Functional Identification of Novel Genes Involved in the Glutathione-Independent Gentisate Pathway in Corynebacterium glutamicum

Corynebacterium glutamicum used gentisate and 3-hydroxybenzoate as its sole carbon and energy source for growth. By genome-wide data mining, a gene cluster designated ncg12918-ncg12923 was proposed to encode putative proteins involved in gentisate/3-hydroxybenzoate pathway. Genes encoding gentisate 1,2-dioxygenase (ncg12920) and fumarylpyruvate hydrolase (ncg12919) were identified by cloning and expression of each gene in Escherichia coli. The gene of ncg12918 encoding a hypothetical protein (Ncg12918) was proved to be essential for gentisate-3-hydroxybenzoate assimilation. Mutant strain RES167Δncg12918 lost the ability to grow on gentisate or 3-hydroxybenzoate, but this ability could be restored in C. glutamicum upon the complementation with pXMJ19-ncg12918. Cloning and expression of this ncg12918 gene in E. coli showed that Ncg12918 is a glutathione-independent maleylpyruvate isomerase. Upstream of ncg12920, the genes ncg12921-ncg12923 were located, which were essential for gentisate and/or 3-hydroxybenzoate catabolism. The Ncg12921 was able to up-regulate gentisate 1,2-dioxygenase, maleylpyruvate isomerase, and fumarylpyruvate hydrolase activities. The genes ncg12922 and ncg12923 were deduced to encode a gentisate transporter protein and a 3-hydroxybenzoate hydroxylase, respectively, and were essential for gentisate or 3-hydroxybenzoate assimilation. Based on the results obtained in this study, a GSH-independent gentisate pathway was proposed, and genes involved in this pathway were identified.     

Loss of Cardiolipin Leads to Perturbation of Mitochondrial and Cellular Iron Homeostasis

Cardiolipin (CL) is the signature phospholipid of mitochondrial membranes, where it is synthesized locally and plays a critical role in mitochondrial bioenergetic functions. The importance of CL in human health is underscored by the observation that perturbation of CL biosynthesis causes the severe genetic disorder Barth syndrome. To fully understand the cellular response to the loss of CL, we carried out genome-wide expression profiling of the yeast CL mutant crd1Δ. Our results show that the loss of CL in this mutant leads to increased expression of iron uptake genes accompanied by elevated levels of mitochondrial iron and increased sensitivity to iron and hydrogen peroxide. Previous studies have shown that increased mitochondrial iron levels result from perturbations in iron-sulfur (Fe-S) cluster biogenesis. Consistent with an Fe-S defect, deletion of ISU1, one of two ISU genes that encode the mitochondrial Fe-S scaffolding protein essential for the synthesis of Fe-S clusters, led to synthetic growth defects with the crd1Δ mutant. We further show that crd1Δ cells have reduced activities of mitochondrial Fe-S enzymes (aconitase, succinate dehydrogenase, and ubiquinol-cytochrome c oxidoreductase), as well as cytosolic Fe-S enzymes (sulfite reductase and isopropylmalate isomerase). Increased expression of ATM1 or YAP1 did not rescue the Fe-S defects in crd1Δ. These findings show for the first time that CL is required for Fe-S biogenesis to maintain mitochondrial and cellular iron homeostasis.     

Functional identification of novel genes involved in the glutathione-independent gentisate pathway in Corynebacterium glutamicum

Corynebacterium glutamicum used gentisate and 3-hydroxybenzoate as its sole carbon and energy source for growth. By genome-wide data mining, a gene cluster designated ncg12918-ncg12923 was proposed to encode putative proteins involved in gentisate/3-hydroxybenzoate pathway. Genes encoding gentisate 1,2-dioxygenase (ncg12920) and fumarylpyruvate hydrolase (ncg12919) were identified by cloning and expression of each gene in Escherichia coli. The gene of ncg12918 encoding a hypothetical protein (Ncg12918) was proved to be essential for gentisate-3-hydroxybenzoate assimilation. Mutant strain RES167Deltancg12918 lost the ability to grow on gentisate or 3-hydroxybenzoate, but this ability could be restored in C. glutamicum upon the complementation with pXMJ19-ncg12918. Cloning and expression of this ncg12918 gene in E. coli showed that Ncg12918 is a glutathione-independent maleylpyruvate isomerase. Upstream of ncg12920, the genes ncg12921-ncg12923 were located, which were essential for gentisate and/or 3-hydroxybenzoate catabolism. The Ncg12921 was able to up-regulate gentisate 1,2-dioxygenase, maleylpyruvate isomerase, and fumarylpyruvate hydrolase activities. The genes ncg12922 and ncg12923 were deduced to encode a gentisate transporter protein and a 3-hydroxybenzoate hydroxylase, respectively, and were essential for gentisate or 3-hydroxybenzoate assimilation. Based on the results obtained in this study, a GSH-independent gentisate pathway was proposed, and genes involved in this pathway were identified.     

