Native Corrinoids from Clostridium cochlearium Are Adeninylcobamides: Spectroscopic Analysis and Identification of Pseudovitamin B₁₂ and Factor A

Abstract

The corrinoids from the obligate anaerobe Clostridium cochlearium were extracted as a mixture of Co_β-cyano derivatives. From 50 g of frozen cells, approximately 2 mg (1.5 μmol) of B₁₂ derivatives was obtained as a crystalline sample. Analysis of the corrinoid sample of C. cochlearium by a combination of high-pressure liquid chromatography and UV-Vis absorbance spectroscopy revealed the presence of three cyano corrinoids in a ratio of about 3:1:1. The spectroscopic data acquired for the sample indicated the main components to be pseudovitamin B₁₂ (Co_β-cyano-7"-adeninylcobamide) (60%) and factor A (Co_β-cyano-7"-[2-methyl]adeninylcobamide) (20%). Authentic pseudovitamin B₁₂ was prepared by guided biosynthesis from cobinamide and adenine. Both pseudovitamin B₁₂ and its homologue, factor A, were subjected to complete spectroscopic analysis by UV-Vis, circular dichroism, mass spectrometry, and by one- and two-dimensional ¹H, ¹³C-, and ¹⁵N nuclear magnetic resonance (NMR) spectroscopy. The third component was indicated by the mass spectra to be an isomer of factor A and is likely (according to NMR) to be 7"-[N⁶-methyl]-adeninylcobamide, a previously unknown corrinoid. C. cochlearium thus biosynthesizes as its native “complete” B₁₂ cofactors the 7"-adeninylcobamides and two homologous corrinoids, in which the nucleotide base is a methylated adenine.

Besides vitamin B₁₂ (cyanocobalamin) itself, a spectrum of corrinoids is provided in nature which differ from vitamin B₁₂ by their cobalt-coordinating axial ligands (29). While the corrin moiety of the naturally occurring corrinoids is completely conserved structurally, a rationalized feature of (the catalytic moieties of) essential cofactors (24), the “complete” B₁₂ derivatives contain a remarkable variety of aromatic nucleotide functions: benzimidazoles (such as 5-methylbenzimidazole, 5,6-dimethylbenzimidazole [DMB], 5-hydroxybenzimidazole, and 5-methoxybenzimidazole), purine bases (such as adenine or 2-methyladenine) (see Fig. 1) or phenols (phenol and p-cresol) (29, 43, 61, 67). In contrast to the situation in the other widespread adenine-containing dinucleotide cofactors, in pseudovitamin B₁₂ (Co_β-cyano-7"-adeninylcobamide) and in factor A (Co_β-cyano-7"-[2-methyl]adeninylcobamide; Fig. 1), the ribose function is part of an unusual α-nucleotide moiety and binds to N7 of the purine ring. This latter property has been suggested by D. C. Hodgkin to be required in adeninylcobamides in order to enable the purine base to coordinate intramolecularly to the corrin-bound cobalt center via its N9 (30, 31, 41).

FIG. 1.

Structural formulae of vitamin B₁₂ derivatives. The general formulae are given on the left; variation of the organic ligand R (Me, methyl; Ado, 5′-deoxy-5′-adenosyl) and/or of the nucleotide base give rise to the different cobamides. (Top) Base-on cobamides vitamin B₁₂ (cyanocobalamin), methylcobalamin, coenzyme B₁₂ (5′-deoxy-5′-adenosylcobalamin), pseudovitamin B₁₂ (Co_β-cyano-7"-adeninylcobamide), Co_β-methyl-7"-adeninylcobamide, factor A (Co_β-cyano-7"-[2-methyl]adeninylcobamide), Co_β-methyl-7"-[2-methyl]adeninylcobamide, and Co_β-cyano-7"-[N⁶-methyl]adeninylcobamide. (Bottom) Base-off cobamides pseudocoenzyme B₁₂ (5′-deoxy-5′-adenosyl-7"-adeninylcobamide) and adenosyl factor A (5′-deoxy-5′-adenosyl-7"-[2-methyl]-adeninylcobamide).

