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PubMed1989

X-ray structure of lipoamide dehydrogenase from Azotobacter vinelandii determined by a combination of molecular and isomorphous replacement techniques.

The crystal structure of lipoamide dehydrogenase from Azotobacter vinelandii has been determined by a combination of molecular replacement and isomorphous replacement techniques yielding eventually a good-quality 2.8 A electron density map. Initially, the structure determination was attempted by molecular replacement procedures alone using a model of human glutathione reductase, which has 26% sequence identity with this bacterial dehydrogenase. The rotation function yielded the correct orientation of the model structure both when the glutathione reductase dimer and monomer were used as starting model. The translation function could not be solved, however. Consequently, data for two heavy-atom derivatives were collected using the Hamburg synchotron facilities. The derivatives had several sites in common, which was presumably a major reason why the electron density map obtained by isomorphous information alone was of poor quality. Application of solvent flattening procedures cleaned up the map considerably, however, showing clearly the outline of the lipoamide dehydrogenase dimer, which has a molecular weight of 100,000. Application of the "phased translation function", which combines the phase information of both isomorphous and molecular replacement, led to an unambiguous determination of the position of the model structure in the lipoamide dehydrogenase unit cell. The non-crystallographic 2-fold axis of the dimer was optimized by several cycles of constrained-restrained least-squares refinement and subsequently used for phase improvement by 2-fold density averaging. After ten cycles at 3.5 A, the resolution was gradually extended to 2.8 A in another 140 cycles. The 2.8 A electron density distribution obtained in this manner was of much improved quality and allowed building of an atomic model of A. vinelandii lipoamide dehydrogenase. It appears that in the orthorhombic crystals used each dimer is involved in contacts with eight surrounding dimers, leaving unexplained why the crystals are rather fragile. Contacts between subunits within one dimer, which are quite extensive, can be divided into two regions separated by a cavity. In one of the contact regions, the level of sequence identity with glutathione reductase is very low but it is quite high in the other. The folding of the polypeptide chain in each subunit is quite similar to that of glutathione reductase, as is the extended conformation of the co-enzyme FAD.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗

PubMed1991

Refined crystal structure of lipoamide dehydrogenase from Azotobacter vinelandii at 2.2 A resolution. A comparison with the structure of glutathione reductase.

The structure of lipoamide dehydrogenase from Azotobacter vinelandii has been refined by the molecular dynamics technique to an R-factor of 19.8% at 2.2 A resolution. In the final model, the root-mean-square deviation from ideality is 0.02 A for bond lengths and 3.2 degrees for bond angles. The asymmetric unit comprises two subunits, each consisting of 466 amino acid residues and the prosthetic group FAD, plus 512 solvent molecules. The last ten amino acid residues of both chains are not visible in the electron density distribution and they are probably disordered. The operation required to superimpose the two chains forming the dimer is a rotation of exactly 180 degrees with no translation component. The final model shows the two independently refined subunits to be very similar, except for six loops located at the surface of the molecule. The structure of each subunit of the enzyme consists of four domains with the catalytic centre located at the subunit interface. The reactive disulphide bridge, 48-53, is oxidized with S gamma of Cys53 located 3.5 A away from carbon C-4a of the isoalloxazine ring. The side-chain of His450' points its N epsilon 2 towards S gamma of Cys48 and is hydrogen bonded to the carboxylate of Glu455'. The FAD is bound in an extended conformation and the isoalloxazine ring is not completely planar with an angle between the pteridine and the benzene ring of 7.3 degrees in the first subunit and of 12.1 degrees in the second one. The overall folding of lipoamide dehydrogenase is very similar to that of glutathione reductase. However, a comparison of the two enzymes, which have only 26% sequence identity, reveals significant conformational differences. These concern the tertiary as well as the quaternary structure of the two molecules. In each subunit of lipoamide dehydrogenase the NAD-binding domain and the interface domain appear to be differently oriented with respect to the FAD-binding domain by 7.1 degrees and 7.8 degrees, respectively. The interface domain contains, in addition, major changes in tertiary structure. Furthermore, the two subunits forming the dimer appear to be shifted with respect to each other by more than 4 A, when the lipoamide dehydrogenase dimer is compared with that of glutathione reductase. In spite of all these changes at the tertiary and quaternary level the active sites of the enzymes, which occur at the dimer interface, appear to be remarkably similar.(ABSTRACT TRUNCATED AT 400 WORDS)

Azotobacter↗

PubMed1992

Evidence of reduced poly-B-hydroxybutyrate biosynthesis in free-living nitrogen-fixing bacteria, Azotobacter chroococcum, following acquired resistance to the fungicide captan.

