplasmic enzyme required 10 mM magnesium for maximal activity and was catalytically optimal at pH 7. 5 and 35 degrees C. The methyltransferase had an apparent molecular mass of 36 kDa for both the native and denatured protein, with a pI of 4.4. Novel N-terminal and internal amino acid sequences were determined as DFVLDNEGNPLENNGGYXYI and RPDFXLEPPYTGPXKARIIRYFY, respectively. For this enzyme, the K(m) for 6,7-dihydroxycoumarin was 500 +/- 21.5 microM, and that for S-adenosyl-L-methionine was 600 +/- 32.5 microM. Catechol, caffeic acid, and 4-nitrocatechol were methyltransferase substrates. Homocysteine was a competitive inhibitor of S-adenosyl-L-methionine, with a K(i) of 224 +/- 20.6 microM. Sinefungin and S-adenosylhomocysteine inhibited methylation, and the enzyme was inactivated by Hg(2+), p-chloromercuribenzoic acid, and N-ethylmaleimide.     

Purification and characterization of recombinant Streptomyces griseus aminopeptidase

Recombinant Streptomyces griseus aminopeptidase (SGAP) was produced using Cangene's expression system, CANGENUS. This heat-stable aminopeptidase with an N-terminal Ala-Pro-Asp-Ile-Pro-Leu-Ala-Asn-Val-Lys-Ala sequence was purified from 16L of Streptomyces lividans fermentation supernatant with high purity and 19.5% recovery rate. This was achieved by the combination of hydrophobic-interaction and size-exclusion chromatographic procedures. The calcium-activated zinc metalloprotein demonstrated no loss of activity at -20 degrees C for at least 8 weeks in both liquid and freeze-dried formulations. The recombinant SGAP showed an apparent molecular mass of 31 kDa by sodium dodecyl sulfate/polyacrylamide gel electrophoresis and 26.8 kDa by gel filtration. The simple, high-yield, inexpensive purification method with few intermediate steps provides a novel and practical procedure for large-scale production of active recombinant S. griseus aminopeptidase.     

Purification and characterization of Streptomyces griseus metalloendopeptidases I and II.

Two metalloendopeptidases, designated as Streptomyces griseus metalloendopeptidases I and II (SGMPI and SGMPII), were isolated from a commercial Pronase P by a method including affinity chromatography on carbobenzoxy-L-alaninyl-triethylenetetraminyl-Sepharose (Z-Ala-T-Sepharose). The two enzymes differed from each other in behavior on ion-exchange chromatography but showed the same amino-terminal sequence at least up to the 20th residue. Their molecular weights were both estimated to be 37,000 by SDS-polyacrylamide gel electrophoresis. Elemental and amino acid composition analyses indicated that both of them contained about 1 g atom of zinc and one cystine residue per mol of protein. Cleavage specificities of the two enzymes toward synthetic peptide-substrates were very similar to those observed with thermolysin. EDTA, o-phenanthroline, and phosphoramidon strongly inhibited these enzymes, while typical serine-protease inhibitors and cysteine-protease inhibitors had no effect. The findings clearly indicate that SGMPI and SGMPII can be classified into the family of zinc-endopeptidases. It was unexpectedly found, however, that these metalloendopeptidases were strongly inhibited by protein serine-protease inhibitors produced by Streptomycetes, such as Streptomyces subtilisin inhibitor (SSI), alkaline protease inhibitor-2c' (API-2c'), and plasminostreptin (PS).     

Purification and partial characterization of multiple bromoperoxidases from Streptomyces griseus

The presence of multiple bromoperoxidases in extracts of Streptomyces griseus Tü 6 was detected. The enzyme pattern varied with the age of the culture. A haem-type bromoperoxidase (BPO 2) was always present. Additionally three nonhaem-type bromoperoxidases (BPO 1a, 1b and 3) were detected and purified to homogeneity. The Mr of non-denatured BPO 1a was 70,000 +/- 10,000 and those of BPO 1b and 3 were 90,000 +/- 5000. BPO 1a and 1b were dimers with subunit Mr values of 34,000 and 43,000, respectively. BPO 3 was a trimer with a subunit Mr of 31,000. The enzymes differed in their isoelectric points, heat stability, and Km values. In immunodiffusion experiments BPO 1a and 3 showed partial identity with the nonhaem-type bromoperoxidase from Streptomyces aureofaciens. The nonhaem-type BPO 1a, 1b and 3 had neither peroxidase nor catalase activity.     

Primary structure of a 7Fe ferredoxin from Streptomyces griseus

The complete primary structure of a Streptomyces griseus (ATCC 13273) 7Fe ferredoxin, which can couple electron transfer between spinach ferredoxin reductase and S. griseus cytochrome P-450_soy for NADPH-dependent substrate oxidation, has been determined by Edman degradation of the whole protein and peptides derived by Staphylococcus aureus V8 proteinase and trypsin digestion. The protein consists of 105 amino acids and has a calculated molecular weight, including seven irons and eight sulfurs, of 12291. The ferredoxin sequence is highly homologous (73%) to that of the 7Fe ferredoxin from Mycobacterium smegmatis. The N-terminal half of the sequence, which is the FeS clusters binding domain, has more than 50% homology with other 7Fe ferredoxins. In particular, the seven cysteines known from the crystal structure of Azotobacter vinelandii ferredoxin I to be involved in binding the two FeS clusters are conserved.     

