Effects of homocitrate, homocitrate lactone, and fluorohomocitrate on nitrogenase in NifV- mutants of Azotobacter vinelandii

Madden, Mark; Paustian, Timothy D.; Ludden, Paul W.; Shah, Vinod K.

doi:10.1128/jb.173.17.5403-5405.1991

Cited by 28 publications

(23 citation statements)

References 23 publications

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“…In previous studies (18,22) NifV Ϫ mutants of Klebsiella pneumoniae and A. vinelandii were found to reduce acetylene levels even though they are unable to fix nitrogen. Thus, strains CA115 and CA116 were tested for acetylene reduction capability and strains CA and CA11.71 were used as positive and negative controls, respectively.…”

Section: N/mentioning

confidence: 99%

Characteristics of orf1 and orf2 in the anfHDGK genomic region encoding nitrogenase 3 of Azotobacter vinelandii

et al. 1996

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In Azotobacter vinelandii, the anfHDGK operon encodes the subunits for the third nitrogenase complex. Two open reading frames (orf1 and orf2) located immediately downstream of anfK were shown to be required for diazotrophic growth under Mo-and V-deficient conditions. We have designated orf1 and orf2 anfO and anfR, respectively. Strains (CA115 and CA116) carrying in-frame deletions in anfO and anfR accumulate the subunits for nitrogenase 3 under Mo-deficient diazotrophic conditions. AnfO and AnfR are required for nitrogenase 3-dependent diazotrophic growth and 15 N 2 incorporation but not for acetylene reduction. AnfO contains a putative heme-binding domain that exhibits similarity to presumed heme-binding domains of P-450 cytochromes. Amino acid substitutions of Cys-158 show that this residue is required for fully functional AnfO as measured by diazotrophic growth under Mo-and V-deficient conditions. The nucleotide sequence of the region located immediately downstream of anfR has been determined. A putative -independent transcription termination site has been identified 250 bp from the 3 end of anfR. A third open reading frame (orf3), located downstream of anfR, does not appear to be required for diazotrophic growth under Mo-and V-deficient conditions.Nitrogen fixation, the reduction of dinitrogen to ammonia, is an enzymatic process that occurs in a wide range of bacterial species, including the soil bacterium Azotobacter vinelandii. This process is catalyzed by the enzyme complex nitrogenase. Nitrogenase is a metalloenzyme composed of two protein components called dinitrogenase reductase and dinitrogenase. A. vinelandii harbors three genetically distinct nitrogenases (3, 4). The first nitrogenase is the molybdenum (Mo)-containing enzyme (nitrogenase 1) that is made in the absence of a fixed nitrogen source when Mo is available (9). The second enzyme, a vanadium (V)-containing nitrogenase (nitrogenase 2), is synthesized under Mo-deficient conditions in the presence of V (4, 9, 12). The third nitrogenase (nitrogenase 3) is expressed only in the absence of Mo and V (3,6,17,30).The protein subunits for the Mo nitrogenase are encoded by the nifHDK operon (10). The structural genes for V nitrogenase comprise two operons, vnfHorfFd and vnfDGK (12). The subunits for the third nitrogenase are encoded by the anfHDGK operon (11).Recently, three other diazotrophs, Clostridium pasteurianum (41), Rhodobacter capsulatus (16, 33), and Rhodospirillum rubrum (4), were found to have alternative nitrogenases that are similar to nitrogenase 3 of A. vinelandii. These nitrogenases are expressed only in the absence of Mo. Nitrogenase 3 is composed of dinitrogenase 3 and dinitrogenase reductase 3. Dinitrogenase reductase 3 is a homodimer (␥ 2 ) encoded by the anfH gene, while dinitrogenase 3 is a hexamer (␣ 2 ␤ 2 ␦ 2 ). The ␣-and ␤-subunits are encoded by the anfD and anfK genes, respectively. The ␦-subunit, a small protein, characteristic of the alternative nitrogenases (33,39,41), is encoded by the anfG gene.In addition to the struct...