Some biological activities of Azotobacter chroococcum, strain Azcap 1, (spontaneous mutant, captan resistant up to 300 micrograms/ml) were assayed on RM medium with and without the presence of the fungicide. Comparisons were also carried out with Az. chroococcum sensitive strains Azwt, Azcan 10 and 14. The hydrolysis of captan, incorporated in agar plates of RM at 100 micrograms/ml, was rapid, since on 4-day plates, no effect was found on the strain Azwt, while on freshly prepared ones its growth was completely blocked. As for Azcap 1, grown on RM only, the behaviour was similar to that of sensitive strains, whereas when grown on captan the results of experiments showed: (i) a lag of approximately 12 h to reach the maximum nitrogen-fixing activity; (ii) delay of 12-24 h in the full consumption of glucose present in the medium, although the invertase activity did not present differences; (iii) high ATP culture content during the 50 h of the experiment; (iv) approximately 6-10-fold lower production of PHB (poly-B-hydroxybutyrate); (v) lack of typical encystment phase, for the tested 96 h and reduced viability in developing colonies on agar RM medium. In contrast, when captan was added to cultural medium at sublethal concentration, 50 micrograms/ml for sensitive strain Azwt and 200 micrograms/ml for Azcap 1, the amount of glutathione produced (to remove the fungicide toxicity) was several times higher for the former.

Azotobacter↗

PubMed1985

Transfer of transposable drug-resistance elements Tn5, Tn7, and Tn76 to Azotobacter beijerinckii: use of plasmid RP4::Tn76 as a suicide vector.

Transposable elements Tn5, Tn7, and Tn76 were transferred to Azotobacter beijerinckii. Evidence was obtained for the transposition of Tn5 but cells of the majority of presumptive transposition isolates had abnormal morphologies and rapidly lost viability when subcultured. Data are presented that indicate that plasmid RP4::Tn76 behaves as a suicide vector upon transfer to this host, allowing the isolation of A. beijerinckii::Tn76 isolates at a high frequency. Nitrogen-fixing mutants and leucine and adenine auxotrophs were isolated from cultures in which the transposition of Tn76 occurred.

Azotobacter↗

PubMed1988

Transformation of Azotobacter vinelandii OP with a broad host range plasmid containing a cloned chromosomal nif-DNA marker.

The non-nitrogen-fixing (Nif-) strain UW10 of Azotobacter vinelandii OP (UW) was naturally induced to competence and transformed with broad host range plasmid pKT210 containing the cloned wild-type nif-10 locus from A. vinelandii UW (Nif+); this marker was unable to complement the nif-10 mutation in trans, but could through recombination with the chromosome. The most frequent type of transformation event observed was recombination between the homologous regions of the plasmid and chromosome (producing Nif+ transformants) with loss of the plasmid vector. At a substantially lower frequency, transformants expressing the plasmid-encoded antibiotic resistance determinants were isolated which were phenotypically Nif-. Agarose gel electrophoresis showed that these transformants contained a plasmid migrating with the same mobility as the original donor plasmid. During culture these transformants acquired a Nif+ phenotype without the loss of the plasmid, as judged by the use of a hybridization probe specific for the cloned nif-DNA fragment. These data indicate that plasmids carrying sequences homologous to chromosomal sequences could be maintained in recombination-proficient A. vinelandii UW. The introduction of plasmids containing sequences homologous to chromosomal sequences was facilitated by prelinearization of the plasmid using a restriction endonuclease generating cohesive ends. Because the site of linearization could be chosen outside the region of shared homology, it was unlikely that the route of plasmid establishment occurred via a homology-facilitated transformation mechanism. The data also indicated that A. vinelandii UW could harbor broad host range cloning vectors based on plasmid RSF1010 without significant impairment of its nitrogen-fixation ability.

