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

Rangaraj, Priya; Ryle, Matthew J.; Lanzilotta, William N.; Ludden, Paul W.; Shah, Vinod K.

doi:10.1074/jbc.274.28.19778

Cited by 28 publications

(31 citation statements)

References 55 publications

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“…Individual alignments showed that, over this stretch, ApbC was 40% identical and 58% similar to MinD from Clostridium acetobutylicum, 20% identical and 34% similar to NifH from Methanococcus voltae, and 17% identical and 29% similar to CooC from Rhodospirillum rubrum. Proteins in each of these families have demonstrated ATPase activity (19,25,44). The first 115 amino acids of ApbC showed no significant homology with other proteins and contained no defined protein motifs.…”

Section: Resultsmentioning

confidence: 99%

Lack of the ApbC or ApbE Protein Results in a Defect in Fe-S Cluster Metabolism in Salmonella enterica Serovar Typhimurium

Skovran

Downs

2003

J Bacteriol

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The isc genes function in the assembly of Fe-S clusters and are conserved in many prokaryotic and eukaryotic organisms. In most bacteria studied, the isc operon can be deleted without loss of cell viability, indicating that additional systems for Fe-S cluster assembly must exist. Several laboratories have described nutritional and biochemical defects resulting from mutations in the isc operon. Here we demonstrate that null mutations in two genes of unknown function, apbC and apbE, result in similar cellular deficiencies. Exogenous ferric chloride suppressed these deficiencies in the apbC and apbE mutants, distinguishing them from previously described isc mutants. The deficiencies caused by the apbC and isc mutations were additive, which is consistent with Isc and ApbC's having redundant functions or with Isc and ApbC's functioning in different areas of Fe-S cluster metabolism (e.g., Fe-S cluster assembly and Fe-S cluster repair). Both the ApbC and ApbE proteins are similar in sequence to proteins that function in metal cofactor assembly. Like the enzymes with sequence similarity to ApbC, purified ApbC protein was able to hydrolyze ATP. The data herein are consistent with the hypothesis that the ApbC and ApbE proteins function in Fe-S cluster metabolism in vivo.Fe-S clusters are cofactors for many proteins and aid in a variety of functions, including electron transfer, protein structure maintenance, substrate binding, and regulation in response to small molecules (reviewed in references 3, 4, 13, 14, 26, and 46). In the last several years, significant strides have been made in our understanding of Fe-S cluster assembly. Expanded efforts in this area were prompted by the discovery of an operon in Azotobacter vinelandii that was involved in Fe-S metabolism (62). Homologues to the genes in this operon (isc) were subsequently demonstrated in Escherichia coli (49,56,57), Salmonella enterica (52), cyanobacteria (51), Pseudomonas aeruginosa (9), Thermotoga maritima (35), mitochondrial eukaryotes (reviewed in reference 34), and amitochondrial anaerobic eukaryotes (55). Genomic sequencing efforts continue to reinforce the conservation of these genes.In A. vinelandii, the isc operon is essential for cell viability (62), but in others, E. coli, S. enterica, and P. aeruginosa, for instance, multiple isc genes or the entire isc gene cluster can be deleted. In each case, the resulting strains are viable but have various growth defects (9,20,49,52,57). In addition, elimination of the isc genes results in reduction but not complete loss of activity of several Fe-S proteins (49, 52, 57), suggesting that additional proteins or systems are involved in the assembly and repair of Fe-S clusters in vivo. One such system is likely to include the Suf proteins, which were hypothesized to have a role in Fe-S cluster metabolism based on their similarity to Isc proteins (41). The suf genes are located in an operon that is regulated by iron and oxidative stress (18,39,41,63) and includes sufA, which has similarity to iscA; sufB, whose gene pr...

