Cloning and expression of the algL gene, encoding the Azotobacter chroococcum alginate lyase: purification and characterization of the enzyme.
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The alginate lyase-encoding gene (algL) of Azotobacter chroococcum was localized to a 3.1-kb EcoRI DNA fragment that revealed an open reading frame of 1,116 bp. This open reading frame encodes a protein of 42.98 kDa, in agreement with the value previously reported by us for this protein. The deduced protein has a potential N-terminal signal peptide that is consistent with its proposed periplasmic location. The analysis of the deduced amino acid sequence indicated that the gene sequence has a high homology (90% identity) to the Azotobacter vinelandii gene sequence, which has very recently been deposited in the GenBank database, and that it has 64% identity to the Pseudomonas aeruginosa gene sequence but that it has rather low homology (15 to 22% identity) to the gene sequences encoding alginate lyase in other bacteria. The A. chroococcum AlgL protein was overproduced in Escherichia coli and purified to electrophoretic homogeneity in a two-step chromatography procedure on hydroxyapatite and phenyl-Sepharose. The kinetic and molecular parameters of the recombinant alginate lyase are similar to those found for the native enzyme. (+info)
Flavodoxin: an allosteric inhibitor of AMP nucleosidase from Azotobacter vinelandii.
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Flavodoxin, which participates in nitrogen fixation, was found to be a potent allosteric inhibitor of AMP nucleosidase [EC 3.2.2.4] from Azotobacter vinelandii. It inhibited the enzyme by decreasing its affinity for ATP without affecting the maximum velocity. The inhibition constant for flavodoxin was estimated to be 10 muM, which is within the range of physiological concentration in the cells. The concentration of flavodoxin able to alter the activity in vitro suggests that this phenomenon could be of significance in the regulation of flavin biosynthesis in vivo. Flavin mononucleotide (FMN), a prosthetic group of flavodoxin, was also found to act as an allosteric inhibitor. Since no inhibitory action of apo-flavodoxin was observed, it was concluded that the FMN chromophore of the flavodoxin is responsible for the inhibition of the enzyme by this protein. (+info)
Interactions of heterologous nitrogenase components that generate catalytically inactive complexes.
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A unique method is described for inhibiting nitrogenase. When Clostridium pasteurianum nitrogenase is assayed in the presence of the Mo-Fe protein of Azotobacter vinelandii, all the characteristic activities of nitrogenase are inhibited. C. pasteurianum nitrogenase is unaffected by the Fe protein of A. vinelandii. The Fe protein, but not the Mo-Fe protein of C. pasteurianum, inhibits A. vinelandii nitrogenase. Both inhibitions described result from the formation of an inactive complex of A. vinelandii Mo-Fe protein and C. pasteurianum Fe protein. Complex formation requires active components, as oxygen-denatured proteins are ineffective. The results for titration of components of the complex against each other and kinetic data each indicate that the inactive complex consists of two molecules of C. pasteurianum Fe protein per molecule of A. vinelandii Mo-Fe protein. The results of kinetic experiments suggest that the Fe protein from each organism competes for the same site(s) on the A. vinelandii Mo-Fe protein. The Fe protein of C. pasteurianum will form an active or an inactive complex with the Mo-Fe proteins from six different organisms. Inhibition by nitrogenase components that form inactive complexes provides numeroius ways to study the mechanism of nitrogenase action. (+info)
Transcription of bacteriophage deoxyribonucleic acid. Comparison of Escherichia coli and Azotobacter vinelandii sigma subunits.
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The effect of the sigma subunit of RNA polymerase on the rate and asymmetry of the in vitro transcription of Escherichia coli and Azotobacter vinelandii phage DNAs has been studied with purified E. coli and A. vinelandii RNA polymerases and hybrid enzymes containing the core subunits of one enzyme and sigma from the other. The effect of sigma on the rate of transcription is characteristic of the template and not of the enzyme and depends on ionic strength. The rate of transcription of A. vinelandii phage A21 DNA is decreased by sigma at high ionic strength, but shows the more characteristic stimulation at KCl concentrations below 0.05 M. In contrast, the stimulation by sigma of T4 DNA transcription increased with an increase in the KCl concentrations. All combinations of core and sigma subunits behaved similarly with respect to stimulation or inhibition by sigma and with respect to asymmetric transcription of S13 replicative form (RF)DNA. However, the heterologous, but not the homologous combinations of core and sigma transcribed A21 symmetrically. S13 RF DNA in the superhelical, but not in the relaxed configuration, is transcribed asymmetrically by the A. vinelandii core enzyme. A role for the core subunits in specific site recognition is indicated by this observation. (+info)
Transcription of Azotobacter phage deoxyribonucleic acid. Salt-dependent equilibrium between steps in initiation.
