Enzymatic synthesis of dihydrosirohydrochlorin (precorrin‐2) and of a novel pyrrocorphin by uroporphyrinogen III methylase

Warren, Martin J.; Stolowich, Neal J.; Santander, Patricio J.; Roessner, Charles A.; Sowa, Blair A.; Scott, A. Ian

doi:10.1016/0014-5793(90)80640-5

Cited by 71 publications

(53 citation statements)

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“…Complete NMR analysis of this product showed it to be identical to the trimethylpyrrocorphin previously synthesized with CysG (36). Urogen I also served as a substrate for P. freudenreichii CobA, with the formation of type I precorrin-2 after 4 h and type I trimethylpyrrocorphin after a prolonged incubation.…”

mentioning

confidence: 62%

“…The tetrapyrrolic products were adsorbed onto a small DEAE-Sephadex column, washed with 0.3 M KCl, and eluted from the column with 20% D 2 O in 2.0 M KCl. The structures of the compounds were determined by comparison of their 13 C-NMR spectra to those of previously isolated tetrapyrroles (22,27,30,34,36).…”

Section: Methodsmentioning

confidence: 99%

“…CobA performs only methylation of C-2 and C-7 of urogen III or its isomer, urogen I (1), whereas CysG performs these methylations and also has an overmethylation property. Previous studies with CysG have resulted in the in vitro synthesis of several tri-and tetra-methylated compounds such as trimethylpyrrocorphins derived from urogen I and urogen III (30,36), a tetramethylpyrrocorphin derived from precorrin-3 (27), and factor S3 (22), a natural product previously isolated from P. freudenreichii.…”

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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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confidence: 62%

Section: Methodsmentioning

confidence: 99%

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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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“…There are two possible explanations for this low level of synthesis. First, CysG A is known to produce a large quantity of the overmethylated compound, a 2,7,12-trimethylpyrrocorphin, which has no known physiological function [8]. By making such large quantities of this compound the enzyme is lowering the precorrin-2 concentration, effectively removing the substrate from the cobalamin biosynthetic pathway.…”

Section: A ) Is Required For Promotion Of Cobalamin Biosynthesismentioning

confidence: 99%

“…Thus two S-adenosyl--methionine (AdoMet)-dependent transmethylations of uro'gen III at positions 2 and 7 produce precorrin-2 (dihydrosirohydrochlorin) [8]. Sirohaem is synthesized via sirohydrochlorin by dehydrogenation of the pyrrocorphin precorrin-2, which is subsequently ferrochelated.…”

Section: Introductionmentioning

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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Synthetic Biology in Metabolic Engineering: From Complex Biochemical Pathways to Compartmentalized Metabolic Processes - a Vitamin Connection

Deery

Frank

Lawrence

et al. 2014

Encyclopedia of Molecular Cell Biology and Molecular Medicine

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Enzymatic synthesis of dihydrosirohydrochlorin (precorrin‐2) and of a novel pyrrocorphin by uroporphyrinogen III methylase

Cited by 71 publications

References 13 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)

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

Synthetic Biology in Metabolic Engineering: From Complex Biochemical Pathways to Compartmentalized Metabolic Processes - a Vitamin Connection

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