Purification of the Azotobacter vinelandii nifV-encoded homocitrate synthase

Zheng, Limin; White, Robert H.; Dean, Dennis R.

doi:10.1128/jb.179.18.5963-5966.1997

Cited by 85 publications

(61 citation statements)

References 22 publications

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“…2 The nifQ gene product has been postulated in molybdenum processing during FeMo-co biosynthesis. The nifV gene encodes homocitrate synthase (19). Recently, NifX has been demonstrated to be required for in vitro FeMo-co synthesis, although its exact role is not known.…”

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confidence: 99%

In Vitro Biosynthesis of Iron-Molybdenum Cofactor and Maturation of the nif-encoded Apodinitrogenase

Rangaraj

Ryle²,

Lanzilotta³

et al. 1999

Journal of Biological Chemistry

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NifH has three different roles in the nitrogenase enzyme system. Apart from serving as the physiological electron donor to dinitrogenase, NifH is involved in iron-molybdenum cofactor (FeMo-co) biosynthesis and in maturation of the FeMo-co-deficient form of apodinitrogenase to a FeMo-co-activable form (apodinitrogenase maturation). The exact roles of NifH in these processes are not well understood. In the present study, the features of NifH required for the aforementioned processes have been investigated by the use of site-specifically altered forms of the enzyme. The ability of six altered forms of NifH inactive in substrate reduction (K15R, D39N, D43N, L127⌬, D129E, and F135Y) to function in in vitro FeMo-co synthesis and apodinitrogenase maturation reactions was investigated. We report that the ability of NifH to bind and not hydrolyze MgATP is required for it to function in these processes. We also present evidence that the ability of NifH to function in these processes is not dictated by the properties known to be required for its function in electron transfer to dinitrogenase. Evidence toward the existence of separate, overlapping sites on NifH for each of its functions (substrate reduction, FeMo-co biosynthesis, and apodinitrogenase maturation) is presented.

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confidence: 99%

In Vitro Biosynthesis of Iron-Molybdenum Cofactor and Maturation of the nif-encoded Apodinitrogenase

Rangaraj

Ryle²,

Lanzilotta³

et al. 1999

Journal of Biological Chemistry

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“…In this model, the [Fe-S]-containing FeMo-co precursor, NifB-co**, would be initially assembled by NifB and then transferred to NifEN for its conversion into the [Fe-S] VK-cluster (13,14). Homocitrate would be provided by the homocitrate-synthase activity of NifV (15,16). Although molybdate satisfies the molybdenum requirement for FeMo-co synthesis in vitro, the physiological source of molybdenum remains unknown.…”

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confidence: 99%

Metal trafficking for nitrogen fixation: NifQ donates molybdenum to NifEN/NifH for the biosynthesis of the nitrogenase FeMo-cofactor

Hernández

Curatti

Aznar

et al. 2008

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

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The molybdenum nitrogenase, present in a diverse group of bacteria and archea, is the major contributor to biological nitrogen fixation. The nitrogenase active site contains an iron-molybdenum cofactor (FeMo-co) composed of 7Fe, 9S, 1Mo, one unidentified light atom, and homocitrate. The nifQ gene was known to be involved in the incorporation of molybdenum into nitrogenase. Here we show direct biochemical evidence for the role of NifQ in FeMo-co biosynthesis. As-isolated NifQ was found to carry a molybdenum-iron-sulfur cluster that serves as a specific molybdenum donor for FeMo-co biosynthesis. Purified NifQ supported in vitro FeMo-co synthesis in the absence of an additional molybdenum source. The mobilization of molybdenum from NifQ required the simultaneous participation of NifH and NifEN in the in vitro FeMo-co synthesis assay, suggesting that NifQ would be the physiological molybdenum donor to a hypothetical NifEN/NifH complex.nif ͉ iron-sulfur cluster ͉ Azotobacter vinelandii B iological nitrogen fixation performed by microorganisms that have nitrogenase(s) accounts for roughly two-thirds of the nitrogen fixed globally. Most nitrogen fixation is carried out by the activity of molybdenum nitrogenase (1), which is widely distributed in nature (2). The molybdenum-nitrogenase enzyme is composed of two proteins (3): a heterotetrameric NifDKprotein component (a 2 ␤ 2 dinitrogenase) and a homodimeric NifH-protein component (dinitrogenase reductase). NifDK contains one iron-molybdenum cofactor (FeMo-co) within the active site of each ␣-subunit (NifD), and one [8Fe-7S] P-cluster at the interface of the ␣-and ␤-subunits in each ␣␤ pair (4). NifH contains a [4Fe-4S] cluster bridging the two subunits and one site for Mg-ATP binding and hydrolysis in each subunit (5).FeMo-co is a unique cofactor ʈ composed of a [Mo-3Fe-3S] partial cubane bridged to a [4Fe-3S] partial cubane by three sulfur ligands and one unidentified light atom in the center of the cofactor (6). The molybdenum atom is also coordinated to the organic acid homocitrate. The biosynthesis of FeMo-co is a complex process that involves the activities of several nitrogen fixation (nif ) gene products that function as molecular scaffolds, enzymes, or escort proteins that carry FeMo-co precursors between assembly sites in the pathway (7,8). Following assembly, FeMo-co is inserted into apo-NifDK, a P-cluster-containing but FeMo-co-deficient form of NifDK that is matured into functional NifDK simply by FeMo-co insertion, to generate the mature dinitrogenase enzyme that is competent for nitrogen fixation.The NifEN scaffold protein is believed to function in the pathway as a central node to which an [Fe-S]-containing FeMo-co precursor, molybdenum, and homocitrate might converge to complete the assembly of FeMo-co (9-12). In this model, the [Fe-S]-containing FeMo-co precursor, NifB-co**, would be initially assembled by NifB and then transferred to NifEN for its conversion into the [Fe-S] VK-cluster (13,14). Homocitrate would be provided by the homocitrate-synthase ac...