Purification and characterization of soybean nodule nitrite reductase

A nodule cytosol nitrite reductase was isolated from soybean [ Glyine max (L.) Mer. cv. Tracy] grown in the presence of nitrate. Enzyme activity increased when increased amounts of nitrate were supplied to the plant. A purification procedure involving ammonium sulfate precipitation, gel filtration, DEAE Sephadex and Blue Sepharose chromatography resulted in an activity capable of forming 6.7 μmol ammonia (mg protein)⁻¹ min⁻¹. This represented a 235-fold increase in specific activity. The molecular weight, estimated by gel filtration, was 55 000. The pH optimum for activity was 7.1. Ammonia formed stoichiometrically as nitrice was consumed. From Lineweaver-Burk plots, K_m values of 0.5m M for nitrite and 0.2m M for methyl viologen were calculated. Spectral data suggest the association of a heme chromophore with the enzyme.     

Purification and characterization of monodehydroascorbate reductase from soybean root nodules.

Soybean (Glycine max (L.) Merr.) root nodules contain the enzymes of the ascorbate-glutathione cycle as an important defense against activated forms of oxygen. A key enzyme in this cycle--monodehydroascorbate reductase (MR)--was purified 646-fold and appeared as a single band on SDS-PAGE with silver or Coomassie blue staining. Purified MR contained 0.7 mol FAD/mol enzyme and had a specific activity of 288 mumol NADH oxidized.min-1.mg protein-1. The enzyme was a single subunit occurring as two isozymes (MR I and MR II) with Mr values of 39,000 and 40,000. Isoelectric focusing revealed that each isozyme consisted of two forms with pl values of 4.6 to 4.7. Ferricyanide and 2,6-dichlorophenol-indophenol were effective as electron acceptors. The purified enzyme did not possess leghemoglobin reductase activity. Inhibition by p-chloromercuribenzoate indicated the involvement of a thiol group in MR activity. The Km values were 5.6, 150, and 7 microM for NADH, NADPH, and monodehydroascorbate, respectively. The pH optimum was 8 to 9. The N-terminal sequence of 10 amino acids of MR II had little homology to known protein sequences.     

Purification and characterization of a novel enoyl coenzyme A reductase from Streptomyces collinus.

A novel NADPH-dependent enoyl reductase, catalyzing the conversion of 1-cyclohexenylcarbonyl coenzyme A (1-cyclohexenylcarbonyl-CoA) to cyclohexylcarbonyl-CoA, was purified to homogeneity from Streptomyces collinus. This enzyme, a dimer with subunits of identical M(r) (36,000), exhibits a Km of 1.5 +/- 0.3 microM for NADPH and 25 +/- 3 microM for 1-cyclohexenylcarbonyl-CoA. It has a pH optimum of 7.5, is most active at 30 degrees C, and is inhibited by both divalent cations and thiol reagents. Two internal peptide sequences were obtained. Ansatrienin A (an antibiotic produced by S. collinus) contains a cyclohexanecarboxylic acid moiety, and it is suggested that the 1-cyclohexenylcarbonyl-CoA reductase described herein catalyzes the final reductive step in the conversion of shikimic acid into this moiety.     

Purification and characterization of dihydrofolate reductase from soybean seedlings

Dihydrofolate reductase (DHFR; EC 1.5.1.3) was purified to homogeneity from soybean seedlings by affinity chromatography on methotrexate-aminohexyl Sepharose, gel filtration on Ultrogel AcA-54, and Blue Sepharose chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the enzyme gave a single protein band corresponding to a molecular weight of 22,000. The enzyme is not a 140,000 Da heteropolymer as reported by others. Amino acid sequence-specific antibodies to intact human DHFR and also antibodies to CNBr-generated fragments of human DHFR bound to the plant enzyme on Western blots and cross-reacted significantly in immunoassays, indicating the presence of sequence homology between the two enzymes. The plant and human enzymes migrated similarly on nondenaturing polyacrylamide electrophoretic gels as monitored by activity staining with a tetrazolium dye. The specific activity of the plant enzyme was 15 units/mg protein, with a pH optimum of 7.4. Km values of the enzyme for dihydrofolate and NADPH were 17 and 30 microM, respectively. Unlike other eukaryotic enzymes, the plant enzyme showed no activation with organic mercurials and was inhibited by urea and KCl. The affinity of the enzyme for folate was relatively low (I50 = 130 microM) while methotrexate bound very tightly (KD less than 10(-10) M). Binding of pyrimethamine to the plant enzyme was weaker, while trimethoprim binding was stronger than to vertebrate DHFR. Trimetrexate, a very potent inhibitor of the human and bacterial enzymes showed weak binding to the plant enzyme. However, certain 2,4-diaminoquinazoline derivatives were very potent inhibitors of the plant DHFR. Thus, the plant DHFR, while showing similarity to the vertebrate and bacterial enzymes in terms of molecular weight and immunological cross-reactivity, can be distinguished from them by its kinetic properties and interaction with organic mercurials, urea, KCl and several antifolates.     