Azotobacter↗

PubMed1991

Tn5 mutagenesis and insertion replacement in Azotobacter vinelandii.

Tn5 insertion mutants of Azotobacter vinelandii were isolated using vectors pJB4JI (IncP) and pGS9 (IncN). A procedure to replace Tn5 (Kmr) by its nontransposing derivative Tn5-131 (Tcr) was developed. For the replacement, a ColEl derivative harboring Tn5-131 (pRZ131) was conjugally mobilized by the IncN plasmid pCU101 into A. vinelandii strains containing Tn5. Both plasmids are unable to be maintained in A. vinelandii, but the transient presence of pRZ131 allows recombination between the incoming and the resident Tn5 elements. Genetic and physical analysis showed that insertion replacements result in lower frequencies of Tn5-associated genomic rearrangements, thereby increasing the stability of Tn5-containing strains.

Azotobacter↗

PubMed1988

Comparison of redox and EPR properties of the molybdenum iron proteins of Clostridium pasteurianum and Azotobacter vinelandii nitrogenases.

Both heterologous crosses of the Clostridium pasteurianum and Azotobacter vinelandii nitrogenase components are completely inactive, although the reasons for this incompatibility are not known. We have compared a number of properties of the MoFe proteins from these organisms (Cp1 and Av1, respectively) in an attempt to find differences that could explain this lack of functional activity. Optical and CD spectroscopic titrations are similar for both Av1 and Cp1, but EPR titrations are significantly different, suggesting different chemical reactivity patterns and/or magnetic interaction behavior. Similarly, reduction measurements on the six-electron-oxidized state of Cp1 and Av1 at controlled potentials indicate a difference in both the relative reduction sequence of the redox centers and the numerical values for their measured midpoint potentials. EPR measurements as a function of temperature also demonstrate that the relaxation behavior of the S = 3/2 MoFe centers associated with the proteins differ markedly. The Cp1 EPR signal only begins to undergo broadening above 65 K, whereas the Av1 signal is severely broadened above 25 K. These variations in the EPR properties for the two proteins are not likely to be due to differences in the stoichiometry and/or geometry of the MoFe cluster units themselves since similar EPR studies of the isolated cofactors showed them to be essentially identical. Thus, the different EPR behavior of the two proteins seems to arise either from protein constraints imposed on identical cofactors, and/or from magnetic interactions due to neighboring metal clusters.

Azotobacter↗

PubMed1993

Further characterization of the redox and spectroscopic properties of Azotobacter vinelandii ferritin.

Bacterial ferritin from Azotobacter vinelandii (AVBF) has many properties in common with and a number of properties distinct from the more thoroughly studied animal ferritins. The most notable differences are the high phosphate content of the mineral core and the presence of heme (12 per AVBF) in AVBF. In both ferritin types, redox reactions are essential to the iron release and deposition function of the ferritins. The heme reduction potential in apo AVBF is pH independent as are both the heme and core reduction potential in holo AVBF. pH measurements confirm the pH independence for heme reduction in apo AVBF; however, they establish the conflicting result that 1.7 +/- 0.2 protons per iron atom are taken up during core reduction. These results are interpreted as a two-step reduction process consisting of a pH independent reduction of heme in holo AVBF followed by a pH dependent reduction of the mineral core. Detailed spectroscopic studies have been undertaken to determine if heme-core interactions are detectable during the redox reactions of AVBF. Optical spectroscopy of the heme groups in apo AVBF demonstrate that all twelve are identical and undergo uniform and rapid reduction. EPR spectroscopy establishes the presence of both low-symmetry, g = 4.3, Fe3+ from the mineral core and low-spin heme with g values of 2.87, 2.32, and 1.46 in holo and identical g values for the low-spin heme in apo AVBF. EPR integration of the heme groups in both apo and holo gave values of 13.2 +/- 1.3 heme spins per AVBF at 4.2, 10, 25, 35, and 45 K. No heme perturbations were detected in holo or apo AVBF by Resonance Raman and circular dichroism spectroscopy. Both reduced and oxidized apo AVBF gave normal fluorescence emission at 330-340 nm when excited at 279 nm. These spectroscopic, redox, and reactivity results provide more detailed properties of AVBF for comparison with other bacterial and animal ferritins.