Three years after the discovery of vitamin B₁₂ (62, 64), pseudovitamin B₁₂ was isolated from an incompletely identified microorganism that was obtained from rumen contents (20). In 1958 the coenzyme form of pseudovitamin B₁₂ (i.e., pseudocoenzyme B₁₂) was discovered and identified by UV and visible (UV-Vis) spectroscopy to be the native cofactor of Clostridium tetanomorphum (2). Factor A was the main component of a mixture of corrinoids which were isolated in a crystalline form from pig manure, bovine gut contents, and feces (25, 26, 29). Pseudovitamin B₁₂ and factor A often occur in anaerobically fermenting systems, such as ruminal (7) and intestinal (12) contents, feces, and sewage sludge (29). The crystal structure of factor A was determined in 1981 and confirmed the presence of an α-nucleotide and binding of N9 of the purine ring to the corrin-bound cobalt center (41). The different nucleotide bases in factor A and in vitamin B₁₂ lead only to small differences in the axial bond lengths, in bond angles, and in the gross three-dimensional (3D) structures of vitamin B₁₂ and factor A. The axial bond lengths of factor A [(Co—C) bond length = 1.86 Å and (Co—N) bond length = 2.12 Å] (41) differ only marginally from those of vitamin B₁₂ [(Co—C) bond length = 1.86 Å and (Co—N) bond length = 2.01 Å] (45). The longer (Co—N) bond in factor A clearly is the consequence of a weaker coordinating adenine heterocycle compared to the more nucleophilic DMB base of vitamin B₁₂.

Pseudovitamin B₁₂ and factor A have been found specifically in a variety of microorganisms, e.g., in Clostridium (2, 28), Propionibacterium (29), and methanogenic (pseudovitamin B₁₂) bacteria (66). Remarkably, Salmonella enterica serovar Typhimurium synthesizes cobalamins under microaerophilic conditions, while pseudovitamin B₁₂ and factor A are found in serovar Typhimurium, when grown strictly anaerobically (39). Whereas aerobic microorganisms have developed a particularly efficient way to biosynthesize the DMB base of their cobalamins from flavine, the biosynthetic access to DMB is now known to be a complex task in obligate anaerobes (60).

Guided biosynthesis opens access to natural but commercially unavailable complete corrinoids and often is also applicable to the synthesis of complete cobamides, whose nucleotide base differs from the known ones, by supplementing the bacterial culture with a suitable nucleotide base precursor (see, for example, reference 29). Early on, the preparation by “guided biosynthesis” of pseudovitamin B₁₂ and factor A was explored, e.g., by means of Escherichia coli, by adding both cobinamide and adenine or 2-methyladenine to the growth medium (27). A range of related fermentation methods for the production of these purinylcobamides were developed, based on other microorganisms, most notably Propionibacterium spp. (13). In line with this, guided biosynthesis has also been employed for the preparation of nonnatural cobamides, such as naphthimidazolyl- and imidazolylcobamides (22, 45, 59, 65), using cultures of Propionibacterium freudenreichii and P. shermanii.

The nature and the role of the nucleotide base in complete corrinoids has attained renewed interest lately (43): electron spin resonance (ESR) spectroscopic work by Stupperich et al. revealed the coordination of histidine to the protein-bound phenolyl-cobamides in the homoacetogenic Sporomusa ovata (67). On the other hand, the bound corrinoid in the corrinoid–iron-sulfur protein involved in the synthesis of acetyl-coenzyme A from Clostridium thermoaceticum was indicated by spectroscopic means to be “base-off,” i.e., to have the nucleotide base of the corrinoid decoordinated in the protein (57). A pioneering structure analysis by Drennan et al. of the B₁₂-binding domain of a methionine synthase from E. coli that depends upon methylcobalamin as a cofactor (21, 51) uncovered the “base-off–His-on” form of the protein-bound complete methylcorrinoid, in which a proteinic histidine residue displaces the cobalt-coordinating DMB base of the corrinoid cofactor. The relevance of the base-off–His-on mode of binding of corrinoids in cobamide-dependent enzymes (46, 72) was supported further by two additional crystal structures, of methylmalonyl coenzyme A mutase (52) and glutamate mutase (58), which both depend upon an adenosylcobamide as a cofactor, such as coenzyme B₁₂ (5′-deoxy-5′-adenosylcobalamin). In contrast, the crystal structure of a diol dehydratase showed the bound coenzyme B₁₂ in its “classic” base-on constitution (63). Accordingly, questions concerning the effect of the nucleotide base on the binding the B₁₂ cofactors by their apoproteins (35, 53, 69) and on the mechanisms of B₁₂-dependent enzymatic reactions have received more attention (15, 43).