Both human ferredoxins 1 and 2 and ferredoxin reductase are important for iron-sulfur cluster biogenesis

Ferredoxins are iron-sulfur proteins that have been studied for decades because of their role in facilitating the monooxygenase reactions catalyzed by p450 enzymes. More recently, studies in bacteria and yeast have demonstrated important roles for ferredoxin and ferredoxin reductase in iron-sulfur cluster assembly. The human genome contains two homologous ferredoxins, ferredoxin 1 (FDX1) and ferredoxin 2 (FDX2--formerly known as ferredoxin 1L). More recently, the roles of these two human ferredoxins in iron-sulfur cluster assembly were assessed, and it was concluded that FDX1 was important solely for its interaction with p450 enzymes to synthesize mitochondrial steroid precursors, whereas FDX2 was used for synthesis of iron-sulfur clusters, but not steroidogenesis. To further assess the role of the FDX-FDXR system in mammalian iron-sulfur cluster biogenesis, we performed siRNA studies on FDX1 and FDX2, on several human cell lines, using oligonucleotides identical to those previously used, along with new oligonucleotides that specifically targeted each gene. We concluded that both FDX1 and FDX2 were important in iron-sulfur cluster biogenesis. Loss of FDX1 activity disrupted activity of iron-sulfur cluster enzymes and cellular iron homeostasis, causing mitochondrial iron overload and cytosolic iron depletion. Moreover, knockdown of the sole human ferredoxin reductase, FDXR, diminished iron-sulfur cluster assembly and caused mitochondrial iron overload in conjunction with cytosolic depletion. Our studies suggest that interference with any of the three related genes, FDX1, FDX2 or FDXR, disrupts iron-sulfur cluster assembly and maintenance of normal cytosolic and mitochondrial iron homeostasis.     

Human ISCA1 Interacts with IOP1/NARFL and Functions in Both Cytosolic and Mitochondrial Iron-Sulfur Protein Biogenesis

Iron-sulfur proteins play an essential role in many biologic processes. Hence, understanding their assembly is an important goal. In Escherichia coli, the protein IscA is a product of the isc (iron-sulfur cluster) operon and functions in the iron-sulfur cluster assembly pathway in this organism. IscA is conserved in evolution, but its function in mammalian cells is not known. Here, we provide evidence for a role for a human homologue of IscA, named IscA1, in iron-sulfur protein biogenesis. We observe that small interfering RNA knockdown of IscA1 in HeLa cells leads to decreased activity of two mitochondrial iron-sulfur enzymes, succinate dehydrogenase and mitochondrial aconitase, as well as a cytosolic iron-sulfur enzyme, cytosolic aconitase. IscA1 is observed both in cytosolic and mitochondrial fractions. We find that IscA1 interacts with IOP1 (iron-only hydrogenase-like protein 1)/NARFL (nuclear prelamin A recognition factor-like), a cytosolic protein that plays a role in the cytosolic iron-sulfur protein assembly pathway. We therefore propose that human IscA1 plays an important role in both mitochondrial and cytosolic iron-sulfur cluster biogenesis, and a notable component of the latter is the interaction between IscA1 and IOP1.     

A Gene Cluster Encoding Steps in Conversion of Naphthalene to Gentisate in Pseudomonas sp. Strain U2

Pseudomonas sp. strain U2 was isolated from oil-contaminated soil in Venezuela by selective enrichment on naphthalene as the sole carbon source. The genes for naphthalene dioxygenase were cloned from the plasmid DNA of strain U2 on an 8.3-kb BamHI fragment. The genes for the naphthalene dioxygenase genes nagAa (for ferredoxin reductase), nagAb (for ferredoxin), and nagAc and nagAd (for the large and small subunits of dioxygenase, respectively) were located by Southern hybridizations and by nucleotide sequencing. The genes for nagB (for naphthalene cis-dihydrodiol dehydrogenase) and nagF (for salicylaldehyde dehydrogenase) were inferred from subclones by their biochemical activities. Between nagAa and nagAb were two open reading frames, homologs of which have also been identified in similar locations in two nitrotoluene-using strains (J. V. Parales, A. Kumar, R. E. Parales, and D. T. Gibson, Gene 181:57–61, 1996; W.-C. Suen, B. Haigler, and J. C. Spain, J. Bacteriol. 178:4926–4934, 1996) and a naphthalene-using strain (G. J. Zylstra, E. Kim, and A. K. Goyal, Genet. Eng. 19:257–269, 1997). Recombinant Escherichia coli strains with plasmids carrying this region were able to convert salicylate to gentisate, which was identified by a combination of gas chromatography-mass spectrometry and nuclear magnetic resonance. The first open reading frame, designated nagG, encodes a protein with characteristics of a Rieske-type iron-sulfur center homologous to the large subunits of dihydroxylating dioxygenases, and the second open reading frame, designated nagH, encodes a protein with limited homology to the small subunits of the same dioxygenases. Cloned together in E. coli, nagG, nagH, and nagAb, were able to convert salicylate (2-hydroxybenzoate) into gentisate (2,5-dihydroxybenzoate) and therefore encode a salicylate 5-hydroxylase activity. Single-gene knockouts of nagG, nagH, and nagAb demonstrated their functional roles in the formation of gentisate. It is proposed that NagG and NagH are structural subunits of salicylate 5-hydroxylase linked to an electron transport chain consisting of NagAb and NagAa, although E. coli appears to be able to partially substitute for the latter. This constitutes a novel mechanism for monohydroxylation of the aromatic ring. Salicylate hydroxylase and catechol 2,3-dioxygenase in strain U2 could not be detected either by enzyme assay or by Southern hybridization. However growth on both naphthalene and salicylate caused induction of gentisate 1,2-dioxygenase, confirming this route for salicylate catabolism in strain U2. Sequence comparisons suggest that the novel gene order nagAa-nagG-nagH-nagAb-nagAc-nagAd-nagB-nagF represents the archetype for naphthalene strains which use the gentisate pathway rather than the meta cleavage pathway of catechol.     

