scholarly journals Altered nitrogenase MoFe proteins from Azotobacter vinelandii. Analysis of MoFe proteins having amino acid substitutions for the conserved cysteine residues within the β-subunit

1991 ◽  
Vol 277 (2) ◽  
pp. 457-464 ◽  
Author(s):  
H D May ◽  
D R Dean ◽  
W E Newton
Keyword(s):  
Signal Intensity ◽  
Azotobacter Vinelandii ◽  
Beta Subunit ◽  
Site Directed Mutagenesis ◽  
Cysteine Residues ◽  
Acetylene Reduction Activity ◽  
Protein Activity ◽  
Whole Cell ◽  
Mofe Protein ◽  
Diazotrophic Growth

The regions surrounding the three strictly conserved cysteine residues (positions 70, 95 and 153) in the beta-subunit of the Azotobacter vinelandii nitrogenase MoFe protein have been proposed to provide P-cluster environments [Dean, Setterquist, Brigle, Scott, Laird & Newton (1990) Mol. Microbiol. 4, 1505-1512]. In the present study, each of these cysteine residues was individually substituted by either serine or alanine by site-directed mutagenesis of the nifK gene, which encodes the MoFe protein beta-subunit. A mutant strain for which the codon for Cys-153 is removed was also isolated. Significant structural or functional roles are indicated for the cysteine residues at positions 70 and 95, where substitution by either serine or alanine eliminates diazotrophic growth of the resulting strains and abolishes or markedly decreases both MoFe-protein acetylene-reduction activity and the intensity of the whole-cell S = 3/2 e.p.r. signal. Changes introduced at position 153 have various effects on the functional properties of the enzyme. The strains produced either by deletion of the Cys-153 residue or its substitution by serine exhibit only a moderate decrease in diazotrophic growth and MoFe-protein activity and no loss of the whole-cell e.p.r.-signal intensity. In contrast, substitution by alanine eliminates diazotrophic growth and very markedly decreases both MoFe-protein activity and e.p.r.-signal intensity. These results are interpreted in terms of a metallocluster-driven protein rearrangement. After purification of the altered MoFe protein, in which serine replaces Cys-153, an investigation of its catalytic and spectroscopic properties confirms that neither the FeMo cofactor, i.e. the substrate-reduction site, nor the component-protein interaction site has been affected. Instead, these data indicate a disruption in electron transfer within the MoFe protein, which is consistent with a role for this residue (and region) at the P clusters.

scholarly journals Site-directed mutagenesis of the Klebsiella pneumoniae nitrogenase. Effects of modifying conserved cysteine residues in the α- and β-subunits

1989 ◽  
Vol 264 (1) ◽  
pp. 257-264 ◽  
Author(s):  
H M Kent ◽  
I Ioannidis ◽  
C Gormal ◽  
B E Smith ◽  
M Buck
Keyword(s):  
Klebsiella Pneumoniae ◽  
Active Component ◽  
Molybdenum Cofactor ◽  
Beta Subunit ◽  
Site Directed Mutagenesis ◽  
Alpha Subunit ◽  
Cysteine Residues ◽  
Directed Mutagenesis ◽  
Diazotrophic Growth ◽  
Iron Molybdenum Cofactor

The five conserved cysteine residues present in the alpha-subunit and the three conserved cysteine residues present in the beta-subunit of nitrogenase component 1 were individually changed to alanine. Mutations in the alpha-subunit at positions 63, 89, 155 and 275 and in the beta-subunit at positions 69, 94 and 152 all resulted in a loss of diazotrophic growth and component 1 activity and loss of the normal e.p.r. signal of the component 1 protein. Component 2 activity was retained. Replacement of cysteine-184 in the alpha-subunit with alanine greatly diminished, but did not eliminate, diazotrophic growth and component 1 activity. Substitution of serine for cysteine at position 152 in the beta-subunit, in contrast with the substitution of alanine at this position, resulted in the formation of active component 1. Replacement of the non-conserved cysteine-112 in the beta-subunit with alanine did not greatly perturb diazotrophic growth or the activity of component 1. Extracts prepared from a mutant, with cysteine-275 of the alpha-subunit replaced by alanine, complemented extracts of a mutant unable to synthesize the iron-molybdenum cofactor of nitrogenase, indicating that the alanine-275 substitution increases the availability of cofactor. Furthermore extracts of this mutant exhibited an e.p.r. signal similar to that of extracted iron-molybdenum cofactor. These data suggest a role for cysteine-275 as a ligand to the cofactor.