Ongoing studies in our laboratories concern adenosylcobamide-dependent glutamate mutase from C. cochlearium (9, 35), as well as from C. tetanomorphum (55, 69), which catalyzes the conversion of l-glutamate to (2S,3S)-3-methyl aspartate, a particularly intriguing mechanistic puzzle (9, 15). In connection with this work, the crystal structure of inactive recombinant glutamate mutase from C. cochlearium, reconstituted with vitamin B₁₂ and methylcobalamin, was determined recently (58). Experiments concerning B₁₂ binding and enzyme catalysis (6, 10, 17, 37, 54, 72) have been carried out with coenzyme B₁₂, although pseudocoenzyme B₁₂ is known to be the natural cofactor in C. tetanomorphum (2). The identity of the native cofactor(s) of the related C. cochlearium was therefore of interest.

We report here the identification of two of the three native corrinoids of C. cochlearium in their stable cyano-Co(III) form by means of a comparison of their UV-Vis, circular dichroism (CD), mass, and nuclear-magnetic-resonance (NMR) spectra with the spectra of authentic samples of pseudovitamin B₁₂ and factor A. Authentic samples of pseudovitamin B₁₂, prepared by guided biosynthesis using Propionibacterium acidi-propionici, and of factor A, were both subjected to detailed spectroscopic analysis. In particular, complete NMR signal assignment of both pseudovitamin B₁₂ and factor A was carried out to achieve the first thorough spectroscopic analysis of purine analogues of vitamin B₁₂. A third corrinoid compound was found, which was tentatively assigned the structure of the novel 7"-[N⁶-methyl]adeninylcobamide. In this way, the corrinoid sample from C. cochlearium was shown to contain three cobamides, pseudovitamin B₁₂, factor A, and an unknown isomer of factor A, in a ratio of ca. 3:1:1.

MATERIALS AND METHODS

Strains and sources of organisms.

The strains used were P. acidi-propionici DSM 20273 and C. cochlearium DSM 1285.

Reagents and solvents.

Crystalline factor A was from the collection of the late W. Friedrich and presumably originated from sewage sludge (P. Renz, personal communication). Cobinamide was prepared by degradation of an aqueous solution of crystalline cyanocobalamin (Hoffmann-LaRoche, Basel, Switzerland) with cerous hydroxide by adaptation of the method described by Renz (59). Separation of cobinamide and α-d-ribazole formed in this reaction was achieved by preparative high-pressure liquid chromatography (HPLC) on an RP-18 column. XAD adsorbent resin (Serdolit AD-4; particle size, 0.1 to 0.2 mm; research grade [Serva, Heidelberg, Germany]) was used. Analytical HPLC was done on an ODS-Hypersil column (5 μm; 250 by 4.6 mm inner diameter, with Phenomenex Security Guard column protection containing two 4-mm C-18 cartridges). The pump used was a Gynkotek model M480G with a vacuum on-line degasser, with a methanol-water (20:80 [vol/vol]) eluent and a flow rate of 1 ml/min. The injection volume was 20 μl, and the detector was a Gynkotek diode array detector UVD340 with detection at 355 nm. All chromatograms were obtained at 18°C, and data were processed by the Gynkotek HPLC data system Gynkosoft 5.50. All other chemicals were of the highest purity and were purchased from Fluka (Neu-Ulm, Germany) Merck (Darmstadt, Germany), or Aldrich (Steinheim, Germany).

Preparation of pseudovitamin B₁₂ by guided biosynthesis.