Azotobacter vinelandii↗

PubMed1996

A kinetic study of iron release from Azotobacter vinelandii bacterial ferritin.

The kinetics of iron release from Azotobacter vinelandii bacterial ferritin (AVBF) was measured by reduction of core iron with S2O4(2-) followed by chelation of Fe2+ with alpha, alpha-bipyridine (bipy). The rate was first order in AVBF and one half order in S2O4(2-), suggesting that SO2- is the active reductant formed by S2O4(2-) = 2SO2-. With zero-order conditions for dithionite and bipy, two consecutive first-order iron release reactions differing by a factor of about 14 were observed with rate constants of 0.0263 and 0.00184 sec-1, respectively, at 25 degrees C and pH 7.0. The faster reaction corresponded to the loss of 1433 iron atoms (91%) and the slower second reaction corresponded to loss of 145 (9%) of the original 1575 iron atoms present. The first reaction increased about twofold with pH variation between 6.5 and 8.0, whereas the second reaction was unchanged in the pH range 5.5-8. Both dramatically increased at pH 5.0. Methyl viologen increased the rate of both reactions about tenfold. The biphasic behavior for iron loss is interpreted as two different populations of iron atoms present in the core of AVBF, the first representing the bulk iron, and the second a group of unique iron atoms released last which may represent iron attached to the interior of the protein shell or iron associated with the heme groups. Kinetic stopped-flow measurements show that the heme is first reduced, followed by reduction of the core iron by reduced heme, suggesting an electron transfer role for heme in AVBF function.

2,2'-Dipyridyl↗

PubMed1992

Identification of six open reading frames from a region of the Azotobacter vinelandii genome likely involved in dihydrogen metabolism.

We reported earlier the identification of two Azotobacter vinelandii open reading frames (ORFs), ORF1 and ORF2, downstream from the hydrogenase structural genes (Chen, J.C. and Mortenson, L.E. (1992) Biochim. Biophys. Acta 1131, 122-124). Sequencing of 6008 base pairs of DNA immediately downstream from ORF2 revealed six additional ORFs (ORF3 through ORF8). All six ORFs are transcribed from the same DNA strand as that of the ORF1 and ORF2. Deduced amino acid sequences of ORF3 through ORF5, and those of ORF4, ORF5, ORF7 and ORF8 have strong homology with genes required for dihydrogen (H2) metabolism in Rhodobacter capsulatus and in Escherichia coli, respectively. ORF4, ORF5, ORF6 and ORF8 would encode for polypeptides containing one or more 'Cys-X-X-Cys' motifs. The predicted products of ORF5 and ORF6 each contain a histidine-rich region, and the product of ORF5 also includes a 'Cys-Thr-Val-Cys-Gly-Cys' region near its amino-terminus. Implications of these findings with respect to metal binding, transport and incorporation, to hydrogenase assembly and to H2 metabolism are discussed.

Amino Acid Sequence↗

PubMed1992

Two open reading frames (ORFs) identified near the hydrogenase structural genes in Azotobacter vinelandii, the first ORF may encode for a polypeptide similar to rubredoxins.

Sequencing of 744 base pairs (bp) of a cloned section of DNA from Azotobacter vinelandii reveals two complete, closely-spaced open reading frames (ORF1 and ORF2). Both ORFs are transcribed from the same DNA strand as that of the structural genes for hydrogenase (hoxK and hoxG, Menon, A.L. et al. (1990) Gene 96, 67-74), and are located downstream from the latter genes. The distance between the end of hoxG and the beginning of ORF1 is approx. 3.0 kilobases (kb). Most of the deduced amino acid sequence of ORF1 shares high homology with rubredoxin sequences. Some of the deduced amino acid sequence of ORF2 shares homology with that of a reported partial ORF from Rhodobacter capsulatus, ORF located within a region of DNA required for dihydrogen oxidation in that organism. Implications of these findings with respect to dihydrogen metabolism are discussed.