scholarly journals Site-directed mutagenesis of the nitrogenase MoFe protein of Azotobacter vinelandii

1987 ◽  
Vol 84 (20) ◽  
pp. 7066-7069 ◽  
Author(s):  
K. E. Brigle ◽  
R. A. Setterquist ◽  
D. R. Dean ◽  
J. S. Cantwell ◽  
M. C. Weiss ◽  
...  
Keyword(s):  
Azotobacter Vinelandii ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Mofe Protein

scholarly journals Mutational Inactivation of a Gene Homologous to Escherichia coli ptsP Affects Poly-β-Hydroxybutyrate Accumulation and Nitrogen Fixation in Azotobacter vinelandii

1998 ◽  
Vol 180 (18) ◽  
pp. 4790-4798 ◽  
Author(s):  
Daniel Segura ◽  
Guadalupe Espín
Keyword(s):  
Escherichia Coli ◽  
Azotobacter Vinelandii ◽  
Growth Defect ◽  
Nucleotide Sequencing ◽  
Acetylene Reduction Activity ◽  
Respiratory Protection ◽  
Phosphotransferase System ◽  
Terminal Domain ◽  
Diazotrophic Growth ◽  
Enzyme I

ABSTRACT Strain DS988, an Azotobacter vinelandii mutant with a reduced capacity to accumulate poly-β-hydroxybutyrate, was isolated after mini-Tn5 mutagenesis of the UW136 strain. Cloning and nucleotide sequencing of the affected locus revealed a gene homologous to Escherichia coli ptsP which encodes enzyme INtr, a homologue of enzyme I of the phosphoenol pyruvate-sugar phosphotransferase system with an N-terminal domain similar to the N-terminal domain of some NifA proteins. Strain DS988 was unable to grow diazotrophically with 10 mM glucose as a carbon source. Diazotrophic growth on alternative carbon sources such as gluconate was only slightly affected. Glucose uptake, as well as glucose kinase and glucose-6-phosphate-dehydrogenase activities that lead to the synthesis of gluconate-6-phosphate, were not affected by the ptsP mutation. The inability of DS988 to grow diazotrophically in 10 mM glucose was overcome by supplying ammonium or other sources of fixed nitrogen. Acetylene reduction activity but not transcription of the nitrogenase structural gene nifH was shown to be impaired in strain DS988 when it was incubated in 10 mM glucose. The diazotrophic growth defect of DS988 was restored either by increasing the glucose concentration to above 20 mM or by lowering the oxygen concentration. These data suggest that a mutation inptsP leads to a failure in poly-β-hydroxybutyrate metabolism and in the respiratory protection of nitrogenase under carbon-limiting conditions.

scholarly journals Involvement of cysteine residues in catalysis and inhibition of human aldose reductase. Site-directed mutagenesis of Cys-80, -298, and -303.

1992 ◽  
Vol 267 (34) ◽  
pp. 24833-24840 ◽  
Author(s):  
J.M. Petrash ◽  
T.M. Harter ◽  
C.S. Devine ◽  
P.O. Olins ◽  
A Bhatnagar ◽  
...  
Keyword(s):  
Aldose Reductase ◽  
Site Directed Mutagenesis ◽  
Cysteine Residues ◽  
Directed Mutagenesis

Modification of a novel x-type high-molecular-weight glutenin subunit gene from Aegilops markgrafii to improve dough strength of wheat flour

2018 ◽  
Vol 69 (9) ◽  
pp. 873
Author(s):  
Xin Ma ◽  
Xuye Du ◽  
Cunyao Bo ◽  
Hongwei Wang ◽  
Anfei Li ◽  
...  
Keyword(s):  
Molecular Weight ◽  
High Molecular Weight ◽  
Wheat Flour ◽  
Cysteine Residue ◽  
Site Directed Mutagenesis ◽  
Cysteine Residues ◽  
Dough Strength ◽  
Typical Structure ◽  
Dough Quality

