scholarly journals Isolation and characterization of nitrogenase MoFe protein from the mutant strain pHK17 of Klebsiella pneumoniae in which the two bridging cysteine residues of the P-clusters are replaced by the non-coordinating amino acid alanine

1996 ◽  
Vol 318 (1) ◽  
pp. 111-118 ◽  
Author(s):  
Faridoon K YOUSAFZAI ◽  
Martin BUCK ◽  
Barry E. SMITH
Keyword(s):  
Klebsiella Pneumoniae ◽  
Mutant Strain ◽  
Specific Activity ◽  
Relative Distribution ◽  
Wild Type ◽  
Surface Charges ◽  
Ph Profile ◽  
Mofe Protein ◽  
Isolation And Characterization ◽  
Specific Activities

Nitrogenase MoFe protein (Kp1) from the mutant strain pHK17 of Klebsiella pneumoniae has been purified to give three catalytically active fractions. In this mutant, each of the two bridging cysteine ligands to the P-clusters, α-Cys-89 and β-Cys-94, has been replaced by a non-coordinating residue, alanine. SDS/PAGE and earlier native gels showed that the three fractions retained the normal α2β2 tetrameric form of wild-type Kp1; therefore we conclude that in each of the fractions the subunits are folded differently, thus resulting in different surface charges and allowing separation of the fractions on ion-exchange chromatography. Earlier EPR and magnetic CD data had shown that the mutant fractions contain P-clusters, and thus the mutated residues are not as essential for maintaining the integrity of the P-clusters as they appear from the X-ray structure. The specific activity of each of the three fractions was less than that of wild-type Kp1, the most active fraction having only 50% of wild-type activity. No change in substrate specificity or in the relative distribution of electrons to various substrates was found. The relationship between ATP hydrolysis and substrate-reducing activity, the EPR spectra of the S = 3/2 spin state of the iron–molybdenum cofactor (FeMoco) and the pH profile of acetylene-reduction activities of the three fractions did not differ significantly from those exhibited by wild-type Kp1. The specific activities of the three mutant fractions and of wild-type Kp1 were linearly proportional to the intensity of the S = 3/2 EPR signal from the FeMoco centres. This implies that those molecules of the three mutant fractions and the wild-type protein that contain EPR-active FeMoco are fully active, i.e. that the Cys to Ala substitution of the P-cluster ligands does not affect the specific activity of the protein. This in turn implies that the P-clusters are not directly associated with the rate-limiting step in enzyme turnover. We conclude that the lower specific activities of the mutant fractions are observed because the fractions are mixtures of species containing a full complement of FeMoco and P-clusters and species lacking some or all of these clusters. On the basis of the Mo contents and EPR spectroscopy of the mutant fractions, we propose that the loss of the P-clusters causes (i) the physical loss or inhibition of binding of some FeMoco; (ii) the EPR and catalytic inactivation of some FeMoco; and/or (iii) the incorporation of a FeMoco-like species into the FeMoco site of the mutant molecules.

scholarly journals The inactive MoFe protein (NifB−Kp1) of the nitrogenase from nif B mutants of Klebsiella pneumoniae. Its interaction with FeMo-cofactor and the properties of the active MoFe protein formed

1984 ◽  
Vol 223 (3) ◽  
pp. 783-792 ◽  
Author(s):  
T R Hawkes ◽  
B E Smith
Keyword(s):  
Klebsiella Pneumoniae ◽  
Signal Intensity ◽  
Specific Activity ◽  
Maximum Activity ◽  
Gene Products ◽  
Wild Type ◽  
Activation Reaction ◽  
Mofe Protein ◽  
Femo Cofactor ◽  
Iron Molybdenum Cofactor

The inactive MoFe protein (NifB-Kp1) of nitrogenase from nifB mutants of Klebsiella pneumoniae may be activated by addition of the iron-molybdenum cofactor (FeMoco) extracted from active MoFe protein (Kp1). However, when apparently saturated with FeMoco, our preparations of NifB-Kp1 yielded activated protein, Kp1-asm, with a specific activity that was at best only 40% of that expected. This was not due to degradation of Kp1-asm, NifB-Kp1 or FeMoco during the activation reaction. Nor could activation be enhanced by addition of other nif-gene products or other proteins. Whereas fully active Kp1 contains 2 FeMoco/molecule, apparent saturation of our NifB-Kp1 preparations required the binding of only 0.4-0.65 FeMoco/molecule. By using chromatography Kp1-asm could be largely resolved from NifB-Kp1 that had not been activated. However, we were unable to isolate fully active MoFe protein (i.e. Kp1-asm containing 2 FeMoco/molecule) from solutions of NifB-Kp1 activated with FeMoco. The maximum activity/ng-atom of total Mo obtained for our purified Kp1-asm was approximately half the maximum activity for FeMoco. Since all NifB-Kp1 preparations contained some Mo, we suggest that FeMoco activated only those NifB-Kp1 molecules already containing one atom of (non-FeMoco) Mo, thus forming Kp1-asm with 2 Mo but only 1 FeMoco/molecule. Kp1-asm was identical with normal Kp1 in terms of its Mr, stability, e.p.r. signals, pattern of substrate reductions, CO inhibition and ATP/2e ratio. In addition, for preparations of differing specific activity, there was a constant and identical relationship between the e.p.r. signal intensity (from FeMoco) and the activity of both Kp1 and Kp1-asm. Assuming the above hypothesis on the structure of Kp1-asm, these data demonstrate that the two FeMoco sites in wild-type Kp1 operate independently.

