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The Biosynthesis of Vitamin B12: a Study by 13C Magnetic Resonance Spectroscopy

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The Biosynthesis of Vitamin B12: a Study by 13C Magnetic Resonance Spectroscopy Proc. Nat. Acad. Sci. USA Vol. 69, No. 9, pp. 2585-2588, September 1972 The Biosynthesis of Vitamin B12: A Study by 13C Magnetic Resonance Spectroscopy (corrin/cyanocobalamin/porphyrins/porphobilinogen/5-aminolevulinic acid) CHARLES ERIC BROW'N*, JOSEPH J. KATZt, AND DAVID SHEMIN* * Division of Biochemistry, Department of Chemistry, Northwestern University, Evanston, Illinois 60201; and t Chemistry Division, Argonne National Laboratory, Argonne, Illinois 60439 Contributed by David Shemin, July 11, 1972 ABSTRACT The origin of the methyl group on C-i unless a methyl group of the B12 contained "C. In this ex- of Ring A of the corrin ring of vitamin B12 was investigated the of the acetic acid formed con- by 'IC magnetic resonance spectroscopy. The proton- periment methyl group decoupled "3C spectra of vitamin B12 synthesized from tained radioactivity (6). The radioactivity was only 8-9% [5..13CJ5.aminolevulinic acid by Propionibacteria were ob- of the calculated value, and it was considered possible that tained by Fourier-transform nuclear magnetic resonance ,it was present in the methyl group at C-i. This low yield of of high resolution, and spectra of high-resolution proton radioactivity could be rationalized because the yield of acetic magnetic resonance of the 'IC-labeled B12 were also taken. The 5-carbon atom of 6-aminolevulinic acid is the source acid was low, and the contribution of the methyl group at C-i of seven or eight known positions of vitamin B12, depending to the general pool of the total acetic acid yield could not be on whether the C-i methyl group is also derived from the ascertained. On the other hand, the finding of methyl- labeled substrate. We have found seven resonances whose labeled acetic acid in this degradation could have arisen if the chemical shifts enable us to identify the position of the "3C atoms in the molecule from the assignment of Dod- labeled carbon atom of the 5-aminolevulinic acid was con- derell and Allerhand. We observed no "IC resonance verted to the methyl group of methionine in the biological corresponding to the C-i methyl group of Ring A. Further- system, and thus indirectly gave rise to labeled methyl more, the proton magnetic resonance spectrum showed groups of B12. no spin-spin splitting of the proton peak at the T values It is of considerable interest to establish the of the assigned for the H atoms in this methyl group. It would origin thus appear that the methyl group at C-i, which has methyl group at C-i, for it would permit differentiation be- been considered to have the same origin as the 6-methine tween the two possible biosynthetic pathways that may be bridge of porphyrins, does not originate from the 5- responsible for the synthesis of the corrin ring, and further- carbon atom of 5-aminolevulinic acid. This finding may more aid in the of the mechanism aid in elucidation of the mechanism of synthesis of eventually understanding the functional isomer of vitamin B12 and porphyrins. of the formation of the functional isomer of the tetracyclic The "IC-labeled vitamin B12 enabled us to make further structure of porphyrins and corrins. The corrin ring system assignments to the 'IC magnetic resonance of the vitamin. of B12 has been considered to arise directly from porpho- bilinogen or from a related porphyrinogen. The corrin moiety of vitamin B12 is structurally (1-3) and Corrin Ring biosynthetically (4-6) similar to the porphyrin ring system / although it is more highly reduced, lacks a bridge carbon 6-aminolevulinic acid ---p Porphobilinogen atom between Rings A and D, and contains an additional II seven "extra" methyl groups. These seven methyl groups Porphyrinogen include the methyl group at C-i, and six methyl groups at The purpose of this investigation is to establish whether or C-2, -5, -7, -12, -15, and -17 (one of the methyl groups at C-12 not the C-S of 5-aminolevulinic acid is the precursor of the arises by decarboxylation of the original acetic acid side methyl group at C-i of B12, as it is for the 5-bridge carbon atom chain), which Bonnet et al. (3) suggested may arise by a C- of porphyrins. Since degradative procedures for B12 are at alkylation of a tetracyclic intermediate (Fig. 1). It has been present inadequate to uncover the origin of the methyl group demonstrated that C-alkylation does indeed occur biologi- at C-i, nuclear magnetic resonance studies were initiated to cally and that at least six of the methyl groups in B12 do arise answer this particular question because this technique allows from the