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Section: Resultsmentioning

confidence: 99%

Lack of the ApbC or ApbE Protein Results in a Defect in Fe-S Cluster Metabolism in Salmonella enterica Serovar Typhimurium

Skovran

Downs

2003

J Bacteriol

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“…Biosynthesis and maturation of the nickel enzymes urease, CO-dehydrogenase, and [NiFe] hydrogenase, as well as the FeMo cofactor of nitrogenase, involve nucleotide-dependent steps mediated by a specific nucleotide-binding protein (25,26,31,41). The nucleotide-dependent steps in the maturation of [NiFe] hydrogenases involve GTP hydrolysis coupled to nickel donation (31) and ATP hydrolysis coupled to the synthesis of CN ligands to iron (9).…”

Section: Discussionmentioning

confidence: 99%

Functional Studies of [FeFe] Hydrogenase Maturation in an Escherichia coli Biosynthetic System

et al. 2006

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H 2 metabolism in microorganisms requires the catalytic activity of hydrogenases, a specialized group of metalloproteins (29,38,51 (8,10,12,28,37,42,45). In contrast, characterization of [FeFe] hydrogenase maturation has been limited by a lack of knowledge of the proteins functioning in the biosynthesis of the catalytic H cluster. Recently, we identified two genes in Chlamydomonas reinhardtii, hydEF and hydG, which encode proteins required for [FeFe] hydrogenase activity by functioning in H cluster biosynthesis and enzyme maturation (40). Both proteins contain conserved motifs found in radical S-adenosylmethionine (SAM)-binding proteins, suggesting that biosynthesis of the H cluster during [FeFe] hydrogenase maturation involves radical-SAM-dependent mechanisms. The radical-SAM domains in both HydE and HydG contain the conserved motif Cx 3 Cx 2 C, with additional motifs in the C-terminal ends that are characteristic of iron-sulfur cluster-binding sites (16). The HydF protein differs from HydE and HydG in that it belongs to the GTPase protein family (27). At the N-terminal end of HydF, there are motifs that match the conserved GTP-binding motifs, with a potential iron-sulfur cluster-binding motif present at the C-terminal end (27).Although the biochemistry of [FeFe] hydrogenase maturation is an emerging field of investigation, the [FeFe] hydrogenases from several organisms have been extensively investigated by biochemical, spectroscopic, and structural methods (reviewed in reference 1). As a result of these investigations, a number of the characteristics of the H cluster and catalytic site have been elucidated. The H cluster is arranged as a unique [2Fe] center connected by a conserved cysteine to a [4Fe4S] cubane cluster (1,35,38). The iron atoms of the [2Fe] center are bridged by a dithio moiety which, based on a combination of structural and theoretical studies, is interpreted as either a propanedithiol or a di(thiomethyl)amine (13,34,35,38). Other features of the [2Fe] center are asymmetric CN and CO ligand pairs at each iron, with an additional CO bridging the two iron atoms (35,38,39). Thus, the HydEF and HydG proteins are thought to function together in a series of undefined steps in synthesis and insertion of the chemically complex H cluster at the [FeFe] hydrogenase catalytic site during maturation.As part of our previous report, we demonstrated that anaerobic coexpression of the C. reinhardtii [FeFe] hydrogenase maturation and structural genes in Escherichia coli resulted in the biosynthesis of an active [FeFe] hydrogenase (40). However, the C. reinhardtii hydEF and hydG clones were unstable and susceptible to rearrangements in E. coli, a problem that

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“…In vitro experiments correlate ATP hydrolysis by NifH to molybdenum incorporation into the FeMo-co precursor (38). However, NifH mutant variants that bind but do not hydrolyze Mg · ATP were functional in FeMo-co synthesis in vitro (60) and in vivo (24). Whether the electron transfer ability of NifH is required for this role is also at disagreement.…”

Section: Role Of Nifen and Its Association With Nifhmentioning

confidence: 99%

Biosynthesis of the Iron-Molybdenum Cofactor of Nitrogenase

Rubio

Ludden

2008

Annu. Rev. Microbiol.

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

Cited by 28 publications

References 55 publications

Lack of the ApbC or ApbE Protein Results in a Defect in Fe-S Cluster Metabolism in Salmonella enterica Serovar Typhimurium

Lack of the ApbC or ApbE Protein Results in a Defect in Fe-S Cluster Metabolism in Salmonella enterica Serovar Typhimurium

Functional Studies of [FeFe] Hydrogenase Maturation in an Escherichia coli Biosynthetic System

Biosynthesis of the Iron-Molybdenum Cofactor of Nitrogenase

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