scholarly journals Purification and characterization of the inactive MoFe protein (NifB-Kp1) of the nitrogenase from nifB mutants of Klebsiella pneumoniae

1983 ◽  
Vol 209 (1) ◽  
pp. 43-50 ◽  
Author(s):  
T R Hawkes ◽  
B E Smith
Keyword(s):  
Klebsiella Pneumoniae ◽  
Double Mutant ◽  
Specific Activity ◽  
Equimolar Amount ◽  
Wild Type ◽  
Purification And Characterization ◽  
Point Mutant ◽  
Mofe Protein ◽  
Iron Molybdenum Cofactor

The inactive MoFe protein of nitrogenase, NifB-Kp1, from two distinct nifB mutants of Klebsiella pneumoniae, Kp5058 (a nifB point mutant) and UNF1718 (a nifB, nifJ double mutant) has been purified and characterized. NifB-Kp1 can be activated by reaction with the iron-molybdenum cofactor, FeMoco, extracted from active MoFe protein. NifB-Kp1 purified from either source had similar properties and was contaminated with an approximately equimolar amount of protein of mol.wt. 21 000. Like active wild-type Kp1, it was an alpha 2 beta 2 tetramer, but it was far less stable than Kp1, deteriorating rapidly at temperatures above 8 degrees C or on mild oxidation. NifB-Kp1 preparations contained 0.4-0.9 Mo and 9.0 +/- 0.9 Fe atoms . mol-1 and, when activated by FeMoco, had a specific activity of approx. 500 units . mg-1. The Mo in our preparations was not associated with the e.p.r. signal normally observed from FeMoco. All preparations exhibited a weak gav. = 1.95 e.p.r. signal which was probably not associated with activatable protein.

scholarly journals Klebsiella pneumoniae nitrogenase MoFe protein: chymotryptic proteolysis affects function by limited cleavage of the β-chain and provides high-specific-activity MoFe protein

1993 ◽  
Vol 291 (1) ◽  
pp. 309-314
Author(s):  
K Fisher ◽  
D J Lower ◽  
R N Pau
Keyword(s):  
Klebsiella Pneumoniae ◽  
Specific Activity ◽  
Gel Filtration ◽  
High Specific Activity ◽  
Visible Spectrum ◽  
Prolonged Treatment ◽  
Beta Chain ◽  
Wild Type ◽  
Alpha Chain ◽  
Mofe Protein

Proteinase treatment with chymotrypsin has been used to probe the structure of native Klebsiella pneumoniae nitrogenase MoFe protein (Kp1). Reaction with chymotrypsin did not bleach Kp1, suggesting that it did not destroy the metal centres, and the Mo and Fe contents of Kp1 were unchanged. High ratios of chymotrypsin to Kp1 (1:1 by mass) cleaved the beta-chain of Kp1 to give 44 and 14 kDa polypeptides, which N-terminal amino acid sequence analysis showed to be derived from cleavage at residue beta-Phe124. A mutant MoFe protein, Kp1Met-124, in which beta-Phe124 is replaced by methionine, was not cleaved by chymotrypsin. Under non-denaturing conditions, the ‘nicked’ beta-chain of the wild-type protein remained associated with the alpha-chain. The alpha-chain was not cleaved by the proteinase treatment. Fission of the wild-type beta-chain was accompanied by loss of enzyme activity, loss of intensity of the g = 3.7 e.p.r. signal derived from dithionite-reduced FeMoco and by changes in the visible spectrum. The e.p.r. spectra of potassium ferricyanide-oxidized native and digested Kp1 show differences in the signals between g = 1.6 and 2.0. After prolonged treatment, the final specific activity of Kp1 was about 25 +/- 5% of the initial activity. This corresponded to 25 +/- 5% of the beta-chain which was resistant to proteolytic action. Brief treatment of Kp1 with a lower concentration of chymotrypsin (chymotrypsin/Kp1 ratio = 1:10 by mass, for 10 min) preferentially cleaved high-molecular-mass polypeptides that routinely contaminate preparations of Kp1 prepared by standard procedures. Treatment with chymotrypsin followed by gel filtration to remove the proteinase and cleaved protein fragments can therefore be used to increase significantly the specific activity of Kp1 preparations and remove contaminating activities, such as the ATPase activity of myokinase.

