Biosynthesis of vitamin B12: order of the later C-methylation steps

Uzar, Horst C.; Battersby, Alan R.

doi:10.1039/c39850000585

Cited by 19 publications

(18 citation statements)

References 3 publications

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“…The C-methyl group, which eventually appears at C-12 of corrins, is the next to be added (6)(7)(8) but this is now known to be inserted at C-11, which leads to structure 8 for the pentamethylated intermediate. Methylation at C-1, the next in sequence, could take place directly at C-1 or first at C-20 followed by a 1,2 shift of the a-methyl group.…”

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confidence: 99%

Biosynthesis of vitamin B12: structure of precorrin-6x octamethyl ester.

Thibaut¹,

Blanche²,

Debussche³

et al. 1990

Proc. Natl. Acad. Sci. U.S.A.

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ABSTRACT'3C-labeled precorrin-6x is biosynthesized by cell-free protein preparations from Pseudomonas denitificans in separate experiments using 8-amino[5-3Cjlevulinic acid and the corresponding -amino[4-'3C]-and bamino[3-'3C~lev-ulinic acid-labeled forms in conjunction with S- [methyl-'3Cjadenosylmethionine for the latter two experiments. These labeled precorrin-6x samples, as their octamethyl esters, are studied by a range of NMR techniques. In addition, nuclear Overhauser effect difference measurements are made on unlabeled precorrin-6x ester to determine connectivities. The structure 6a so established for precorrin-6x ester (i) confirms the results reported in the preceding paper that precorrin-6x has a ring-contracted macrocycle, still carries the C-12 acetate residue, and stands at the oxidation level of a dehydrocorrin; (ii) reveals the unexpected methylation at C-11 not C-12, leading to a structure with separated chromophores; and (iii) implies that methyl migration from C-11 to C-12 occurs when precorrin-6x is converted into hydrogenobyrinic acid. Proposals for the biosynthesis ofthe corrin macrocycle ofhydrogenobyrinic acid and vitamin B12 are made.The isolation of precorrin-6x reported in the preceding paper (1) has changed the entire direction of research on vitamin B12, in that views that had almost become dogma (e.g., no involvement of external redox reagents and early decarboxylation of the C-12 acetate before ring contraction) are now swept away. The discovery of this precursor opened the way to further progress in delineating the biosynthetic pathway to hydrogenobyrinic acid (structure la), cobyrinic acid (structure lb), and thus to vitamin B12 itself. We now report our studies, which have established the unexpected structure 6a for precorrin-6x octamethyl ester (Scheme I).Accurate mass measurement (electron impact) on precorrin-6x octamethyl ester proved its composition to be C52H70N4016 (found: 1006.4786; requires 1006.4724, error 6.2 ppm). This molecular formula corresponds to a ringcontracted macrocycle with seven double bonds, which if conjugated would result in a purple or blue pigment. Yet precorrin-6x ester is only pale yellow and shows Amax (CH2Cl2), 361 (log E 4.30), 371 (sh 4.27), and 430 (sh 3.74) with Am,, at 298 nm (3.52); these data prove that the molecule has separated chromophores.13C-labeled precorrin-6x was produced biosynthetically from 13C-labeled precorrin-3 (structure 3b), which in turn had been generated via its aromatized form (structure 2b) as in the preceding paper (1) from 8-amino[5-13C]levulinic acid (ALA) containing 99 atom % 13C ([5-13C]ALA; structure 4b). The resultant precorrin-6x octamethyl ester (structure 6b) was examined in C62H6 by proton-noise decoupled`C NMR.Seven signals appeared from 13C-enriched centers (Table 1), thus confirming the loss of C-20 prior to formation of precorrin-6x (1); all the signals were doublets save one, for C-15, which was a triplet, the characteristic pattern (2) for a type III tetrapyrrole macrocycle. This is as expected (1) sin...

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confidence: 99%

Biosynthesis of vitamin B12: structure of precorrin-6x octamethyl ester.

Thibaut¹,

Blanche²,

Debussche³

et al. 1990

Proc. Natl. Acad. Sci. U.S.A.

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“…This shift allows the double bonds to move into conjugation, thus setting up the characteristic corrin chromophore of hydrogenobyrinic acid. precorrin-3 (9). There was a gap, however, before precorrin-6A (20) could be reached.…”

Section: The Pathway Beyond Precorrin-6bmentioning

confidence: 96%

“…For cobyrinic and B12, researchers were interested in ning down the order of attachment o1 remaining five C methyl groups added precorrin 3. Experiments based on labeled methyl groups (9) proved that is the fourth methylation site. The me was then extended (8, 10, 1 1) to show C-12(a is the fifth site followed by C-1, C-5 and C-15 being last with roughly ilar timing.…”

Section: The Pigments Of Lifementioning

confidence: 99%

“…The me was then extended (8, 10, 1 1) to show C-12(a is the fifth site followed by C-1, C-5 and C-15 being last with roughly ilar timing. The 12,-methyl of coby acid (10) is not SAM-derived; it is foi by decarboxylation of the 12-acetate .pinf the after 13C-due still present in precorrin-3 (9).…”

Section: The Pigments Of Lifementioning

confidence: 99%

“…The soluble protein preparation from this engineered bacterial strain contained large amounts of the enzymes encoded by the eight overexpressed genes. This cellfree system could convert precorrin-3 (9) into hydrogenobyrinic acid (11) in high yield provided that the necessary cofactors, including SAM and reduced nicotinamide adenine dinucleotide phosphate (NADPH) (14), were added. When this incubation was run without added NADPH, no hydrogenobyrinic acid was formed, although a yellow pigment was produced in its place.…”

Section: The Great Surprisementioning

confidence: 99%

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How Nature Builds the Pigments of Life: the Conquest of Vitamin B ₁₂

Battersby¹

1994

Science

Self Cite

162

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In part because humans cannot synthesize vitamin B12 and must obtain it from organisms that produce it and because B12 deficiency leads to pernicious anemia, it has been important to understand how microorganisms build this quite complex substance. As shown here, an interdisciplinary attack was needed, which combined the strengths of genetics, molecular biology, enzymology, chemistry, and spectroscopy. This allowed the step-by-step synthetic pathway of B12 to be elucidated, and this approach has acted as a model for future research on the synthesis of substances in living organisms. One practical outcome of such an approach has been the improved availability of B12 for animal feedstuffs and human health.

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