The biosynthesis of porphyrins, chlorophylls, and vitamin B12

Leeper, Finian J.

doi:10.1039/np9890600171

Cited by 95 publications

(32 citation statements)

References 80 publications

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“…The enzyme hydroxymethylbilane synthase (HMBS, EC 4.3.1.8) is involved in the early stages of the biosynthesis of the porphyrins and related macrocycles (Battersby et al, 1980;Leeper, 1985aLeeper, ,b, 1987Leeper, , 1989. It assembles four porphobilinogen (PBG) (I) units in a head-to-tail fashion, the first of these being covalently bound to the enzyme via a dipyrromethane cofactor (Jordan & Warren, 1987;Hart et al, 1987Hart et al, , 1990) (Scheme 1).…”

Section: Introductionmentioning

confidence: 99%

Studies on the mechanism of hydroxymethylbilane synthase concerning the role of arginine residues in substrate binding

Lander

Pitt

Alefounder

et al. 1991

Biochemical Journal

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The role of conserved arginine residues in hydroxymethylbilane synthase was investigated by replacing these residues in the enzyme from Escherichia coli with leucine residues by using site-directed mutagenesis. The kinetic parameters for these mutant enzymes and studies on the formation of intermediate enzyme-substrate complexes indicate that several of these arginine residues are involved in binding the carboxylate side chains of the pyrromethane cofactor and the growing oligopyrrole chain.

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Section: Introductionmentioning

confidence: 99%

Studies on the mechanism of hydroxymethylbilane synthase concerning the role of arginine residues in substrate binding

Lander

Pitt

Alefounder

et al. 1991

Biochemical Journal

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“…Earlier structural work had established that the first three methyl groups are introduced at positions C-2, C-7, and C-20 in that order (1) to form the last known partially methylated intermediate, precorrmn-3. This has been isolated, so far, as the octamethyl ester of its aromatized form (structure 2a) but it is highly probable that precorrin-3 itself has the dihydro structure 3a (2) and the dihydro form can be generated from added aromatic form in the enzymic incubation mixture.…”

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confidence: 99%

Biosynthesis of vitamin B12: isolation of precorrin-6x, a metal-free precursor of the corrin macrocycle retaining five S-adenosylmethionine-derived peripheral methyl groups.

Thibaut¹,

Debussche²,

Blanche³

1990

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

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ABSTRACT-Aminolevulinic acid and trimethylisobacteriochlorin are converted by cell-free protein preparations from Pseudomonas denitrificans into a metal-free pigment, precorrin-6x. This pigment, which accumulates when the cell-free system lacks NADPH, can be enzymically converted in high yield (>50%) into hydrogenobyrinic acid by the complete enzyme preparation. Double-labeling experiments establish that precorrin-6x carries five C-methyl groups, which appear at C-1, C-2, C-7, C-12a, and C-17 ofthe hydrogenobyrinic acid formed enzymically from precorrin-6x. This precursor of the corrin macrocycle is at the dehydrocorrin level of oxidation, has undergone ring contraction and extrusion of C-20, but still carries the acetic acid side chain at C-12. It is demonstrated that the conversion of precorrin-6x into hydrogenobyrinic acid specifically requires an NADPH-dependent reduction step.Cobyrinic acid (structure 5a), a known precursor of vitamin B12, is biosynthesized from uroporphyrinogen III (structure 1) by a multistep process including eight C-methylations (Scheme I). Earlier structural work had established that the first three methyl groups are introduced at positions C-2, C-7, and C-20 in that order (1) to form the last known partially methylated intermediate, precorrmn-3. This has been isolated, so far, as the octamethyl ester of its aromatized form (structure 2a) but it is highly probable that precorrin-3 itself has the dihydro structure 3a (2) and the dihydro form can be generated from added aromatic form in the enzymic incubation mixture. Pulse-labeling experiments (3) revealed that the fourth methyl is introduced at C-17 and extension of this approach showed that C-12 is methylated next followed by C-1 with C-5 and C-15 last (4-7). Finally, the ring contraction steps leading to the corrin macrocycle were found to involve extrusion of C-20 and its attached methyl group, with these appearing, respectively, as the carboxyl and methyl groups of acetic acid (8, 9).Hydrogenobyrinic acid (structure 4a), the cobalt-free analogue of cobyrinic acid (structure 5a), had been thought not to occur in nature (10, 11) but was recently isolated from Pseudomonas denitrificans grown in a cobalt-containing medium (12). Labeling studies (12) showed that (i) the methylation sequence from precorrmn-3 forward to hydrogenobyrinic acid is the same as that outlined above for cobyrinic acid and (ii) the methyl group at C-20 of precorrin-3 (structure 3a) is lost during its conversion into hydrogenobyrinic acid (structure 4a), presumably as acetic acid. These results clearly indicate that the cobalt-free corrinoid system is constructed by the same sequence of reactions as its cobaltcontaining analogue but without cobalt insertion for the whole pathway.We now outline experiments leading to the isolation of an additional precursor ofthe corrin macrocycle that carries five peripheral methyl groups. MATERIALS AND METHODSStarting Materials. Labeled samples of trimethylisobacteriochlorin (structures 2b and 2c) were obtained by incubation of s...

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“…1+ (13) ,P Me M4c A Scheme II C-12 acetate residue initiates the methyl migration from C-11 to C-12. This aspect and several other key features of the biosynthetic scheme can be studied experimentally.…”

mentioning

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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The biosynthesis of porphyrins, chlorophylls, and vitamin B12

Cited by 95 publications

References 80 publications

Studies on the mechanism of hydroxymethylbilane synthase concerning the role of arginine residues in substrate binding

Studies on the mechanism of hydroxymethylbilane synthase concerning the role of arginine residues in substrate binding

Biosynthesis of vitamin B12: isolation of precorrin-6x, a metal-free precursor of the corrin macrocycle retaining five S-adenosylmethionine-derived peripheral methyl groups.

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

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