The metallo-sulphur centres of the nitrogenase MoFe protein (Kp1) from wild-type and mutant strains of Klebsiella pneumoniae.

1993 ◽  
Vol 51 (1-2) ◽  
pp. 348
Author(s):  
B.E. Smith ◽  
M. Buck ◽  
K.Y. Faridoon ◽  
C.A. Gormal ◽  
B.D. Howes ◽  
...  
Keyword(s):  
Klebsiella Pneumoniae ◽  
Wild Type ◽  
Mofe Protein ◽  
Mutant Strains

The metallo-sulphur centres of the nitrogenase MoFe protein (Kp1) from wild-type and mutant strains of Klebsiella pneumoniae.

1993 ◽  
Vol 51 (1-2) ◽  
pp. 357
Author(s):  
B.E. Smith ◽  
M. Buck ◽  
K.Y. Faridoon ◽  
C.A. Gormal ◽  
B.D. Howes ◽  
...  
Keyword(s):  
Klebsiella Pneumoniae ◽  
Wild Type ◽  
Mofe Protein ◽  
Mutant Strains

scholarly journals Nitrogenase from nifV mutants of Klebsiella pneumoniae contains an altered form of the iron-molybdenum cofactor

1984 ◽  
Vol 217 (1) ◽  
pp. 317-321 ◽  
Author(s):  
T R Hawkes ◽  
P A McLean ◽  
B E Smith
Keyword(s):  
Klebsiella Pneumoniae ◽  
Active Site ◽  
Molybdenum Cofactor ◽  
Wild Type ◽  
H2 Evolution ◽  
Mofe Protein ◽  
Fe Protein ◽  
Metal Contents ◽  
Altered Form ◽  
Iron Molybdenum Cofactor

When the iron-molybdenum cofactor (FeMoco) was extracted from the MoFe protein of nitrogenase from a nifV mutant of Klebsiella pneumoniae and combined with the FeMoco-deficient MoFe protein from a nifB mutant, the resultant MoFe protein exhibited the NifV phenotype, i.e. in combination with wild-type Fe protein it exhibited poor N2-fixation activity and its H2-evolution activity was inhibited by CO. These data provide strong evidence that FeMoco contains the active site of nitrogenase. The metal contents and e.p.r. properties of FeMoco from wild-type and nifV mutants of K. pneumoniae are very similar.

Isolation and Characterization of Wild-Type Lipoxygenase LOXPsa1 from Pleurotus sapidus

2014 ◽  
Vol 69 (3-4) ◽  
pp. 149-154 ◽  
Author(s):  
Ina Plagemann ◽  
Ulrich Krings ◽  
Ralf G. Berger
Keyword(s):  
De Novo ◽  
Specific Activity ◽  
Maximal Activity ◽  
Wild Type ◽  
Isolation And Characterization ◽  
Peptide Mass ◽  
Purification Scheme ◽  
Pleurotus Sapidus ◽  
Nano Liquid Chromatography

The lipoxygenase LOXPsa1 of Pleurotus sapidus, originally investigated because of its ability to oxidize (+)-valencene to the valuable grapefruit aroma (+)-nootkatone, was isolated from the peptidase-rich lyophilisate using a three-step purification scheme including preparative isoelectric focusing and chromatographic techniques. Nano-liquid chromatography electrospray ionization tandem mass spectrometry (nLC-ESI-MS=MS) of the purified enzyme and peptide mass fingerprint analysis gave 38 peptides of the lipoxygenase from P. sapidus. Nearly 50% of the 643 amino acids long sequence encoded by the cDNA was covered. Both terminal peptides of the native LOXPsa1 were identified by de novo sequencing, and the postulated molecular mass of 72:5 kDa was confirmed. With linoleic acid as the substrate, the LOXPsa1 showed a specific activity of 113 U mg-1 and maximal activity at pH 7.0 and 30 °C, respectively.

Isolation and characterization of electrophoretic variants of human prostatic acid phosphatase.