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The transcription of Azotobacter phage A21 DNA by Escherichia coli or Azotobacter vinelandii RNA polymerase differs from that of some other DNAs in its inhibition by moderate concentrations of KCl. This characteristic results in an apparent low template activity for this DNA as compared with T4 DNA under standard assay conditions. From an analysis of the dependence of the various steps in initiation on KCl it is concluded that the effect is exerted on an equilibrium between an inactive polymerase-DNA complex and an active preintitiation complex. This salt-sensitive equilibrium favors the inactive complex at a lower KCl concentration than with other templates. It can be approached from other low or high salt concentrations at a measurably slow rate. (+info)
Evidence for a two-electron transfer using the all-ferrous Fe protein during nitrogenase catalysis.
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The nitrogenase-catalyzed H(2) evolution and acetylene-reduction reactions using Ti(III) and dithionite (DT) as reductants were examined and compared under a variety of conditions. Ti(III) is known to make the all-ferrous Fe protein ([Fe(4)S(4)](0)) and lowers the amount of ATP hydrolyzed during nitrogenase catalysis by approximately 2-fold. Here we further investigate this behavior and present results consistent with the Fe protein in the [Fe(4)S(4)](0) redox state transferring two electrons ([Fe(4)S(4)](2+)/[Fe(4)S(4)](0)) per MoFe protein interaction using Ti(III) but transferring only one electron ([Fe(4)S(4)](2+)/[Fe(4)S(4)](1+)) using DT. MoFe protein specific activity was measured as a function of Fe:MoFe protein ratio for both a one- and a two-electron transfer reaction, and nearly identical curves were obtained. However, Fe protein specific activity curves as a function of MoFe:Fe protein ratio showed two distinct reactivity patterns. With DT as reductant, typical MoFe inhibition curves were obtained for operation of the [Fe(4)S(4)](2+)/[Fe(4)S(4)](1+) redox couple, but with Ti(III) as reductant the [Fe(4)S(4)](2+)/[Fe(4)S(4)](0) redox couple was functional and MoFe inhibition was not observed at high MoFe:Fe protein ratios. With Ti(III) as reductant, nitrogenase catalysis produced hyperbolic curves, yielding a V(max) for the Fe protein specific activity of about 3200 nmol of H(2) min(-1) mg(-1) Fe protein, significantly higher than for reactions conducted with DT as reductant. Lag phase experiments (Hageman, R. V., and Burris, R. H. (1978) Proc. Natl. Acad. Sci. U. S. A. 75, 2699-2702) were carried out at MoFe:Fe protein ratios of 100 and 300 using both DT and Ti(III). A lag phase was observed for DT but, with Ti(III) product formation, began immediately and remained linear for over 30 min. Activity measurements using Av-Cp heterologous crosses were examined using both DT and Ti(III) as reductants to compare the reactivity of the [Fe(4)S(4)](2+)/[Fe(4)S(4)](1+) and [Fe(4)S(4)](2+)/[Fe(4)S(4)](0) redox couples and both were inactive. The results are discussed in terms of the Fe protein transferring two electrons per MoFe protein encounter using the [Fe(4)S(4)](2+)/[Fe(4)S(4)](0) redox couple with Ti(III) as reductant. (+info)
Purification and properties of nitrogenase from the cyanobacterium, Anabaena cylindrica.
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The nitrogenase complex was isolated from nitrogen-starved cultures of Anabaema cylindrica. Sodium dithionite, photochemically reduced ferredoxin, and NADPH were found to be effective election donors to nitro genase in crude extracts whereas hydrogen and pyruvate were not. The Km for acetylene in vivo is ten-fold higher than the Km in vitro, whereas this pattern does not hold for the non-heterocystous cyanobacterium, Plectonema boryanum. This indicates that at least one mechanism of oxygen protection in vivo involves a gas diffusion barrier presented by the heterocyst cell wall. The Mo-Fe component was purified to homogeneity. Its molecular weight (220,000), subunit composition, isoelectric point (4.8), Mo, Fe, and S2- content (2, 20 and 20 mol/mol component), and amino acid composition indicate that this component has similar properties to Mo-Fe-containing components isolated from other bacterial sources. The isolated components from A. cylindrica were found to cross-react, to varying degrees, with components isolated from Azotobacter vinelandii, Rhodospirillum rubrum, and P. boryanum. (+info)
Studies on the product binding sites of the Azotobacter vinelandii ribonucleic acid polymerase.
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During chain elongation RNA polymerase exists as a ternary DNA-enzyme-RNA complex in which a discrete length of the nascent RNA chain proximal to the 3'-OH terminus will be bound to the product binding site (Krakow, J. S., and Fronk, E. (1969) J. Biol. Chem. 244, 5988). We have utilized the poly[d(A-T)]-directed reaction to determine the length of the nascent poly[r(A-U)] protected from attack by pancreatic ribonuclease. Following release of the ribonuclease resistant oligo[r(A-U)] from the ternary complex, its size was determined by ion exchange chromatography on DEAE-cellulose, gel filtration on Bio-Gel P-10, and the ratio of 3'-terminal uridine to internal 2':3'-UMP following alkaline hydrolysis. The results indicate that the length of the nascent protected fragment is approximately 12 residues. (+info)