Structural genes for the vanadium nitrogenase from Azotobacter chroococcum.

Robson, Richard M.; Woodley, Paul; Pau, Richard N.; Eady, Robert R.

doi:10.1002/j.1460-2075.1989.tb03495.x

Cited by 92 publications

(55 citation statements)

References 44 publications

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“…In contrast to the reddish-brown color of ⌬nifB Av1, which j The ␣-subunit of Av1 V is known to migrate faster than the ␤-subunit on an SDS͞PAGE, despite a larger molecular weight of the ␣-subunit (16,27). Faster migration of the ␣-subunit has also been observed in the case of the VFe protein of Azotobacter chroococcum, designated Ac1 V (44).…”

Section: Resultsmentioning

confidence: 98%

Nitrogenase reactivity with P-cluster variants

Corbett

Fay

et al. 2005

Proc. Natl. Acad. Sci. U.S.A.

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Nitrogenase is a multicomponent metalloenzyme that catalyzes the conversion of atmospheric dinitrogen to ammonia. For decades, it has been generally believed that the [8Fe-7S] P-cluster of nitrogenase component 1 is indispensable for nitrogenase activity. In this study, we identified two catalytically active P-cluster variants by activity assays, metal analysis, and EPR spectroscopic studies. Further, we showed that both P-cluster variants resemble [4Fe-4S]-like centers based on x-ray absorption spectroscopic experiments. We believe that our findings challenge the dogma that the standard P-cluster is the only cluster species capable of supporting substrate reduction at the FeMo cofactor and provide important insights into the general mechanism of nitrogenase catalysis and assembly.MoFe protein ͉ VFe protein N itrogenase catalyzes one of the most remarkable chemical transformations in biological systems, the reduction of atmospheric dinitrogen to a bioavailable form, ammonia. Three classes of nitrogenase systems, namely, the Mo-, V-, and Fe-only nitrogenases, have been identified (for recent reviews see refs. 1-11). f Although encoded by different structural genes, all three nitrogenases are comprised of two essential component metalloproteins, component 1 (MoFe, VFe, or FeFe protein) and component 2 (Fe protein). g The homodimeric Fe protein of the Mo-nitrogenase has one [4Fe-4S] cluster bridged between the two subunits, whereas the ␣ 2 ␤ 2 -tetrameric MoFe protein contains two unique metal clusters per ␣␤-subunit: the [8Fe-7S] P-cluster (14), which is located at the ␣␤-interface, and the [Mo-7Fe-9S-X-homocitrate] h FeMo cofactor (FeMoco), which is situated within the ␣-subunit. ATP-dependent electron transfer is believed to proceed from the [4Fe-4S] cluster of the Fe protein to the P-cluster of the MoFe protein and finally to FeMoco where substrate reduction takes place. The Fe protein of the V-nitrogenase shares a high degree of homology with the Fe protein of the Mo-nitrogenase and, apart from the presence of an additional ␥-subunit, the VFe protein is also believed to be highly homologous to the MoFe protein with respect not only to the structural genes encoding the ␣-and ␤-subunits, but also to the redox centers contained within the protein, designated the P-cluster and the FeV cofactor (FeVco), respectively, in this case (4, 10, 16). The structurally homologous heterometal centers, i.e., FeMoco and FeVco, are also categorically termed cofactors.It is generally believed that the structural integrity of the P-cluster is indispensable for nitrogenase reactivity. However, the exact mechanism by which the P-cluster carries out its function in substrate reduction and, in particular, the oxidation states and structural conformations of the P-cluster involved in this process, remain largely a puzzle (17). Two types of cofactordeficient MoFe or VFe protein variants, generated by nifH or nifB deletion, i respectively, have been used to study the features of P-clusters without the interference of the cofactor centers...

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Section: Resultsmentioning

confidence: 98%

Nitrogenase reactivity with P-cluster variants

Corbett

Fay

et al. 2005

Proc. Natl. Acad. Sci. U.S.A.

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“…One of the two components of the vnf-encoded complex is an iron (Fe) protein (dinitrogenase reductase 2, encoded by vnfH) that is essentially identical in sequence to the product of nifH, and is cross-functional with it as an electron donor in Anabaena variabilis (Pratte et al, 2006). The other component is a vanadium-iron (V-Fe) protein (dinitrogenase 2, for which vnfDG encodes the fused α-/δ-subunit and vnfK encodes the β-subunit) (Robson et al, 1986(Robson et al, , 1989Joerger et al, 1990;Zehr et al, 2003;Raymond et al, 2004;Boison et al, 2006). In A. variabilis, the vnfEN genes are also essential for N 2 fixation (functioning as a scaffold for catalytic cluster formation), although these genes are apparently absent in many strains of bacteria that utilize this system (e.g.…”

Section: Introductionmentioning

confidence: 99%

“…The three major enzyme complexes for N 2 fixation can be viewed in terms of a cascade of efficiency, with the Mo-dependent (nif-encoded) complex being most efficient, followed by the V-dependent (vnf-encoded) complex, and finally the Fe-dependent (anf-encoded) complex (Robson et al, 1986(Robson et al, , 1989Hales et al, 1986aHales et al, , 1986bChisnell et al, 1988;Eady, 1989Eady, , 2003Eady et al, 1988;Joerger et al, 1990;Walmsley & Kennedy, 1991;Raina et al, 1993;Bellenger et al, 2011). Paradoxically, Mo is the least abundant of the three crucial biometals in the continent crust, whereas V is approximately two orders of magnitude more abundant, and Fe is by far the most abundant of the three (Erickson, 1973;Wedepohl, 1995).…”