1983 ◽  
Vol 29 (11) ◽  
pp. 1886-1889 ◽  
Author(s):  
M D Hibbard ◽  
R C McCarthy ◽  
H Markowitz
Keyword(s):  
Enzyme Activity ◽  
Acid Phosphatase ◽  
Specific Activity ◽  
Prostatic Acid Phosphatase ◽  
Ph Gradient ◽  
Electrophoretic Variants ◽  
Isolation And Characterization ◽  
Immunochemical Determination ◽  
Km Value ◽  
Specific Activities

Abstract Prostatic acid phosphatase (EC 3.1.3.2) purified from benign hypertrophic prostate tissue was fractionated by preparative slab isoelectric focusing over a pH gradient of 3.16 to 7.16. Twenty-two of 29 fractions contained enzyme activity. We further examined each active fraction by determining the Michaelis-Menten constant and specific activity. The protein concentration used in the latter determination was estimated either spectrophotometrically or immunochemically by three different radioimmunoassays for the enzyme. Determination of specific activities for each fraction directly correlated enzyme activity with an immunochemical determination, which indicated the immunochemical relationships among different molecular species of the enzyme. We found that the Michaelis-Menten constants for the isolated fractions were similar to the Km value for purified, unfractionated enzyme. Most fractions analyzed by each immunoassay had similar specific activities; the few fractions with discrepant specific activities were found at either end of the pH gradient. The similarity in specific activities among the fractions indicates that RIAs involving polyclonal antisera detect all of the electrophoretic variants of the enzyme.

scholarly journals Isolation and characterization of a high iturin yielding Bacillus velezensis UV mutant with improved antifungal activity

PLoS ONE ◽  
2020 ◽  
Vol 15 (12) ◽  
pp. e0234177
Author(s):  
Young Tae Kim ◽  
Sung Eun Kim ◽  
Won Jung Lee ◽  
Zhao Fumei ◽  
Min Sub Cho ◽  
...  
Keyword(s):  
Biological Control ◽  
Antifungal Activity ◽  
Mutant Strain ◽  
Fusarium Wilt ◽  
Potential Candidate ◽  
Wild Type ◽  
Bacillus Velezensis ◽  
Sclerotinia Rot ◽  
Isolation And Characterization

To isolate Bacillus velezensis mutants with improved antifungal activity for use in the biological control of phytopathogenic fungi, wild-type Bacillus velezensis KRF-001 producing iturin, surfactin, and fengycin was irradiated by ultraviolet (UV) rays. The in vitro and in vivo antifungal activities of UV mutants and characterization of the cyclic lipopeptides produced by a selected mutant were examined. A mutant strain yielding high levels of iturin showed over 2-fold higher antifungal activity than the wild-type against Fusarium oxysporum. A potent suppressive effect of the mutant was also observed on spore germination of Botrytis cinerea, the causative agent of cucumber gray mold, at different butanol extract concentrations. Further analysis of the mutant by real-time PCR and high-performance liquid chromatography revealed increased expression of iturin and surfactin biosynthesis genes as well as enhanced production of iturin and surfactin metabolites. However, the amounts of fengycin obtained from the mutant strain BSM54 were significantly lesser than those of iturin and surfactin. Particularly, iturin A production by the mutant was 3.5-fold higher than that of the wild-type, suggesting that the higher antifungal activity of the mutant against F. oxysporum resulted from the increased expression of biosynthesis genes associated with iturin production. The commercial greenhouse experiment using soil naturally infested with Sclerotinia sclerotiorum (sclerotinia rot) and F. oxysporum (fusarium wilt) showed that the mutant strain reduced sclerotinia rot and fusarium wilt diseases (P = 0.05) more effectively than the wild-type and commercially available product Cillus® in Korea. These results suggest that the mutant with high iturin yield is a potential candidate for the development of a biological control agent in agriculture.

Effect of the carbon source and cyclic AMP on isocitrate dehydrogenase, succinate dehydrogenase, and malate dehydrogenase in Klebsiella pneumoniae C3

1982 ◽  
Vol 28 (10) ◽  
pp. 1101-1106 ◽  
Author(s):  
A. Juarez ◽  
R. Parés ◽  
J. Vives-Rego
Keyword(s):  
Klebsiella Pneumoniae ◽  
Stationary Phase ◽  
Succinate Dehydrogenase ◽  
Malate Dehydrogenase ◽  
Isocitrate Dehydrogenase ◽  
Specific Activity ◽  
Exponential Growth Phase ◽  
Specific Activities ◽  
In The Beginning ◽  
Excess Glucose

When strain C3 of Klebsiella pneumoniae is grown on a minimal medium with excess glucose, isocitrate dehydrogenase, malate dehydrogenase, and succinate dehydrogenase specific activities increase in the last period of the exponential growth phase and in the beginning of the stationary phase. Glucose exhaustion does not alter the development of malate dehydrogenase and succinate dehydrogenase, but specific activities are higher than those obtained with excess glucose. In contrast, glucose exhaustion can be correlated with a decrease of isocitrate dehydrogenase specific activity in the stationary phase. Induction of strain C3 isocitrate dehydrogenase by glucose in complex medium and repression by cAMP in mineral medium were observed. Glucose induction and the NADP/NADPH ratio are suggested as regulatory mechanisms controlling isocitrate dehydrogenase synthesis in the Enterobacteriaceae, but the former appears to be restricted to some Klebsiella strains.

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