Purification and specificity of carboxypeptidase from Streptomyces griseus K-1.

A carboxypeptidase of St. griseus K-1 (CPase S) was found to possess the specificities of both mammalian pancreatic CPase A and B. Three adsorbents for affinity chromatography were prepared by coupling l-Leu, d-Leu, and d-Arg with CH-Sepharose 4B. d-Arg-CH-Sepharose and l-Leu-CH-Sepharose retained the purified CPase S but d-Leu-CH-Sepharose did not. The activities of CPase S toward CGL and BGA were eluted in the same position. CPase S migrated as a single band on polyacrylamide gel electrophoresis and the two activities were both extracted from this band on the gel.     

Molecular characterization of a gene encoding a photolyase from Streptomyces griseus.

By using a synthetic DNA probe derived from an amino acid sequence in the most conserved region of three known photolyases (Escherichia coli, Anacystis nidulans and Saccharomyces cerevisiae), we isolated a DNA fragment containing two long open reading frames (ORFs) from a genomic DNA library of Streptomyces griseus. One ORF encodes a polypeptide of 455 amino acids (Mr 50594), which exhibits substantial similarities with the other three photolyases. Photoreactivation-repair deficient E. coli cells could be converted into photoreactivatable ones by introduction of plasmids harboring this ORF, indicating that this is the photolyase gene of S. griseus. The deduced aa sequence of Streptomyces photolyase was most similar to that of E. coli. The putative DNA binding site as well as cofactor binding regions were proposed.     

Purification and characterization of soybean root nodule ferric leghemoglobin reductase

A ferric leghemoglobin reductase from the cytosol of soybean (Glycine max) root nodules was purified to homogeneity and partially characterized. The enzyme is a flavoprotein with flavin adenine dinucleotide as the prosthetic group and consists of two identical subunits, each having a molecular mass of 54 kilodaltons. The pure enzyme shows a high activity for ferric leghemoglobin reduction with NADH as the reductant in the absence of any exogenous mediators. The enzyme also exhibits NADH-dependent 2,6-dichloroindophenol reductase activity. A sequence of the first 50 N-terminal amino acids of the purified protein was obtained. Comparisons with known protein sequences have shown that the sequence of the ferric leghemoglobin reductase is highly related to those of the flavin-nucleotide disulfide oxido-reductases, especially dihydrolipoamide dehydrogenase of the pyruvate dehydrogenase complex.     

Purification and Properties of a Lytic Enzyme from Streptomyces griseus H-402

A lytic enzyme which was capable of lysing cells of Streptococcus mutans was purified from the culture filtrate of Streplomyces griseus H–402 by Amberlite CG–50 treatment, CM-cellulose and hydroxylapatite column chromatographies, and Sephadex G–150 gelfiltration. The lytic enzyme was obtained in a crystalline form which was homogeneous in polyacrylamide gel electrophoresis. The molecular weight was estimated to be 2×10⁴ by the thin-layer gel-filtration method on Sephadex G–75, and 2.3 × 10⁴ by the method of polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The enzyme was found to be a N-acetylmuramidase whose activity was lost by N-bromosuccinimide as an inhibitor.     

Purification and characterization of a novel (R)-imine reductase from Streptomyces sp. GF3587

The (R)-imine reductase (RIR) of Streptomyces sp. GF3587 was purified and characterized. It was found to be a NADPH-dependent enzyme, and was found to be a homodimer consisting of 32 kDa subunits. Enzymatic reduction of 10 mM 2-methyl-1-pyrroline (2-MPN) resulted in the formation of 9.8 mM (R)-2-methylpyrrolidine ((R)-2-MP) with 99% e.e. The enzyme showed not only reduction activity for 2-MPN at neutral pH (6.5-8.0), but also oxidation activity for (R)-2-MP under alkaline pH (10-11.5) conditions. It appeared to be a sulfhydryl enzyme based on the sensitivity to sulfhydryl specific inhibitors. It was very specific to 2-MPN as substrate.     

Streptomyces griseus aminopeptidase is a calcium-activated zinc metalloprotein. Purification and properties of the enzyme

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 degrees 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.     

Purification and characterization of human placental ferredoxin

A ferredoxin-type iron-sulfur protein was isolated from human placenta mitochondria. The properties of the purified protein were very similar to those of adrenal ferredoxin (adrenodoxin), and immunological cross-reactivity with polyclonal antibodies to bovine adrenodoxin was observed. The N-terminal amino acid sequence and the visible absorption spectrum were identical to bovine adrenodoxin. The molecular mass as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Mr approximately 13,500), however, is slightly smaller than that of adrenodoxin, and the C-terminal sequence is different. Human placental ferredoxin can substitute for bovine adrenodoxin in reactions reconstituted with bovine adrenal enzymes which catalyze the side chain cleavage of cholesterol to pregnenolone and the 11 beta-hydroxylation of deoxycorticosterone to corticosterone.