High-molecular-weight glutenin subunits (HMW-GS) in bread wheat are major determinants of dough viscoelastic properties and the end-use quality of wheat flour. Cysteine residues, which form intermolecular disulphide bonds in HMW-GS, could improve the strength of gluten. To our knowledge, the number and position of cysteine residues in HMW-GS are conserved between wheat (Triticum aestivum) and Aegilops markgrafii. In the present study, we modified a gene (1Cx1.1) from Ae. markgrafii for an HMW-GS that possessed the typical structure and conserved number of cysteines. Site-directed mutagenesis was carried out in 1Cx1.1 to investigate how the position of cysteine residues in HMW-GS affects the mixing properties of dough. Six HMW-GS containing an extra cysteine residue were expressed in Escherichia coli, and the proteins were purified at sufficient scale for incorporation into flour to test dough quality. There were large differences in dough property among samples containing different modified subunits. Cysteine substituting in the N-terminal or repetitive-domain of HMW-GS could significantly improve dough quality. The results showed that the strategy was useful for providing genetic resources for gene engineering, and hence could be valuable for improving the processing quality of wheat.

scholarly journals Key Role of Cysteine Residues in Catalysis and Subcellular Localization of Sulfur Oxygenase-Reductase of Acidianus tengchongensis

2005 ◽  
Vol 71 (2) ◽  
pp. 621-628 ◽  
Author(s):  
Zhi-Wei Chen ◽  
Cheng-Ying Jiang ◽  
Qunxin She ◽  
Shuang-Jiang Liu ◽  
Pei-Jin Zhou
Keyword(s):  
Cytoplasmic Membrane ◽  
Sulfur Oxidation ◽  
Site Directed Mutagenesis ◽  
Cysteine Residues ◽  
Wild Type ◽  
Immune Electron Microscopy ◽  
Mutant Proteins ◽  
Close Proximity ◽  
Catalytic Sites

ABSTRACT Analysis of known sulfur oxygenase-reductases (SORs) and the SOR-like sequences identified from public databases indicated that they all possess three cysteine residues within two conserved motifs (V-G-P-K-V-C31 and C101-X-X-C104; numbering according to the Acidianus tengchongensis numbering system). The thio-modifying reagent N-ethylmaleimide and Zn2+ strongly inhibited the activities of the SORs of A. tengchongensis, suggesting that cysteine residues are important. Site-directed mutagenesis was used to construct four mutant SORs with cysteines replaced by serine or alanine. The purified mutant proteins were investigated in parallel with the wild-type SOR. Replacement of any cysteine reduced SOR activity by 98.4 to 100%, indicating that all the cysteine residues are crucial to SOR activities. Circular-dichroism and fluorescence spectrum analyses revealed that the wild-type and mutant SORs have similar structures and that none of them form any disulfide bond. Thus, it is proposed that three cysteine residues, C31 and C101-X-X-C104, in the conserved domains constitute the putative binding and catalytic sites of SOR. Furthermore, enzymatic activity assays of the subcellular fractions and immune electron microscopy indicated that SOR is not only present in the cytoplasm but also associated with the cytoplasmic membrane of A. tengchongensis. The membrane-associated SOR activity was colocalized with the activities of sulfite:acceptor oxidoreductase and thiosulfate:acceptor oxidoreductase. We tentatively propose that these enzymes are located in close proximity on the membrane to catalyze sulfur oxidation in A. tengchongensis.

scholarly journals Site-directed mutagenesis of Azotobacter vinelandii ferredoxin I: [Fe-S] cluster-driven protein rearrangement.

1990 ◽  
Vol 87 (2) ◽  
pp. 598-602 ◽  
Author(s):  
A. E. Martin ◽  
B. K. Burgess ◽  
C. D. Stout ◽  
V. L. Cash ◽  
D. R. Dean ◽  
...  
Keyword(s):  
Azotobacter Vinelandii ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Ferredoxin I
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