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

Abstract

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...

Precorrin-6x reductase from Pseudomonas denitrificans: purification and characterization of the enzyme and identification of the structural gene

“…When NADPH is omitted from the complete cell-free system, hydrogenobyrinic acid is no longer produced and precorrin-6x accumulates instead. This observation has been considered as the first evidence that the pathway to corrins most likely involves an NADPH-dependent reduction step with precorrin-6x as the substrate (22).…”

“…RSF1010 derivatives were introduced into SC510 Rif' through conjugation with pRK2073 as a helper vector as previously described (2,6). P. denitrificans strains were grown in 250-mi Erlenmeyer flasks containing 25 ml of PS4 medium (6) (20,22). After chromatography through a column of Sephadex G-25 in buffer A, the protein fraction (520 mg of protein) was incubated anaerobically at 30°C for 3 h with [1,11,17-methyl-"'C]precorrin-6x (630 nmol, 4.0 nCi nmol-1) (compound 2b in Fig.…”

“…1), the cobalt-free analog of cobyrinic acid, was recently isolated from Pseudomonas denitrificans (5) and was shown to be constructed from precorrin-3 by the same methylation sequence as cobyrinic acid (5). More recently, a previously undescribed biosynthetic precursor of hydrogenobyrinic acid, named precorrin-6x, was isolated from P. denitrificans (22), and the complete structure of its methylester derivative was established (compound 2c in Fig. 1) (19).…”

“…The structure of precorrin-6x implies that upon conversion to hydrogenobyrinic acid, this intermediate undergoes (i) reduction to the oxidation level of a corrin, (ii) S-adenosyl-L-methionine (SAM)-dependent methylation at C-5 and C-15, (iii) decarboxylation of the acetic acid residue at C-12, and (iv) NADPH, cell-free protein extracts from P. denitrificans normally produce hydrogenobyrinic acid from trimethylisobacteriochlorin (compound 4 in Fig. 1) (5,22). The aromatic trimethylisobacteriochlorin is first reduced by the enzymatic incubation mixture to the dihydro form, precorrin-3, the true intermediate of the pathway (compound 1 in Fig.…”

Biosynthesis of vitamin B12 in Pseudomonas denitrificans: the biosynthetic sequence from precorrin-6y to precorrin-8x is catalyzed by the cobL gene product

“…Previous studies on a recombinant strain of Pseudomonas denitrificans have led to the isolation (12) and structure elucidation (9) of precorrin-6x (see structures in Fig. 1), a precursor of the corrin hydrogenobyrinic acid (3,6).…”