P. acidi-propionici was grown in a 100-liter fermentor which contained 1 kg of casein hydrolysate, 500 g of yeast extract, 170 g of NaH₂PO₄ · H₂O, and 180 g of K₃PO₄ · H₂O. The solution was sterilized at 120°C for 30 min. The medium was completed by adding autoclaved aqueous solutions of 750 g of glucose (1.5 liters), 60 g of MgCl₂ · 6H₂O, 860 mg of FeCl₂ · 4H₂O, and 1.7 g of CoCl₂ · 6H₂O (all 50 ml), as well as a sterile filtered solution of 400 mg of Ca-panthotenate, 30 mg of D-biotin, 1 g of cobinamide, and 10 g of adenine. The pH was adjusted to 6 to 7 by the addition of 400-ml batches of a solution containing 80 g of KHCO₃ and 20 g of NaHCO₃, and the temperature was kept at 37°C. The medium was inoculated with a 2-liter freshly grown culture of P. acidi-propionici. To prevent acidification of the batches during cell growth, the medium was further buffered with bicarbonate solution to keep the pH in the range of 6 to 7 and was supplied batchwise with aqueous glucose solution (250 g/500 ml). After 5 days of incubation, approximately 3 kg (wet weight) of cell material was harvested by centrifugation. The cells were resuspended in 5 liters of 0.1 M acetic acid (pH adjusted to 5.0 by NaOH) made 2 mM in cyanide by the addition of KCN and kept in closed half-filled, 1-liter bottles. The suspensions were incubated at 100°C for 20 min. A slightly red-colored cobamide-containing supernatant was obtained after centrifugation. The cell paste was reextracted once as described above. Portions of the pooled supernatants were run over a column of neutral aluminium oxide (3 by 10 cm) and loaded onto an XAD-4 column (3 by 10 cm; XAD grain diameter, 0.1 to 0.2 mm) to adsorb the cobamide. Prior to use, the XAD column was washed with 0.1 M KOH in methanol and then equilibrated with water. The cobamide-loaded column was rinsed with 10 bed volumes of water before eluting the cobamide with 80% methanol. The pooled methanol fractions were flash evaporated to dryness at 40°C. The residue was completely dissolved in approximately 20 ml of water. Further purification was performed by preparative HPLC (65) using an automated gradient controller and an RP-18 column (Nucleosil 120 C₁₈; 1 by 25 cm [Marchery & Nagel]). Degassed methanol and 0.1% acetic acid were mixed as solvent components A and B and applied in two different systems at a flow rate of 3 ml/min. Solvent system I (23% A–77% B) led to the elution of pseudovitamin B₁₂ after about 15 min; this was observed with a two-wavelength detector operating at 254 and 546 nm. Solvent system II (70% A–30% B, reached by a linear gradient within 10 min) was applied for approximately 30 min in order to elute the matrix. A linear program reversal reestablished the original conditions. The corrinoid-containing HPLC fractions were combined and flash evaporated to dryness. By crystallization from water-acetone, 230 mg (171 μmol) of pure pseudovitamin B₁₂ (dried at 2 Pa, 6 h) was obtained. The sample of pseudovitamin B₁₂ was subjected to UV-Vis, CD, mass, and NMR spectroscopic analysis as described below.

Isolation and purification of the corrinoids from C. cochlearium.

A culture of C. cochlearium was grown anaerobically as reported elsewhere (49). Cells were harvested aerobically and stored at −80°C. The isolation and purification of the corrinoids from C. cochlearium in the cyano form were carried out as described above for the isolation of pseudovitamin B₁₂. The amounts of solvents used in the purification procedure were adapted to the amount of C. cochlearium cell material. From 50 g of frozen cells a sample of approximately 2 mg of crystalline corrinoids was obtained (corresponding to 30 nmol of corrinoids/g [wet weight]). This sample of corrinoids from C. cochlearium was subsequently analyzed by HPLC and by UV-Vis, mass, and NMR spectroscopy. HPLC chromatograms of the sample showed three fractions: I (retention time [RT] = 22.96 min, 63% relative integral area [RIA]), II (RT = 24.35 min, 22% RIA), and III (RT = 36.10 min, 15% RIA). All three components had highly similar UV-Vis spectra (200 to 600 nm) which showed no significant difference from those of pseudovitamin B₁₂ and of factor A. By peak matching, fraction I was identified with pseudovitamin B₁₂ and fraction III was identified with factor A (see Fig. 3).