methyl group of methionine (5, 6). simultaneous observations on all the carbon atoms that arise It has been suggested that the methyl group at C-i does from the 'IC-labeled precursor. not arise by C-alkylation but that its origin is the same as that of the 5-methine bridge carbon atom of porphyrins (6). This MATERIALS AND METHODS suggestion arose from structural considerations and from an experiment in which B12 was biosynthesized from [5-'4C]a- Preparation of [5-13CJ5-aminolevulinic acid aminolevulinic acid. It has been demonstrated that [5..'4C]_ [2-"1CIMalonic acid, 75% enriched in "IC, was purchased 5-aminolevulinic acid used for B12 or porphyrin synthesis from Merck, Sharp, and Dohme of Canada Ltd., esterified (7), only labels carbon atoms 4,9,14, and 16 and the bridge carbon and used for the synthesis of 5-aminolevulinic acid essentially atoms; therefore, the labeled B12 degraded by a Kuhn- by the method of Shermin (8). The dimethylmalonic ester Roth oxidation cannot yield [14C~methyl-labeled acetic acid was condensed with #-carbmethoxypropionyl chloride in 258 Downloaded by guest on September 29, 2021 2586 Biochemistry: Brown et at. Proc. Nat. Acad. Sci. USA 69 (1972) and p-aminobenzoic acid and 5 mmol each of K2HPO4 and NaH2PO4. To each liter of the inoculated medium about 50 mg of the [5-13C] -aminolevulinic acid was added. Usually 8 liters of medium were used for B12 production. The cultures were grown anaerobically for 3 days at 300, then aerated for 2 days at 370 essentially by the method of Menon and Shemin (9). The pH was maintained at 6.85 i 0.05 in a pH- stat by addition of concentrated NH40H or 6 N HCl. About 50-60 g (wet weight) of cells per liter were obtained. The amount of B12 was determined by a bioassay (10) in Difco assay media. Isolation of vitamin B12 Cells were collected by centrifugation and suspended in an equal volume of water, which was acidified to pH 2.2-2.5 with 6 N H2SO4 and broken by sonication. The suspension was adjusted to pH 5 with 5 N NaOH and centrifuged. Vitamin B12 was isolated from the supernatant solution by extraction with a solvent mixture of 55% o-cresol and 45% CCl4 (v/v), and back-extracted into water by the procedure of Bray and Shemin (6). The crude B12 was purified by ion-exchange FIG. 1. Structure of Vitamin B12 (cyanocobalamin). The closed circles (0) represent the positions derived from the 8- chromatography on a Dowex-50 (Na+) pH 7 and a Dowex-2 carbon atom of p-aminolevulinic acid. (OH-) pH 9.5 column (11), and finally crystallized from an acetone-water mixture. The purity of the product was de- termined by thin-layer chromatography (12) and UV and visi- ble spectroscopy. The yield of pure crystalline B12 was about anhydrous dioxane with the aid of sodium hydride, and the 5 mg/liter. oximation reaction on the subsequent,-ketoadipic acid was accomplished with isoamyl nitrite. The identification and Spectra purity of the [5-11C]6-aminolevulinic acid was verified by The nuclear magnetic resonance (NMR) studies on 6-amino- 1HMR (magnetic resonance) spectroscopy and by thin-layer levulinic acid were performed on a Varian T-60 NMR spec- chromatography. trometer. The 13CMR spectra for the B12 were obtained on a Bruker HFX-90 MHz NMR spectrometer, adapted for Synthesis of [13CJB12 Fourier pulse operation. Proton magnetic resonance spectra Production of Vitamin B12. Propionibacterium shermanji were recorded on Varian 100 MHz (37°C) and 220 MHz (ATCC 9615) maintained in stab cultures (Difco tomato- (21°C) NMR spectrometers. juice agar) were grown anaerobically for 3 days at 30° in the The '1CMR spectra were recorded on a 12 mM solution medium described below. 25 ml of these starter cultures was of the 'IC-labeled B12 at 250 in D20. The scan covered 5000 Hz, used to inoculate 1 liter of medium containing 20 g of yeast and CS2 in a coaxial capillary was the external reference. extract, 46 g of glucose, 10 mg of Co(NO3)2. 6H20, 25 mg of 52,600 Pulses at a recycle time of 0.4 sec were collected. Nat- dimethylbenzimidazole, 50 mg of L-methionine, and 1 mg each ural abundance of '3C could not be observed on commercial of riboflavin, calcium pantothenate, biotin, niacin, thiamine, B12 under these operating conditions. C-)I(f) G. 0 2.--NC t Goa)aiCD°6 ai FIG. 2. Proton-decoupled 13CMR spectrum of "8C-labeled cyanocobalamin. The range of the spectrum is 0-206 ppm upfield from CS2. Conditions are given in text. Downloaded by guest on September 29, 2021 Proc. Nat. Acad. Sci. USA 69 (1972) A 13CMR Study of B12 Biosynthesis 2587 RESULTS and especially no resonance could be seen in the 154- The concentration of 13C in the isolated B12 was determined 162 ppm region, the range assigned by Doddrell and Allerhand by mass spectroscopy to be 5.7%.
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