Controlled Expression of
            <i>nif</i>
            and
            <i>isc</i>
            Iron-Sulfur Protein Maturation Components Reveals Target Specificity and Limited Functional Replacement between the Two Systems

Santos, Patricia C. Dos; Johnson, Deborah C.; Ragle, Brook E.; Unciuleac, Mihaela-Carmen; Dean, Dennis R.

doi:10.1128/jb.01734-06

Cited by 82 publications

(58 citation statements)

References 34 publications

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“…Additional in vivo experiments showing that NifU and NifS were necessary for the assembly of the [Fe 4 -S 4 ] cluster of NifH (18,19,46) provided physiological evidence to further support the roles of NifU and NifS in NifH maturation.…”

Section: Nifu and Nifs Proteinsmentioning

confidence: 99%

Biosynthesis of the Iron-Molybdenum Cofactor of Nitrogenase

Rubio

Ludden

2008

Annu. Rev. Microbiol.

385

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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Section: Nifu and Nifs Proteinsmentioning

confidence: 99%

Biosynthesis of the Iron-Molybdenum Cofactor of Nitrogenase

Rubio

Ludden

2008

Annu. Rev. Microbiol.

385

311

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“…The Lasergene 7 software was used to analyze DNA and protein sequences. When necessary, A. vinelandii genomic DNA was extracted, amplified, and sequenced as described previously (5).…”

Section: Methodsmentioning

confidence: 99%

“…The parent plasmid used for deletion, insertion, or amino acid substitutions was pDB1577, which contains the intact nfuA gene, as well as 570-bp downstream of the gene, in the pUC-7 vector. Abundant production and controlled expression of NifUS and their variants were achieved by placing the desired genes under the control of the inducible arabinose regulatory elements as described previously (5). Plasmid pDB1598 was used to inactivate nfuA in DJ1626, a strain where nifUS is placed under the control of the araBAD promoter with concomitant in-frame deletion of the nif-regulated nifUS (5).…”

Section: C155amentioning

confidence: 99%

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A Proposed Role for the Azotobacter vinelandii NfuA Protein as an Intermediate Iron-Sulfur Cluster Carrier

Bandyopadhyay

Naik

O’Carroll

et al. 2008

Journal of Biological Chemistry

Self Cite

129

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Iron-sulfur clusters ([Fe-The in vivo maturation of simple [Fe-S] proteins is proposed to require preassembly of [Fe-S] species on molecular scaffolds. The first [Fe-S] cluster assembly system to be described is the NIF 2 system from Azotobacter vinelandii. This system consists of a cysteine desulfurase, encoded by nifS, which supplies the S for [Fe-S] cluster formation, and a proposed scaffold protein, encoded by nifU (1). The NIF system is specialized for the maturation of [Fe-S] proteins involved in nitrogen fixation.A. vinelandii also contains a second [Fe-S] protein maturation system designated ISC. The ISC system is required for the general maturation of cellular [Fe-S] proteins involved in intermediary metabolism, such as aconitase (2). The ISC system is more complicated than the NIF system as it includes the products of eight contiguous genes : iscR, iscS, iscU, iscA, hscB, hscA, fdx, and iscX (3). Although the NIF and ISC systems exhibit physiological target specificity, each can partially replace the function of the other, when expressed at high levels (4, 5).Even though the NIF and ISC systems are differentiated by their apparent target specificities, they share a number of common structural and functional features. For example, NifS and IscS have similar sequences, and they both exhibit cysteine desulfurase activity (2). IscU also shares considerable sequence identity when compared with the N-terminal domain of NifU, including conservation of three cysteine residues that are likely to provide the nucleation site(s) for [Fe-S] cluster assembly (2, 6).NifU is a modular protein that contains three distinct domains (Fig. 1). The central domain contains a stable redoxactive [2Fe-2S] cluster with an as-yet-unknown function (7). In vitro and in vivo experiments have established that labile [Fe-S] clusters can be assembled on both the N-terminal and C-terminal domains of NifU, and such cluster-loaded forms of NifU can be used for activation of the nitrogenase 9). Thus, NifU contains two different sites upon which labile [Fe-S] clusters can be assembled in vitro, but the functional relationship between these sites is not yet known.There are no genes within the ISC transcriptional unit that encode proteins with sequence similarity to the C-terminal domain of NifU. However, located elsewhere on the A. vinelandii genome is a gene, designated nfuA, whose product encodes a protein having a C-terminal sequence similar to the C-terminal domain of NifU (Fig. 1). The sequence conservation between NifU and NfuA includes two cysteine residues that are required for the in vitro assembly of [Fe-S] clusters within the NifU C-terminal domain. Like NifU, NfuA also appears to be a modular protein because the amino acid sequence within its N-terminal region shares some sequence similarity with another protein involved in [Fe-S] protein maturation designated IscA (Fig. 1). IscA is a nonessential protein encoded within the ISC transcriptional unit, and it has been proposed to serve as an alternative [Fe-S] cluster ass...

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“…Aconitase can be measured qualitatively in a gel-based assay (zymogram) 21 . A non-radioactive method, which also uses regulatable promoters, has recently been developed for a bacterial system, Azotobacter vinelandii 41,42 . This method allows determination of both Fe/S cluster amount and type, but might not be applicable for eukaryotic systems due to inherently lower protein amounts.…”

Section: Introductionmentioning

confidence: 99%