Amino Acid Sequence↗

PubMed1982

Regulation and biochemical characterization of the glutamine synthetase of azotobacter vinelandii.

We have investigated the regulation of the activity and synthesis of the glutamine synthetase (l-glutamate:ammonia ligase (ADP-forming), EC (6.3.1.2) of Azotobacter vinelandii. Synthesis of the enzyme was not repressed by NH+4 and/or a number of amino acids in the growth medium; however, biosynthetic activity was rapidly lost through adenylylation in response to ammonium ion. The enzyme could be prepared as a 'relaxed, divalent-cation-free form which was catalytically inactive. The 'taut', active form could be restored with 1-5 mM Mg2+, Mn2+, Ca2+ or CO2+ and taut-vs.-relaxed difference spectra unique to each divalent cation were generated. Mg2+ and CO2+ each supported biosynthetic catalysis, but with different substrate Km and Vmax values. L-Alanine, glycine and L-aspartate were the most potent of several inhibitors of the biosynthetic and the gamma-glutamyl transferase activities; only aspartate and AMP behaved differentially toward glutamine synthetase adenylylation state: the more highly adenylylated enzyme was more severely affected. Any two of alanine, glycine or AMP showed cumulative inhibition, while the inhibitory effects of groups of three effectors were not cumulative. The Co2+-supported biosynthetic activity of Al vinelandii glutamine synthetase was markedly less sensitive to inhibition my glycine and alanine and was stimulated up to 50% by 1-10 mM aspartate.

Ammonia↗

PubMed1987

Spectroscopic studies of the seven-iron-containing ferredoxins from Azotobacter vinelandii and Thermus thermophilus.

The seven-iron-containing ferredoxins from Azotobacter vinelandii and Thermus thermophilus have been investigated by low-temperature magnetic circular dichroism (MCD) and electron paramagnetic resonance (EPR) spectroscopies and room temperature ultraviolet-visible absorption spectroscopy. The results confirm the presence of one trinuclear and one tetranuclear iron-sulfur cluster in both ferredoxins and facilitate comparison of the electronic and magnetic properties of the oxidized and reduced [3Fe-xS] clusters. MCD magnetization data are consistent with an S = 2 ground state for both reduced [3Fe-xS] clusters, but indicate differences in the rhombicity of the zero-field splittings. The data permit rationalization of the absence of a delta M = 4 EPR transition for the reduced [3Fe-xS] cluster in A. vinelandii ferredoxin I. Spectroscopic studies of anaerobically isolated A. vinelandii ferredoxin I do not support the hypothesis that the [3Fe-xS] cluster arises as a result of aerial oxidative damage to a [4Fe-4S] cluster during isolation. The possibility that two distinct forms of [3Fe-xS] clusters can exist in A. vinelandii ferredoxin I was investigated by spectroscopic studies as a function of pH. The results reveal two distinct and interconvertible forms of the reduced [3Fe-xS] cluster, but do not permit rationalization of the inconsistencies in the structural data that have been reported for the oxidized clusters.

Azotobacter↗

PubMed1988

Aerobic, inactive forms of Azotobacter vinelandii hydrogenase: activation kinetics and insensitivity to C2H2 inhibition.

Azotobacter vinelandii hydrogenase (EC class 1.12), either purified or membrane-associated, was obtained aerobically in an inactive state. The kinetics of activation by treatment with a reductant (H2 or dithionite) were determined. Three distinct phases of the activation were observed. Aerobically prepared, inactive hydrogenase was insensitive to acetylene inhibition, but could be rendered acetylene-sensitive by reduction with dithionite. These findings indicate that acetylene inhibition of hydrogenase requires catalytically active enzyme.

Acetylene↗

PubMed1989

Redox properties of the diheme cytochrome c4 from Azotobacter vinelandii and characterisation of the two hemes by NMR, MCD and EPR spectroscopy.

