Genetic structure and regulation of the cysG gene in Salmonella typhimurium

Goldman, Barry S.; Roth, John R.

doi:10.1128/jb.175.5.1457-1466.1993

Cited by 38 publications

(34 citation statements)

References 40 publications

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“…The addition of a methyl group to one of these carbons converts it to an sp 3 center with a concomitant upfield shift to 40 to 60 ppm. Thus, after a 4-h incubation of a TB1(pISA417) lysate with unlabeled SAM and urogen III derived from [3][4][5][6][7][8][9][10][11][12][13] C]ALA, 13 C-NMR spectroscopy of the product revealed the presence of two new sp 3 carbons (Fig. 4A), which is consistent with the addition of two methyl groups to urogen III.…”

Section: Resultsmentioning

confidence: 58%

“…Control lysates of TB1(pUC19), which presumably have a low level of endogenous CysG expressed from the chromosomal gene, had no urogen III methyltransferase activity detectable by the NMR assay even after a 15-h incubation. The best substrates for analysis of urogen III methyltransferase activity are urogens derived from [3][4][5][6][7][8][9][10][11][12][13] C]ALA in which the carbons that become methylated are 13 C labeled (Fig. 4) and appear in a 13 C-NMR spectrum in the sp 2 region as a single peak at around 120 ppm.…”

Section: Resultsmentioning

confidence: 99%

“…This compound is not an intermediate in the vitamin B 12 pathway and has not yet been fully characterized, but it was of interest to test if P. freudenreichii CobA as well as CysG could use precorrin-3 as a substrate. Thus, precorrin-2 methyltransferase (CobI, which methylates precorrin-2 at C-20 to form precorrin-3) (32) was added to an incubation mixture containing urogen III derived from [3][4][5][6][7][8][9][10][11][12][13] C]ALA, SAM, and the TB1(pISA417) lysate. The product isolated from this reaction mixture had an NMR spectrum identical to that of the tetramethylated compound formed by CysG under similar conditions.…”

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confidence: 99%

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Cloning, sequencing, and expression of the uroporphyrinogen III methyltransferase cobA gene of Propionibacterium freudenreichii (shermanii)

et al. 1995

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We cloned, sequenced, and overexpressed cobA, the gene encoding uroporphyrinogen III methyltransferase in Propionibacterium freudenreichii, and examined the catalytic properties of the enzyme. The methyltransferase is similar in mass (27 kDa) and homologous to the one isolated from Pseudomonas denitrificans. In contrast to the much larger isoenzyme encoded by the cysG gene of Escherichia coli (52 kDa), the P. freudenreichii enzyme does not contain the additional 22-kDa peptide moiety at its N-terminal end bearing the oxidase-ferrochelatase activity responsible for the conversion of dihydrosirohydrochlorin (precorrin-2) to siroheme. Since it does not contain this moiety, it is not a likely candidate for synthesis of a cobalt-containing early intermediate that has been proposed for the vitamin B 12 biosynthetic pathway in P. freudenreichii. Uroporphyrinogen III methyltransferase of P. freudenreichii not only catalyzes the addition of two methyl groups to uroporphyrinogen III to afford the early vitamin B 12 intermediate, precorrin-2, but also has an overmethylation property that catalyzes the synthesis of several tri-and tetra-methylated compounds that are not part of the vitamin B 12 pathway. The enzyme catalyzes the addition of three methyl groups to uroporphyrinogen I to form trimethylpyrrocorphin, the intermediate necessary for biosynthesis of the natural products, factors S1 and S3, previously isolated from this organism. A second gene found upstream from the cobA gene encodes a protein homologous to CbiO of Salmonella typhimurium, a membrane-bound, ATP-dependent transport protein thought to be part of the cobalt transport system involved in vitamin B 12 synthesis. These two genes do not appear to constitute part of an extensive cobalamin operon.Uroporphyrinogen (urogen) III methyltransferase, a key enzyme in the biosynthetic pathways of vitamin B 12 and siroheme, catalyzes the S-adenosyl-L-methionine (SAM)-dependent bismethylation of its substrate, urogen III, resulting in the formation of dihydrosirohydrochlorin (known as precorrin-2 in the vitamin B 12 pathway). In the biosynthesis of vitamin B 12 in the anaerobe Propionibacterium freudenreichii, labeling experiments have indicated that cobalt is inserted soon after the formation of precorrin-2 (3, 20), and the recent isolation of a cobalt-containing tetramethylated corphinoid, possibly an intermediate, from this organism supports these observations (3). Cobalt insertion in the vitamin B 12 pathway of the aerobe Pseudomonas denitrificans, however, occurs at a much later stage with insertion into hydrogenobyrinic acid diamide (6).Urogen III methyltransferase has been purified to homogeneity from several different organisms, and the nucleotide sequences of the corresponding genes have been determined, revealing that the enzyme exists in at least two forms. One form, encoded by the cysG gene, is required for siroheme and, thus, for cysteine synthesis in Escherichia coli (14,31,35) and siroheme and vitamin B 12 synthesis in Salmonella typhimurium (8, 10). The ...