Purification and characterisation of a gentisate 1,2-dioxygenase fromRalstonia solanacearum GMI 1000

The gene encoding gentisate 1,2-dioxygenase from a soil-borne Gram-negative bacterium,Ralstonia solanacearum GMI 1000, was cloned and overexpressed inEscherichia coli. The resulting product incorporated a (His) 6 tag was purified to homogeneity from the harvested cell extracts by affinity chromatography. SDS-PAGE showed that the polypeptide exhibited an approximate molecular mass of 38 kDa. The optimal temperature and pH for gentisate cleavage catalysed by the enzyme were 30 °C and 8.0, respectively. TheK _m of the enzyme was determined to be 56 μM. ThepI is 4.6–4.8. Moreover, site-directed mutagenesis revealed that His105, His 107, and His 146 are the crucial residues involved in the catalytic activity of gentisate 1,2-dioxygenase fromRalstonia solanacearum GMI 1000.     

Metabolic regulation and chromosomal localization of carbaryl degradation pathway in Pseudomonas sp. strains C4, C5 and C6

Pseudomonas sp. strains C4, C5 and C6 degrade carbaryl (1-naphthyl N-methylcarbamate) via 1-naphthol, 1,2-dihydroxynaphthalene, salicylate and gentisate. Carbon source-dependent metabolic studies suggest that enzymes responsible for carbaryl degradation are probably organized into ‘upper’ (carbaryl to salicylate), ‘middle’ (salicylate to gentisate) and ‘lower’ (gentisate to TCA cycle) pathway. Carbaryl and 1-naphthol were found to induce all carbaryl pathway enzymes, while salicylate and gentisate induce middle and lower pathway enzymes. The strains were found to harbor plasmid(s), and carbaryl degradation property was found to be stable. Genes encoding enzymes of the degradative pathway such as 1-naphthol 2-hydroxylase, salicylaldehyde dehydrogenase, salicylate 5-hydroxylase and gentisate 1,2-dioxygenase were amplified from chromosomal DNA of these strains. The gene-specific PCR products were sequenced from strain C6, and phylogenetic tree was constructed. Southern hybridization and PCR analysis using gel eluted DNA as template supported the presence of pathway genes onto the chromosome and not on the plasmid(s).     

Functional characterization of a gene cluster involved in gentisate catabolism in Rhodococcus sp. strain NCIMB 12038

Rhodococcus sp. strain NCIMB 12038 utilizes naphthalene as a sole source of carbon and energy, and degrades naphthalene via salicylate and gentisate. To identify the genes involved in this pathway, we cloned and sequenced a 12-kb DNA fragment containing a gentisate catabolic gene cluster. Among the 13 complete open reading frames deduced from this fragment, three (narIKL) have been shown to encode the enzymes involved in the reactions of gentisate catabolism. NarI is gentisate 1,2-dioxygenase which converts gentisate to maleylpyruvate, NarL is a mycothiol-dependent maleylpyruvate isomerase which catalyzes the isomerization of maleylpyruvate to fumarylpyruvate, and NarK is a fumarylpyruvate hydrolase which hydrolyzes fumarylpyruvate to fumarate and pyruvate. The narX gene, which is divergently transcribed with narIKL, has been shown to encode a functional 3-hydroxybenzoate 6-monooxygenase. This led us to discover that this strain is also capable of utilizing 3-hydroxybenzoate as its sole source of carbon and energy. Both NarL and NarX were purified to homogeneity as His-tagged proteins, and they were determined by gel filtration to exist as a trimer and a monomer, respectively. Our study suggested that the gentisate degradation pathway was shared by both naphthalene and 3-hydroxybenzoate catabolism in this strain.