Biosynthesis of vitamin B12: isolation of 15,23-dihydrosirohydrochlorin, a biosynthetic intermediate: structural studies and incorporation experiments

Battersby, Alan R.; Frobel, Klaus; Hammerschmidt, Friedrich; Jones, Christopher

doi:10.1039/c39820000455

Cited by 40 publications

(34 citation statements)

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“…They do not prove it, however, since seco-F430 3 could be an isolation artefact which the cells are capable of converting to F430 1. For example, sirohydrochlorin is converted to vitamin Blz by Propionibacterium shermanii although 15,23-dihydrosirohydrochlorin rather than sirohydrochlorin is the actual intermediate in the biosynthesis of vitamin B1 [28]. Dihydrosirohydrochlorin is readily oxidized to sirohydrochlorin during purification but can be reduced back to the dihydro form by the cells [28].…”

Section: Discussionmentioning

confidence: 99%

“…7. All available evidence indicates that F430, siroheme, and vitamin BIZ share the same biosynthetic pathway up to 15,23-dihydro-sirohydrochlorin 7 [28] as the last common intermediate [6,9,26,271. The subsequent steps, leading to seco-F430 3, must involve (not necessarily in that order): amidation of the acetate side chain in ring A and B, insertion of nickel, rearrangement of the n-system with concomitant reduction of two (C = C) double bonds, and formation of the lactam ring attached to ring B.…”

Section: Discussionmentioning

confidence: 99%

“…This finding can be taken as evidence that NIX is a prophinoid since only porphinoids are synthesized from 8 mol GAla/mol. Dihydrosirohydrochlorin [28], which is assumed to be an intermediate in F430 biosynthesis [9,26,271, contains two methyl groups derived from S-adenosylmethionine. Cells of M .…”

Section: Conditions For N I X Accumulationmentioning

confidence: 99%

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Biosynthesis of coenzyme F430 in methanogenic bacteria

Pfaltz

Kobelt

Hüster

et al. 1987

European Journal of Biochemistry

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Coenzyme F430 is a hydroporphinoid nickel complex present in all methanogenic bacteria. It is part of the enzyme system which catalyzes methane formation from methyl-coenzyme M. We describe here that under certain conditions a second nickel porphinoid accumulates in methanogenic bacteria. The compound was identified at 15,l 73-seco-F430-173-acid. The structural assignment rests on 14C-labelling experiments, fast-atom-bombardment mass spectra, 'H-NMR spectra of the corresponding hexamethyl ester, and ultraviolet/visible spectral comparison with model compounds. In cell extracts and in intact cells of methanogenic bactera, 15,173-seco-F430-173-acid was converted to F430. These findings indicate that the new nickel-containing porphinoid is an intermediate in the biosynthesis of coenzyme F430.Coenzyme F430 [l] is a yellow non-fluorescent nickel porphinoid found in all methanogenic bacteria [2 -51. Its structure has been recently determined [6-91. As shown in Fig. 1, coenzyme F430 1 possesses a highly saturated ligand system with a chromophore not previously encountered among natural tetrapyrroles. It may be considered a tetrahydro derivative of a corphin [6, 101, combining structural elements of both porphyrins and corrins. The isolated imine double bond, the lactam ring attached to ring B, and the sixmembered carbocyclic ring, built from the propionate side chain in ring D, further distinguish coenzyme F430 from other natural prophinoids. Suggestions that the parent coenzyme is larger, containing coenzyme M and a lumazine derivative covalently bound to the porphinoid ligand skeleton, in the meantime, have been ruled out [7, 11, 121. [19, 201. Coenzyme F430 is present in the cells also in a non-proteinbound, free form [7, 11, 211. Free F430 has been shown to be the precursor of bound F430 in the biosynthesis of methylcoenzyme M reductase [21]. The free and the enzyme-bound form (the latter determined after dissociation from the protein) have identical structures [7, 111. The biosynthesis of coenzyme F430 has been partially unravelled. The pathway starts from glutamate [22], which via glutamyl-tRNA and glutamate I-semialdehyde is converted to 5-aminolevulinic acid (6Ala) [23]. From there the biosynthesis proceeds along the well established pathway to uroporphyrinogen I11 [24], the common precursor of all natural tetrapyrroles [25]. Analogous ot the biosynthesis of vitamin BI2 and siroheme [25], uroporphyrinogen I11 is then methylated at positions 2 and 7 by S-adenosylmethionine [6, 261 in a sequence of reactions which, according to the available evidence, lead to 15,23-dihydro-sirohydrochlorin [9, 26 -281. The subsequent steps converting dihydro-sirohydrochlorin to coenzyme F430 and the intermediates involved are still unknown.In this communication we report the isolation of a nickelcontaining biosynthetic precursor of coenzyme F430 which was found to be a 15,173-seco derivative of F430, possessing a free propionate side chain in ring D in place of the sixmembered carbocyclic ring (3 Fig. 1). MATERIALS AND ...