FIG. 3.

(Top) HPLC chromatogram of the corrinoid sample isolated from C. cochlearium. (Bottom) UV-Vis spectra of the new corrinoid (fraction II of the HPLC chromatogram) (see Materials and Methods for technical details).

Spectroscopic data.

UV-Vis and CD spectra were measured on Hitachi U-3000 and on Jasco-J715 instruments, respectively. Fast-atom-bombardment (FAB) MS spectra were recorded on a Finnigan MAT95 spectrometer (nitrobenzyl alcohol matrix, Cs⁺ bombardment). NMR spectra were obtained on a Varian 500 Unity Plus spectrometer equipped with field gradient facilities, a 5-mm indirect detection and 5-mm broadband direct detection probe (499.887 MHz, ¹H; 125.15 MHz, ¹³C; and 50.66 MHz, ¹⁵N). NMR solutions had a sample size of 0.7 ml at 26°C. The solvents included 90% 10 mM phosphate buffer (pH 5.2) and 10% D₂O. ¹H, ¹³C, and ¹⁵N signal assignments were from ¹H NMR spectra with water presaturation; ¹H-broad-band-decoupled ¹³C NMR spectra; ¹H,¹³C gradient-enhanced heteronuclear single-quantum coherence experiments (PFG-HSQC) (19, 38); ¹H,¹⁵N gradient-enhanced heteronuclear single-quantum coherence experiments (PFG-HSQC) (19, 38), 2D gradient-enhanced heteronuclear multiple-bond coherence experiments (PFG-HMBC) (5, 38), 2D total correlation spectroscopy (watergate-TOCSY) (3, 14, 18, 56), and 2D rotating frame Overhauser enhancement spectroscopy (watergate-ROESY) (4, 11, 56). All 2D NMR experiments were parametrized as described earlier (42). The 2D gradient-enhanced HSQC-TOCSY (PFG-HSQC-TOCSY) experiment (8, 34) was parametrized as described elsewhere (68).

Pseudovitamin B₁₂.

For UV-Vis analyses (c = 6 · 10⁻⁴ M), with λ_max expressed in nanometers and where s denotes the shoulders, the λ_max (log ɛ) values were 276(4.26), 306(3.92), 321(3.89), 343s(4.11), 360(4.44), 410(3.53), 479s(3.67), 518(3.87), and 548(3.90). For CD analyses (c = 6 · 10⁻⁴ M), where the wavelengths of the extrema λ_max and λ_min and of the zero passages λ₀ are given in nanometers, the molar decadic CD is indicated by Δɛ, and s denotes the shoulder the λ_max/λ_min(Δɛ) values were as follows: 578(1.48), 530s(−2.29), 488(−6.39), 429(14.13), 362(−11.21), 353(−7.97), 349(−8.38), 333(−4.77), 323(−6.82), 297(−0.35), 284s(−1.09), 269(−4.08), 258(−3.23), 247(−6.29), and 235(1.82). The λ₀ values were 559, 462, 381, 238, and 232. For FAB-MS analyses, the positive-ion spectra, expressed as m/z (relative percent intensity) were 1,347.8(8), 1,346.8(29), 1,345.8(76), 1,344.8(100, MH⁺), 1,343.8(9), 1,342.8(10), 1,320.8(7), 1,319.8(15), 1,318.8(27, MH⁺-CN), 1,317.8(19),and 1,316.8(23). For NMR analyses (c = 10 mM; see Fig. 4 for a 500-MHz ¹H NMR spectrum), a complete listing of assigned ¹H, ¹³C, and ¹⁵N signals is given in Tables 1 and 2. For the atom numbering of B₁₂ derivatives, see Fig. 2.

FIG. 4.

The 500-MHz ¹H NMR spectra of aqueous solutions of pseudovitamin B₁₂, factor A, and the corrinoid sample isolated from C. cochlearium are shown. Two signals, tentatively assigned to 7"-[N⁶-methyl]adeninylcobamide, are labeled with a question mark.

TABLE 1.