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“…Analogs of the nifE and nifN genes exist in the vnf regulon (vnfEN (12)), probably serving a similar role during FeV-co synthesis. The gene product of nifV is homocitrate synthase (13). Homocitrate is a structural component of FeMo-co (14) and most probably FeV-co, because nifV is also required for full functionality of the vnfdinitrogenase (15).…”

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A Vanadium and Iron Cluster Accumulates on VnfX during Iron-Vanadium-cofactor Synthesis for the Vanadium Nitrogenase inAzotobacter vinelandii

Rüttimann-Johnson

Staples

Rangaraj

et al. 1999

Journal of Biological Chemistry

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The vnf-encoded nitrogenase from Azotobacter vinelandii contains an iron-vanadium cofactor (FeV-co) in its active site. Little is known about the synthesis pathway of FeV-co, other than that some of the gene products required are also involved in the synthesis of the ironmolybdenum cofactor (FeMo-co) of the widely studied molybdenum-dinitrogenase. We have found that VnfX, the gene product of one of the genes contained in the vnf-regulon, accumulates iron and vanadium in a novel V-Fe cluster during synthesis of FeV-co. The electron paramagnetic resonance (EPR) and metal analyses of the V-Fe cluster accumulated on VnfX are consistent with a VFe 7-8 S x precursor of FeV-co. The EPR spectrum of VnfX with the V-Fe cluster bound strongly resembles that of isolated FeV-co and a model VFe 3 S 4 compound. The V-Fe cluster accumulating on VnfX does not contain homocitrate. No accumulation of V-Fe cluster on VnfX was observed in strains with deletions in genes known to be involved in the early steps of FeV-co synthesis, suggesting that it corresponds to a precursor of FeV-co. VnfX purified from a nifB strain incapable of FeV-co synthesis has a different electrophoretic mobility in native anoxic gels than does VnfX, which has the V-Fe cluster bound. NifB-co, the Fe and S precursor of FeMo-co (and presumably FeV-co), binds to VnfX purified from the nifB strain, producing a shift in its electrophoretic mobility on anoxic native gels. The data suggest that a precursor of FeV-co that contains vanadium and iron accumulates on VnfX, and thus, VnfX is involved in the synthesis of FeV-co.

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Purification of the Azotobacter vinelandii nifV-encoded homocitrate synthase

Cited by 85 publications

References 22 publications

In Vitro Biosynthesis of Iron-Molybdenum Cofactor and Maturation of the nif-encoded Apodinitrogenase

In Vitro Biosynthesis of Iron-Molybdenum Cofactor and Maturation of the nif-encoded Apodinitrogenase

Metal trafficking for nitrogen fixation: NifQ donates molybdenum to NifEN/NifH for the biosynthesis of the nitrogenase FeMo-cofactor

A Vanadium and Iron Cluster Accumulates on VnfX during Iron-Vanadium-cofactor Synthesis for the Vanadium Nitrogenase inAzotobacter vinelandii

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