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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Biosynthesis of coenzyme F430 in methanogenic bacteria

Pfaltz

Kobelt

Hüster

et al. 1987

European Journal of Biochemistry

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“…Precorrin-2 and precorrin-3 are highly oxygen-sensitive intermediates and are normally isolated in their aromatized forms as the esters sirohydrochlorin methyl ester (compound 6) and factor III methyl ester (compound 7). Working in strictly anaerobic conditions, Battersby and co-workers purified and identified as 15,23-dihydrosirohydrochlorin octamethyl ester (compound 3) the methyl ester derivative of precorrin-2 (2). An equivalent structural study on precorrin-3 has not been reported but, since C methylation does not affect oxidation level, it is very likely that the true trimethylated intermediate is a dihydro derivative of factor III (compound 4 or a double-bond tautomer of it).…”

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confidence: 99%

Assay and purification of S-adenosyl-L-methionine:precorrin-2 methyltransferase from Pseudomonas denitrificans

Thibaut¹,

Couder²,

Crouzet³

et al. 1990

J Bacteriol

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S-Adenosyl-L-methionine:precorrin-2 methyltransferase (SP2MT), which catalyzes the C-20 methylation of precorrin-2 to precorrin-3, was purified to homogeneity from extracts of a recombinant strain of Pseudomonas denitrificans derived from a cobalamin-overproducing strain. Ammonium sulfate fractionation followed by chromatography on DEAE-Trisacryl, hydroxyapatite, and Mono Q HR purified the enzyme about 110-fold, with a 28% yield. For enzyme purification and characterization, a coupled-enzyme assay was developed which generated in situ the highly oxygen-sensitive substrate, precorrin-2, from 8-aminolevulinic acid. Evidence is given that the chemically reduced form of sirohydrochlorin (dihydrosirohydrochlorin) is methylated at C-20 to precorrin-3 by pure SP2MT. No subsequent SP2MT-dependent methylation reaction of precorrin-3 was detected. The native enzyme has an apparent molecular weight of 53,000, as estimated by gel filtration, and consists of two identical subunits of Mr 26,000, as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Stepwise Edman degradation provided the N-terminal sequence of the first 17 amino acids.

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“…Work on B12 and siroheme synthesis suggests that there should be three steps involved in converting URO III to siroheme (2,28,29,35 (33). However, hemH mutants have been isolated in S. typhimunum, and none require cysteine for growth (42 (4,39).…”

Section: Resultsmentioning

confidence: 99%

Genetic structure and regulation of the cysG gene in Salmonella typhimurium

Goldman

Roth

1993

J Bacteriol

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Siroheme, a cofactor of both sulfite and nitrite reductase in Sabnonella typhimurium, requires the cysG gene for its synthesis. Three steps are required to synthesize siroheme from uroporphyrinogen m, the last common intermediate in the heme and siroheme pathways. All previously characterized cysG mutants were shown to be defective for the synthesis of cobalamin (B12), which shares a common precursor with siroheme. Since few cysG auxotrophs had been previously analyzed and since there is no evidence of siroheme mutants outside of the cysG region, we sought to expand the analysis of the region by isolating more mutations and studying the transcriptional regulation of the cysG gene using IacZ fusions. We isolated and analyzed 66 cysG auxotrophs. All were defective for both siroheme and cobalamin synthesis. Five exceptional mutants were partially defective for the synthesis of both and appear to be leaky. Complementation tests with tandem duplications suggest that the mutations causing the Cys auxotrophy affect only one cistron. The cysG gene is transcribed in a clockwise direction; this was demonstrated by a method that permits determining the orientation of two genes of unknown orientation provided their relative map order is known. The cysG gene was not part of the cysteine regulon, but had a substantial basal level of expression which was induced fivefold when cells were grown anaerobically on nitrite. Finally, we used Mud-generated duplications to genetically determine the organization of the cysG and nirB genes.

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Biosynthesis of vitamin B12: isolation of 15,23-dihydrosirohydrochlorin, a biosynthetic intermediate: structural studies and incorporation experiments

Cited by 40 publications

References 2 publications

Biosynthesis of coenzyme F430 in methanogenic bacteria

Biosynthesis of coenzyme F430 in methanogenic bacteria

Assay and purification of S-adenosyl-L-methionine:precorrin-2 methyltransferase from Pseudomonas denitrificans

Genetic structure and regulation of the cysG gene in Salmonella typhimurium

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