Biosynthesis of vitamin B ₁₂ : Factor IV, a new intermediate in the anaerobic pathway

Abstract

The structure of a novel tetradehydrocorrin, factor IV, isolated from Propionibacterium shermanii has been established by multidimensional NMR spectroscopy. Incorporation of radiolabeled factor IV into cobyrinic acid established the biointermediacy of this cobalt complex, whose structure has implications for the mechanisms of the anaerobic pathway to B 12 .In the aerobic bacterium Pseudomonas denitrificans, the biosynthetic pathway to vitamin B 12 , featuring an oxygenrequiring step, has been established (for reviews, see refs. 1 and 2), but it is now clear that a second, anaerobic route to corrin has existed in nature for ca. 4 billion years (2, 3). Thus, the obligate anaerobes, such as methanogenic bacteria, and the semianaerobic Propionibacterium shermanii are able to synthesize B 12 by processes that differ at several pivotal steps from the aerobic pathway. The Ps. denitrificans route uses a twostage ring contraction sequence on the metal-free substrate precorrin-3, using O 2 (4) to fashion ␥-lactone and tertiary hydroxyl functions at C-1 and C-20 in precorrin-3x (Scheme I) as a prelude to a pinacol-like rearrangement, whereas cobalt is inserted late, i.e., not until all S-adenosylmethionine (AdoMet)-derived methyl groups are in place and amidation of the carboxylates has begun (1, 2).In contrast, it has been found (5) that in Pr. shermanii cobalt is inserted early into precorrin-3, which is ring-contracted without O 2 as cofactor (Scheme I), and at a later, unknown stage, a unique exchange of carbonyl oxygen at the C-27 (ring A) acetate occurs (6, 7); this is another event that is not paralleled in aerobic metabolism (8). In this report, we describe the isolation of a new B 12 intermediate from Pr. shermanii whose surprising structure provides circumstantial but compelling evidence for those mechanisms operating during anaerobic biosynthesis that mediate ring contraction and loss of the 2-carbon fragment from the western side. MATERIALS AND METHODSPreparation of Factor IV from epi-Factor II. A cell-free extract of Pr. shermanii (from 50 g of wet cells) was prepared by sonication (50 min at 0-4ЊC) in phosphate buffer (pH 7.6; 50 ml). Glutathione (5 mg), ATP (22 mg), NAD (5 mg), NADH (10 mg), AdoMet (75 mol), EDTA (3.5 mol), Co 2ϩ (4 mol), and Co 3ϩ (1.8 mol) were added to the supernatant of the centrifuged suspension (17,000 ϫ g; 25 min at OЊC). For preparative isolation, 3-epi; 8-epi factor II ( Fig. 1; 500 mol) was added, and the incubation was carried out at 31ЊC for 16 hr. Sodium cyanide (5 mg) was then added, and the tetrapyrrolic mixture was adsorbed on DEAE-Sephadex A-25 (Pharmacia) followed by extraction and esterification by MeOH͞

Characterization of the Enzyme CbiH60 Involved in Anaerobic Ring Contraction of the Cobalamin (Vitamin B12) Biosynthetic Pathway

“…This is consistent with the enzyme CbiH 60 catalyzing both the C17 methylation and the ring contraction event. The carbon assignments (supplemental Table S3) are in close agreement with the previously published spectra of cobalt factor IV heptamethyl ester (5). Together with the EPR analysis, these data clearly demonstrate that CbiH 60 is able to convert cobalt(II) factor III into cobalt(II)-precorrin-4 in the presence of SAM and DTT.…”

“…The product of the reaction is thought to be cobalt factor IV. However, this reaction is also known to proceed with cobalt-precorrin 3 as a substrate, with cobalt-precorrin 4 as the product (5,15). The low yield of the overall reaction, typically below 5%, has meant that the reaction has remained poorly characterized and has not been observed with a purified enzyme system.…”

“…The low yield of the overall reaction, typically below 5%, has meant that the reaction has remained poorly characterized and has not been observed with a purified enzyme system. It has been suggested that the central cobalt ion of the intermediate may play a role in the catalytic mechanism underpinning ring contraction (5). The poor yield of either cobalt-precorrin-4 or cobalt factor IV has also meant that the remaining steps in the anaerobic synthesis of the corrin ring have not been fully elucidated.…”

“…The earlier intermediates of the cobalamin pathway are given the term precorrin-n, where n refers to the number of methyl groups that have been added to the developing corrin frame. Along the early steps of the anaerobic pathway, however, some of the intermediates were found to be more oxidized and are termed factors (5,(7)(8)(9), where, for instance, factor II would represent the oxidized version of precorrin-2. Overall, the two pathways for cobalamin synthesis are quite similar because the sequences in which the modifications that take place during corrin-ring synthesis, such as the addition of the peripheral methyl groups and amidations, are identical (6).…”

“…Like all other modified tetrapyrroles, cobalamin is derived from a common precursor, uroporphyrinogen III, which is synthesized from 5-aminolevulinic acid in three highly conserved steps (3). Cobalamin differs from other modified tetrapyrroles in that its macrocycle is reduced in size because of the loss of the methylene bridge between pyrrole rings A and D (4,5). This ring contraction is necessary to ensure the tight coordination of the central cobalt ion, which is found at the epicenter of the corrin structure.…”