Section: Introductionmentioning

confidence: 99%

Lichen-symbiotic cyanobacteria associated withPeltigerahave an alternative vanadium-dependent nitrogen fixation system

Hodkinson

Allen

Forrest

et al. 2014

European Journal of Phycology

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In past decades, environmental nitrogen fixation has been attributed almost exclusively to the action of enzymes in the wellstudied molybdenum-dependent nitrogen fixation system. However, recent evidence has shown that nitrogen fixation by alternative pathways may be more frequent than previously suspected. In this study, the nitrogen fixation systems employed by lichen-symbiotic cyanobacteria were examined to determine whether their diazotrophy can be attributed, in part, to an alternative pathway. The mining of metagenomic data (generated through pyrosequencing) and PCR assays were used to determine which nitrogen-fixation systems are present in cyanobacteria from the genus Nostoc associated with four samples from different geographical regions, representing different lichen-forming fungal species in the genus Peltigera. A metatranscriptomic sequence library from an additional specimen was examined to determine which genes associated with N 2 fixation are transcriptionally expressed. Results indicated that both the standard molybdenum-dependent system and an alternative vanadium-dependent system are present and actively transcribed in the lichen symbiosis. This study shows for the first time that an alternative system is utilized by cyanobacteria associated with fungi. The ability of lichen-associated cyanobacteria to switch between pathways could allow them to colonize a wider array of environments, including habitats characterized by low temperature and trace metal (e.g. molybdenum) availability. We discuss the implications of these findings for environmental studies that incorporate acetylenereduction assay data.

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“…Dinitrogenase-1 is a tetramer (Mr, -240,000) consisting of two pairs of nonidentical subunits (a2P2). Dinitrogenase-2 and dinitrogenase-3, on the other hand, are probably hexamers, each containing three pairs of nonidentical subunits (a2P282) (25,39).The structural genes encoding nitrogenase-1 and nitrogenase-3 are organized as single operons (nifHDK and anf HDGKorflord2, respectively), while those encoding nitrogenase-2 form two independently regulated operons, vnfHorf Fd and vn.fDGK (8,25,26,39 …”

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confidence: 99%

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Nucleotide sequence and mutational analysis of the vnfENX region of Azotobacter vinelandii

Wolfinger

Bishop

1991

J Bacteriol

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The nucleotide sequence (3,600 bp) of a second copy of nifENX-like genes in Azotobacter vinelandii has been determined. These genes are located immediately downstream from vnfA and have been designated vnJENX. The vnJENX genes appear to be organized as a single transcriptional unit that is preceded by a potential RpoN-dependent promoter. While the nifEN genes are thought to be evolutionarily related to nifDK, the vaJEN genes appear to be more closely related to niJEN than to either nifDK, vnJDK, or anfDK. Mutant strains (CA47 and CA48) carrying insertions in vnfE and vnfN, respectively, are able to grow diazotrophically in molybdenum (Mo)-deficient medium containing vanadium (V) (Vnf+) and in medium lacking both Mo and V (Anf).However, a double mutant (strain DJ42.48) which contains a nijEN deletion and an insertion in vnJE is unable to grow diazotrophically in Mo-sufficient medium or in Mo-deficient medium with or without V. This suggests that NifE and NifN substitute for VnfE and VnfN when the vnjEN genes are mutationally inactivated. AnfA is not required for the expression of a vnJN-lacZ transcriptional fusion, even though this fusion is expressed under Mo-and V-deficient diazotrophic growth conditions. Azotobacter vinelandii is able to grow diazotrophically using any of three genetically distinct nitrogenases depending on the presence or absence of molybdenum (Mo) or vanadium (V) in the growth medium. The well-characterized Mo-containing nitrogenase (nitrogenase-1) is synthesized under conditions of Mo sufficiency. Under conditions where V replaces Mo, an alternative V-containing nitrogenase (nitrogenase-2) is expressed, and in the absence of both Mo and V, an alternative nitrogenase (nitrogenase-3) that does not appear to contain either Mo or V is made (18, 23). Each of these nitrogenase complexes is composed of two protein components, dinitrogenase reductase and dinitrogenase. Dinitrogenase reductase-1 is a dimer of two identical subunits with an Mr of approximately 60,000 (10, 11). Dinitrogenase reductase-2 and dinitrogenase reductase-3 are also dimers of two identical subunits (13,20). Dinitrogenase-1 is a tetramer (Mr, -240,000) consisting of two pairs of nonidentical subunits (a2P2). Dinitrogenase-2 and dinitrogenase-3, on the other hand, are probably hexamers, each containing three pairs of nonidentical subunits (a2P282) (25,39).The structural genes encoding nitrogenase-1 and nitrogenase-3 are organized as single operons (nifHDK and anf HDGKorflord2, respectively), while those encoding nitrogenase-2 form two independently regulated operons, vnfHorf Fd and vn.fDGK (8,25,26,39

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Structural genes for the vanadium nitrogenase from Azotobacter chroococcum.

Cited by 92 publications

References 44 publications

Nitrogenase reactivity with P-cluster variants

Nitrogenase reactivity with P-cluster variants

Lichen-symbiotic cyanobacteria associated withPeltigerahave an alternative vanadium-dependent nitrogen fixation system

Nucleotide sequence and mutational analysis of the vnfENX region of Azotobacter vinelandii

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