From biphasic stopped-flow kinetic studies it has been established that the two heme centres of cytochrome c4 from Azotobacter vinelandii undergo redox change with [Co(terpy)2]3+/2+ (260 mV) at different rates. Rate constants for oxidation and reduction at pH 7.5 give reduction potentials for the two heme centres in agreement with previous values from spectrophotometric titrations (263 and 317 mV). From NMR studies on the fully reduced protein two sharp methyl methionine resonances are observed at -3.16 and -3.60 ppm, consistent with axial methionine coordination. On titration with [Fe(CN)6]3- the -3.16 ppm resonance is the first to disappear, and is assigned to the less positive reduction potential. Line-broadening effects are observed on partial oxidation, which are dominated by intermolecular processes in an intermediate time-range exchange process. The hemes of the oxidised protein are distinguishable by EPR g-values of 3.64 and 3.22. The former is of interest because it is at an unusually low field for histidine/methionine coordination, and has an asymmetric or ramp shape. The latter assigned to the low potential heme is similar to that of a cytochrome c551. The MCD spectra of the fully oxidised protein are typical of low-spin Fe(III) heme centres, with a negative peak at 710 nm characteristic of methionine coordination, and an NIR peak at 1900 nm characteristic of histidine/methionine (axial) coordination. Of the four histidines per molecule only two undergo diethyl pyrocarbonate (DEPC) modification.

Azotobacter↗

PubMed1991

Physical, chemical and immunological properties of the bacterioferritins of Escherichia coli, Pseudomonas aeruginosa and Azotobacter vinelandii.

The 70-amino-acid-residue N-terminal sequence of the bacterioferritin (BFR) of Azotobacter vinelandii was determined and shown to be highly similar to the N-terminal sequences of the Escherichia coli and Nitrobacter winogradskyi bacterioferritins. Electrophoretic and immunological analyses further indicate that the bacterioferritins of E. coli, A. vinelandii and Pseudomonas aeruginosa are closely related. A novel, two-subunit assembly state that predominates over the 24-subunit form of BFR at low pH was demonstrated. The results indicate that the bacterioferritins form a family of proteins that are distinct from the ferritins of plants and animals.

Amino Acid Sequence↗

PubMed1991

Kinetic analysis of the interaction of nitric oxide with the membrane-associated, nickel and iron-sulfur-containing hydrogenase from Azotobacter vinelandii.

The effects of nitric oxide (NO) on the membrane-associated form of the nickel and iron-sulfur-containing hydrogenase from Azotobacter vinelandii have been investigated. In the presence of H2 and an electron acceptor (turnover conditions), NO acts as a noncompetitive inhibitor vs. methylene blue (Ki = 12 microM). There is no element of competition between NO and H2, implying that the site of NO action is not the H2-activating site of the hydrogenase. When the membrane-associated hydrogenase is incubated under non-turnover conditions, the enzyme is irreversibly inactivated by NO in a time-dependent process. The inactivation is a non-saturable, pseudo-first-order process which is consistent with a direct chemical reaction between NO and the hydrogenase. Kinetic evidence is presented which is compatible with an interaction between NO and a redox-active component other than the H2-activating site on the enzyme. The complex inhibition pattern of NO has been interpreted in terms of two distinct interactions of NO with iron-sulfur centers of the hydrogenase.

Azotobacter↗

PubMed1993

The in-vivo identification of the MoFe protein (FeMo cofactor) of nitrogenase in Klebsiella pneumoniae and of the Mo-storage protein in Azotobacter vinelandii via the nuclear quadrupole interaction of 99Mo(beta-)99Tc.

The expression of the MoFe protein of nitrogenase in Klebsiella pneumoniae was identified in vivo via the nuclear quadrupole interaction (NQI) of 99Mo(beta-)99Tc using perturbed angular correlations of gamma-rays. The NQI parameters were: omega approx. 360 Mrad/s and eta approx. 1. In addition, the NQI of the 'Mo-storage protein' in Azotobacter vinelandii cells which had been grown in the presence of NH4+ (13 mM), i.e. under conditions of strict repression of nitrogenase synthesis, was determined: omega approx. 190 Mrad/s, eta approx. 0.25. Under these conditions, the characteristic signal of the MoFe protein (FeMo cofactor) was absent.

Azotobacter vinelandii↗