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Section: N/mentioning

confidence: 99%

Characteristics of orf1 and orf2 in the anfHDGK genomic region encoding nitrogenase 3 of Azotobacter vinelandii

et al. 1996

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“…Since NifV Ϫ mutants of K. pneumoniae (27) and A. vinelandii (24) are able to reduce acetylene even though they are unable to grow diazotrophically, it was of interest to test the AnfG and VnfG mutant strains for their abilities to reduce acetylene. The results of a typical experiment are given in Table 2.…”

Section: Vol 177 1995mentioning

confidence: 99%

The genes encoding the delta subunits of dinitrogenases 2 and 3 are required for mo-independent diazotrophic growth by Azotobacter vinelandii

et al. 1995

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vnfG and anfG encode the ␦ subunits of alternative nitrogenases 2 and 3 in Azotobacter vinelandii, respectively. As a first step towards elucidating the role of these subunits, diazotrophic growth and acetylene reduction studies were conducted on mutants containing alterations in the genes encoding these subunits. Mutants containing a stop codon (C36stop) or an in-frame deletion in anfG were unable to grow in N-free, Mo-deficient medium (Anf ؊ Anf ؊ strain with this mutation was unable to grow under these conditions. This shows that the vnfG gene product is required for nitrogenase 2-dependent growth. Strains with mutations in vnfG and anfG reduced acetylene to different degrees. This indicates that the ␦ subunits are not required for acetylene reduction by nitrogenases 2 and 3.

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“…First, it was shown that a small organic molecule, later shown to be homocitrate (4), is required for the in vitro biosynthesis of FeMo cofactor (3). Second, the addition of homocitrate to cultures of Klebsiella pneumoniae or Azotobacter vinelandii nifV mutants cured both their diazotrophic growth and their biochemical phenotypes (5,12). Third, [ 3 H]homocitrate could be incorporated into FeMo cofactor during its in vitro formation and an organic constituent extracted from purified MoFe protein was identified by nuclear magnetic resonance spectroscopic analysis as homocitrate (6).…”

mentioning

confidence: 99%

“…It might also be possible to use substituted forms or analogs of the homocitrate precursors to form homocitrate analogs. The availability of homocitrate analogs, as well as of radioactively labeled homocitrate, would be useful in expanding the approach described by Madden et al (11,12) to monitor the fate of homocitrate during FeMo cofactor formation and to determine the role of homocitrate in nitrogenase catalysis. (iii) Although it is clear that NifV catalyzes homocitrate formation, it is not yet known how Mo becomes coordinated to homocitrate, nor is it known how homocitrate and Mo become incorporated into FeMo cofactor.…”

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Purification of the Azotobacter vinelandii nifV-encoded homocitrate synthase

1997

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The nifV gene product (NifV) from Azotobacter vinelandii was recombinantly expressed at high levels in Escherichia coli and purified. NifV is a homodimer that catalyzes the condensation of acetyl coenzyme A (acetyl-CoA) and ␣-ketoglutarate. Although ␣-ketoglutarate supports the highest level of activity, NifV will also catalyze the condensation of acetyl-CoA and certain other keto acids. E. coli cells in which a high level of nifV expression is induced excrete homocitrate into the growth medium.

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Effects of homocitrate, homocitrate lactone, and fluorohomocitrate on nitrogenase in NifV- mutants of Azotobacter vinelandii

Cited by 28 publications

References 23 publications

Characteristics of orf1 and orf2 in the anfHDGK genomic region encoding nitrogenase 3 of Azotobacter vinelandii

Characteristics of orf1 and orf2 in the anfHDGK genomic region encoding nitrogenase 3 of Azotobacter vinelandii

The genes encoding the delta subunits of dinitrogenases 2 and 3 are required for mo-independent diazotrophic growth by Azotobacter vinelandii

Purification of the Azotobacter vinelandii nifV-encoded homocitrate synthase

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