¹H and ¹³C NMR chemical shifts and signal assignments for pseudovitamin B₁₂, factor A, and vitamin B₁₂^a

Assignment	Pseudovitamin B12a		Factor Aa		Vitamin B12b
	δ(1H) (ppm)	δ(13C)c (ppm)	δ(1H) (ppm)	δ(13C) (ppm)	δ(1H) (ppm)	δ(13C) (ppm)
β-CN		124.2		NM
C1		87.9		88.5		87.9
C1A	0.33	22.2	0.34	22.8	0.34	22.2
C2		50.4		50.9		50.1
C21	2.27	45.9	2.27	46.2	2.29	45.6
C22		179.5		180.0		178.6
C2A	1.29	19.8	1.29	20.0	1.29	19.6
C3	3.94	59.5	3.96	60.2	4.06	59.2
C31	1.86(Hsi)	28.5	1.85(Hsi)	29.3	1.84	28.8
C31	1.98(Hre)	28.5	1.95(Hre)	29.3		28.8
C32	2.40	38.2	2.34(Hre)	38.7	2.39	37.8
C32	2.40	38.2	2.42(Hsi)	38.7	2.47	37.8
C33		182.1		182.1		180.7
C4		183.2		183.7		182.8
C5		111.7		112.4		110.3
C51	2.31	18.9	2.34	19.0	2.43	18.2
C6		167.6		167.9		168.1
C7		53.8		54.6		54.2
C71	2.08(Hre)	46.5	2.05(Hre)	47.1	2.09	45.9
C71	2.44(Hsi)	46.5	2.42(Hsi)	47.1	2.48	45.9
C72		179.4		179.5		177.9
C7A	1.66	22.1	1.70	22.4	1.76	21.9
C8	3.25	59.5	3.24	59.9	3.31	58.5
C81	0.87(Hre)	29.1	0.95(Hre)	29.3		28.8
C81	1.78(Hsi)	29.1	1.85(Hsi)	29.3	1.91	28.8
C82	1.22(Hre)	35.1	1.22(Hre)	34.9	0.91	34.6
C82	1.72(Hsi)	35.1	1.79(Hsi)	34.9	1.74	34.6
C83		181.9		181.6		180.0
C9		176.9		177.4		176.4
C10	5.94	97.3	5.94	97.6	5.98	97.7
C11		179.3		179.5		179.7
C12		51.0		51.6		50.9
C12A	1.38	22.0	1.36	22.4	1.34	22.1
C12B	1.06	34.4	1.05	34.8	1.09	34.1
C13	3.20	57.1	3.20	57.4	3.22	56.5
C131	1.87	31.3	1.85	31.2	1.87	30.8
C132	2.54	37.8	2.53	38.4	2.52	37.5
C133		182.1		182.1		181.0
C14		169.6		170.5		168.8
C15		105.9		106.4		106.9
C151	2.44	18.1	2.44	18.1	2.46	18.0
C16		181.8		182.7		181.7
C17		62.4		63.0		62.0
C171	1.68(Hsi)	36.0	1.69(Hsi)	36.5	1.71	35.3
C171	2.51(Hre)	36.0	2.52(Hre)	36.5	2.40	35.3
C172	1.92(Hre)	35.7	1.94(Hre)	35.8	2.01	34.3
C172	2.37(Hsi)	35.7	2.40(Hsi)	35.8	2.55	34.3
C173		178.3		178.9		177.5
C17B	1.25	19.6	1.24	20.0	1.28	18.8
C18	2.64	42.0	2.64	42.4	2.65	41.9
C181	2.57	34.9	2.58	35.0	2.57	35.0
C181	2.62	34.9	2.62	35.0	2.64	35.0
C182		179.5		180.1		178.5
C19	3.97	78.4	3.97	78.9	4.00	77.7
C175	2.78	48.71	2.77	49.0	2.85	48.3
C175	3.48	48.71	3.50	49.0	3.50	48.3
C176	4.16	76.12	4.17	76.1	4.19	75.8
C177	1.14	22.03	1.14	22.3	1.15	21.8
C1R	6.40	92.2	6.37	93.0	6.25	89.8
C2R	4.14	73.34	4.15	74.3	4.17	71.7
C3R	4.57	75.75	4.59	76.9	4.62	75.9
C4R	3.91	85.86	3.92	85.9	3.93	84.9
C5R	3.62	63.3	3.63	63.5	3.64	63.3
C5R	3.81	63.3	3.81	63.5	3.82	63.3
O2R	5.61		5.62
C2N	8.00	156.6		166.3		135.8(DMB-C5N)
C4N		113.1		111.8		139.5(DMB-C9N)
C5N		160.7		161.5		132.8(DMB-C8N)
C6N		155.1		155.7		114.3(DMB-C7N)
C8N	7.06	147.7	7.03	147.7	7.10	144.7(DMB-C2N)
C21N			2.26	28.4	2.16	22.8(DMB-C10N)

