Evidence that the CysG protein catalyzes the first reaction specific to B12 synthesis in Salmonella typhimurium, insertion of cobalt

Fazzio, Thomas G.; Roth, John R.

doi:10.1128/jb.178.23.6952-6959.1996

Cited by 44 publications

(32 citation statements)

References 43 publications

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“…Moreover, K. pneumoniae can anaerobically biosynthesize vitamin B 12 . Comparative genomic analysis revealed that the genes involved are KPN_03184 to KPN_03200 (10,39). Meanwhile, reactions belonging to incomplete pathways were removed from the reconstruction.…”

Section: Metabolic Network Reconstructionmentioning

confidence: 99%

An Experimentally Validated Genome-Scale Metabolic Reconstruction of Klebsiella pneumoniae MGH 78578, i YL1228

et al. 2011

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Klebsiella pneumoniae is a Gram-negative bacterium of the family Enterobacteriaceae that possesses diverse metabolic capabilities: many strains are leading causes of hospital-acquired infections that are often refractory to multiple antibiotics, yet other strains are metabolically engineered and used for production of commercially valuable chemicals. To study its metabolism, we constructed a genome-scale metabolic model (iYL1228) for strain MGH 78578, experimentally determined its biomass composition, experimentally determined its ability to grow on a broad range of carbon, nitrogen, phosphorus and sulfur sources, and assessed the ability of the model to accurately simulate growth versus no growth on these substrates. The model contains 1,228 genes encoding 1,188 enzymes that catalyze 1,970 reactions and accurately simulates growth on 84% of the substrates tested. Furthermore, quantitative comparison of growth rates between the model and experimental data for nine of the substrates also showed good agreement. The genome-scale metabolic reconstruction for K. pneumoniae presented here thus provides an experimentally validated in silico platform for further studies of this important industrial and biomedical organism.Klebsiella pneumoniae, a Gram-negative bacterium of the family Enterobacteriaceae, is a microorganism with significance in both medicine and biotechnology. K. pneumoniae is a common opportunistic human pathogen, causing pneumonia, urinary tract infections, and bacteremia (25). Most clinical K. pneumoniae isolates are multidrug resistant, and up to 20% of them are extended-spectrum beta-lactamase-producing strains. The situation is worsened by the recent spreading of carbapenem-resistant NDM-1 strains, which leave very few antibiotics effective and therefore pose a serious threat to human health (23). In biotechnology, K. pneumoniae is capable of metabolizing glycerol as a sole source of carbon to produce 1,3-propanediol, a chemical that has many industrial applications (16,51). Proper functioning of the metabolic network in K. pneumoniae underlies its ability both to cause disease and to serve as a useful platform strain for metabolic engineering.The study of metabolism in many organisms has greatly benefitted from in silico genome-scale reconstruction of their metabolic networks combined with flux balance analysis (FBA), a computational technique that incorporates many different components of metabolism and their couplings to provide insight into steady-state flux distributions among the different pathways. Such system level analyses are critical since they can reveal emergent, network level properties not readily apparent from more-focused investigation of individual genes or pathways.The metabolic network reconstruction of Escherichia coli K-12 represents the best-developed genome-scale network to date because E. coli is arguably the most studied and best characterized microorganism in terms of genome annotation, functional characterization, and knowledge of growth behavior (11). Metabolic network r...

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Section: Metabolic Network Reconstructionmentioning

confidence: 99%

An Experimentally Validated Genome-Scale Metabolic Reconstruction of Klebsiella pneumoniae MGH 78578, i YL1228

et al. 2011

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“…1). The CysG protein performs the two methylation reactions first, making the intermediate tetrapyrrole (dihydrosirohydrochlorin) available as a substrate for B 12 biosynthesis (4,24,27,28). We postulate that the CysF protein may first reduce, or insert iron into, Uro-III thereby preventing its flux into the B 12 biosynthetic pathway; alternatively, the CysF protein may perform only a single methylation, which may yield a product capable of functioning in NirBD but not competent for B 12 biosynthesis.…”

Section: Discussionmentioning

confidence: 99%

“…The methylation functions of CysG are catalyzed by the C-terminal portion of the enzyme; proteins homologous to this domain perform methylation of Uro-III and other tetrapyrroles (20). Metal insertion appears to be catalyzed by the N-terminal portion of CysG (4).…”