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Section: Resultsmentioning

confidence: 58%

Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 99%

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Cloning, sequencing, and expression of the uroporphyrinogen III methyltransferase cobA gene of Propionibacterium freudenreichii (shermanii)

et al. 1995

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“…Formation of functional YSiR will need, in addition to the putative a and 13 subunits, the enzymatic machinery for siroheme production. One, two, or even three gene products might be necessary for this (14,57), and since S. cerevisiae does not possess nitrite reductase, mutants with mutations in the corresponding genes are expected to appear simply as methionine auxotrophs, defective in sulfite reduction. As Strains and media.…”

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confidence: 99%

Two divergent MET10 genes, one from Saccharomyces cerevisiae and one from Saccharomyces carlsbergensis, encode the alpha subunit of sulfite reductase and specify potential binding sites for FAD and NADPH

1994

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The yeast assimilatory sulfite reductase is a complex enzyme that is responsible for conversion of sulfite into sulfide. To obtain information on the nature of this enzyme, we In Saccharomyces cerevisiae, sulfate taken up from the surrounding environment is converted into sulfite through three enzymatic steps. Sulfite is further reduced to sulfide, which in turn combines with O-acetyl homoserine to form homocysteine and eventually methionine. A central step in this assimilation pathway is conversion of sulfite into sulfide, a six-electron transfer reaction catalyzed by the yeast assimilatory sulfite reductase (YSiR). YSiR was found to be NADPH dependent (35) and to contain the prosthetic groups flavin adenine dinucleotide (FAD), flavin mononucleotide (FMN), an iron-sulfur cluster and a nonflavin chromophore (61) later identified as siroheme (46), a uroporphyrinogen III derivative also found in bacterial sulfite reductases (33,34), and nitrite reductase (21). Some methionine auxotrophs of S. cerevisiae were found to possess a defective sulfite reductase devoid of FAD or of FAD and FMN (62). The mutants devoid only of FAD were later assigned to the metlO complementation group and shown to be affected solely in the sulfite reduction step (30,31). The results of Kobayashi and Yoshimoto (25) suggested that YSiR has an ct212 structure and that the molecular mass of

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“…Furthermore, because mutations in S. typhimurium cysG that affect sirohaem synthesis also seem to affect cobalamin biosynthesis in parallel, it has been argued that the two domains of CysG must be essential for both activities [21]. This paper investigates the contribution made by the two functional domains of CysG towards sirohaem and cobalamin biosynthesis and whether the two regions of the protein act independently.…”

Section: Scheme 1 Biosynthesis Of Sirohaem and Cobyric Acid From Uro'mentioning

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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Genetic structure and regulation of the cysG gene in Salmonella typhimurium

Cited by 38 publications

References 40 publications

Cloning, sequencing, and expression of the uroporphyrinogen III methyltransferase cobA gene of Propionibacterium freudenreichii (shermanii)

Cloning, sequencing, and expression of the uroporphyrinogen III methyltransferase cobA gene of Propionibacterium freudenreichii (shermanii)

Two divergent MET10 genes, one from Saccharomyces cerevisiae and one from Saccharomyces carlsbergensis, encode the alpha subunit of sulfite reductase and specify potential binding sites for FAD and NADPH

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

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