Purification and characterization of the nifN and nifE gene products from Azotobacter vinelandii mutant UW45.

Paustian, Timothy D.; Shah, Vinod K.; Roberts, Gary P.

doi:10.1073/pnas.86.16.6082

Cited by 55 publications

(56 citation statements)

References 33 publications

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“…nifV mutants, unlike wild-type K. pneumoniue, fail to accumulate homocitrate in their growth medium and the addition of homocitrate to cultures corrects the nifV-phenotype [43]. Homocitrate had not been previously found in bacteria, but in yeasts and fungi its formation is catalysed by homocitrate synthase which condenses acetyl-CoA and 2-oxoglutarate.…”

Section: N I Pmentioning

confidence: 99%

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Metalloclusters of the nitrogenases

Smith

Eady

1992

European Journal of Biochemistry

123

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Biological nitrogen fixation, the reduction of dinitrogen to ammonia, is catalysed by nitrogenases. These enzymes are found in relatively few groups of bacteria and until recently it appeared that N2 fixation occurred by a single route involving a molybdenum-containing enzyme. However, it is now clear that Mo is not an essential metal for N2 fixation. The soil bacterium Azotobacter chroococcum has an Mo-containing nitrogenase with properties very similar to those found in many other diazotrophic organisms, but it also has a vanadium-containing nitrogenase which functions in the presence of V if Mo is not available. The closely related species A . vinelandii has, in addition to Mo and V nitrogenases, a third nitrogenase which functions only under conditions where Mo and V levels are very low [l].The three nitrogenase systems of A. vinelandii are genetically distinct, being encoded by different structural genes. These genes for the three nitrogenase systems have been cloned and sequenced and have considerable similarity. The nitrogenases have been purified and shown to have similar requirements for activity, viz. a low-potential electron donor, MgATP and the absence of oxygen. They can each be separated into two essential component proteins. All three have a distinct Fe-containing protein (component 2) which, together with either an MoFe protein, a VFe protein, or a homologous protein which contains Fe but only low levels of Mo or V (component l), make up the active nitrogenases.Studies on the structure and function of nitrogenase proteins have benefitted significantly from information provided from studies on the biochemical genetics of nitrogen fixation. The genetics of nitrogen fixation is complex. In Klebsiella pneumoniae, which is the most intensively studied diazotroph and for which there is no evidence for the presence of additional Mo-independent nitrogenases, the 20 nif' genes required solely for nitrogen fixation and its regulation are clustered on a 23-kb region of the chromosome. The entire n[f gene cluster has been sequenced [2]. In addition to the structural genes of nitrogenase (nif'HDK), genes involved in the activation of nitrogenase Fe protein ( n i j w , iron-molybdenum cofactor (FeMoco) biosynthesis (nijii V3ENH) (see below), electron donation ( n i j l f l , several genes of unknown function and the regulatory genes nif'AL are located in this cluster (see Fig. 1 and [3, 41).A . vinelandii and A . chroococcum have a comparable gene cluster, except that they contain additional open reading frames (ORFs) of unknown function and nifABQ map outside the major nif'gene region. The Azotobacter nitrogen fixation genes associated with Mo-independent nitrogenases have also been cloned and sequenced. They include the structural genes of the V nitrogenase (vnf: vanadium nitrogen fixation) and, in A . vinelandii only, the structural genes of the third nitrogenase (an8 alternative nitrogen fixation). In addition to these structural genes, specific regulatory nif'A-like genes and a reiteration of nif'EN-like ...

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Section: N I Pmentioning

confidence: 99%

“…The requirement for homocitrate in this synthetic system was demonstrated using desalted extracts of mutants which show very low levels of FeMoco synthesis [43]. A low-molecular-mass component present in the growth medium of diazotrophic cultures of A .…”

Section: Homocitratementioning

confidence: 99%

Metalloclusters of the nitrogenases

Smith

Eady

1992

European Journal of Biochemistry

123

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“…NifEN is a 200-kDa α 2 β 2 heterotetramer that contains [Fe-S] clusters (56). The function of NifEN is essential to FeMo-co biosynthesis.…”

Section: Properties Of the Nifen Proteinmentioning

confidence: 99%

Biosynthesis of the Iron-Molybdenum Cofactor of Nitrogenase

Rubio

Ludden

2008

Annu. Rev. Microbiol.

381

311

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The iron-molybdenum cofactor (FeMo-co), located at the active site of the molybdenum nitrogenase, is one of the most complex metal cofactors known to date. During the past several years, an intensive effort has been made to purify the proteins involved in FeMo-co synthesis and incorporation into nitrogenase. This effort is starting to provide insights into the structures of the FeMo-co biosynthetic intermediates and into the biochemical details of FeMo-co synthesis. 93

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“…2 The nifN and nifE gene products together form a tetrameric protein that shows a high sequence similarity to the nifK and nifD gene products (20). Thus, NifNE has been postulated to form a scaffold upon which FeMo-co is synthesized (21,22). The metabolic product of NifB, NifB cofactor (23), has been shown to function as a specific iron and sulfur donor to FeMo-co during FeMo-co biosynthesis (24).…”

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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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Purification and characterization of the nifN and nifE gene products from Azotobacter vinelandii mutant UW45.

Cited by 55 publications

References 33 publications

Metalloclusters of the nitrogenases

Metalloclusters of the nitrogenases

Biosynthesis of the Iron-Molybdenum Cofactor of Nitrogenase

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

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