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confidence: 99%

“…Since cobalamin contains a central cobalt atom, it is likely that an iron-free intermediate in siroheme biosynthesis is released from the CysG enzyme, making it available to cobalamin biosynthetic enzymes (encoded by the cbi and cob genes). While it has been proposed that CysG also performs cobalt insertion (4), this activity has also been ascribed to the CbiK protein, encoded within the cbi/cob operon (19). Unlike Salmonella, E. coli cannot synthesize B 12 de novo (14) and uses CysG activity solely for siroheme biosynthesis.…”

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confidence: 99%

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Alternative Pathways for Siroheme Synthesis inKlebsiella aerogenes

2001

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Siroheme, the cofactor for sulfite and nitrite reductases, is formed by methylation, oxidation, and iron insertion into the tetrapyrrole uroporphyrinogen III (Uro-III). The CysG protein performs all three steps of siroheme biosynthesis in the enteric bacteria Escherichia coli and Salmonella enterica. In either taxon, cysG mutants cannot reduce sulfite to sulfide and require a source of sulfide or cysteine for growth. In addition, CysG-mediated methylation of Uro-III is required for de novo synthesis of cobalamin (coenzyme B 12 ) in S. enterica. We have determined that cysG mutants of the related enteric bacterium Klebsiella aerogenes have no defect in the reduction of sulfite to sulfide. These data suggest that an alternative enzyme allows for siroheme biosynthesis in CysG-deficient strains of Klebsiella. However, Klebsiella cysG mutants fail to synthesize coenzyme B 12 , suggesting that the alternative siroheme biosynthetic pathway proceeds by a different route. Gene cysF, encoding an alternative siroheme synthase homologous to CysG, has been identified by genetic analysis and lies within the cysFDNC operon; the cysF gene is absent from the E. coli and S. enterica genomes. While CysG is coregulated with the siroheme-dependent nitrite reductase, the cysF gene is regulated by sulfur starvation. Models for alternative regulation of the CysF and CysG siroheme synthases in Klebsiella and for the loss of the cysF gene from the ancestor of E. coli and S. enterica are presented.

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“…It has been suggested that the observed cobalt chelation activity might reflect a function required for corrin biosynthesis [14,20] and it…”

Section: Abbreviations Used : Adomet S-adenosyl-l-methionine ; Cysgmentioning

confidence: 99%

Effect of mutations in the transmethylase and dehydrogenase/chelatase domains of sirohaem synthase (CysG) on sirohaem and cobalamin biosynthesis

Woodcock

Raux

Levillayer

et al. 1998

Biochemical Journal

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The Escherichia coli CysG protein (sirohaem synthase) catalyses four separate reactions that are required for the transformation of uroporphyrinogen III into sirohaem, initially two S-adenosyl-L-methionine-dependent transmethylations at positions 2 and 7, mediated through the C-terminal, or CysGA, catalytic domain of the protein, and subsequently a ferrochelation and dehydrogenation, mediated through the N-terminal, or CysGB, catalytic domain of the enzyme. This report describes how the deletion of the NAD+-binding site of CysG, located within the first 35 residues of the N-terminus, is detrimental to the activity of CysGB but does not affect the catalytic activity of CysGA, whereas the mutation of a number of phylogenetically conserved residues within CysGA is detrimental to the transmethylation reaction but does not affect the activity of CysGB. Further studies have shown that CysGB is not essential for cobalamin biosynthesis because the presence of the Salmonella typhimurium CobI operon with either cysGA or the Pseudomonas denitrificans cobA are sufficient for the synthesis of cobyric acid in an E. coli cysG deletion strain. Evidence is also presented to suggest that a gene within the S. typhimurium CobI operon might act as a chelatase that, at low levels of cobalt, is able to aid in the synthesis of sirohaem.

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Evidence that the CysG protein catalyzes the first reaction specific to B12 synthesis in Salmonella typhimurium, insertion of cobalt

Cited by 44 publications

References 43 publications

An Experimentally Validated Genome-Scale Metabolic Reconstruction of Klebsiella pneumoniae MGH 78578, i YL1228

An Experimentally Validated Genome-Scale Metabolic Reconstruction of Klebsiella pneumoniae MGH 78578, i YL1228

Alternative Pathways for Siroheme Synthesis inKlebsiella aerogenes

Effect of mutations in the transmethylase and dehydrogenase/chelatase domains of sirohaem synthase (CysG) on sirohaem and cobalamin biosynthesis

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