^a1H NMR, δ with δ(H₂O)_int = 4.67 ppm; ¹³C NMR, δ with δ(TSP)_ext = 0.0 ppm. NM, not measured. 

^bAll chemical shifts are from Calafat and Marzilli (16). ¹H chemical shifts are referenced to H₂O by subtracting 0.11 ppm to the reported values (relative to TSP). 

^c31P-¹³C couplings: 1, J_CP = 4.8 Hz; 2, J_CP = 6.2 Hz; 3, J_CP no information because of signal overlap; 4, J_CP < 1 Hz; 5, J_CP = 2.8 Hz; 6, J_CP = 7.0 Hz. 

TABLE 2.

Amide group ¹H and ¹⁵N NMR chemical shifts for pseudovitamin B₁₂ and factor A

Assignment	Pseudovitamin B12a		Factor Aa [δ(1H)] (ppm)
	δ(1H) (ppm)	δ(15N) (ppm)
N23	6.96(HZ)/7.66(HE)	110.8	6.97(HZ)/7.67(HE)
N34	6.79(HZ)/7.47(HE)	104.7	6.76(HZ)/7.47(HE)
N73	6.84(HZ)/7.27(HE)	109.5	6.89(HZ)/7.27(HE)
N84	6.36(HE)/6.85(HZ)	102.6	6.33(HE)/6.81(HZ)
N134	6.87(HZ)/7.59(HE)	105.3	6.87(HZ)/7.59(HE)
N174	8.05	113.4	8.09
N183	6.92(HZ)/7.78(HE)	106.3	6.92(HZ)/7.79(HE)

^a1H NMR, δ with δ(H₂O)_int = 4.67 ppm; ¹⁵N NMR, δ with δ[NH₃(l)]_ext = 0.0 ppm. 

FIG. 2.

Atom numbering of pseudovitamin B₁₂ (and factor A) used for the description of NMR results (44). The atom numbering for the DMB of vitamin B₁₂ is also shown.

Factor A.

For UV-Vis analyses (c = 6 · 10⁻⁴ M), the λ_max(log s(4.07), 361(4.41), 408(3.50), 480s(3.62), 517(3.84), and 548(3.87). For CD analyses (c = 6 · 10⁻⁴ M), the λ_max/λ_min(Δɛ) values were 580(0.62), 541(−2.60), 533(−2.44), 492(−5.84), 430(13.18), 387s(0.38), 362(−11.66), 352s(−7.86), 334(−4.38), 324(−6.78), 313s(−4.58), 296(−1.39), 292(−1.43), 284(−0.58), 271(−2.39), 258(−0.96), 247(−3.98), and 235(3.50). The λ₀ values were 563, 461, 382, 241, and 230. For FAB-MS analyses, the m/z (relative percent intensity) values were 1,361.5(8), 1,360.6(25), 1,359.6(77), 1,358.5(100.0, MH⁺), 1,357.5 (7), 1,333.6(13), 1,332.6(28, MH⁺-CN), 1,331.6(16), and 1,330.6(16). For NMR analyses (c = 10 mM; see Fig. 4 for a 500-MHz ¹H NMR spectrum), a complete listing of assigned ¹H, ¹³C, and ¹⁵N signals is given in Tables 1 and 2. For the atom numbering of B₁₂ derivatives, see Fig. 2.

Sample of cyano corrinoids from C. cochlearium.

For HPLC analyses, the three fractions were as follows: I, RT = 23 min (65%); II, RT = 24.4 min (20%); III, RT = 36.1 min (15%). For UV/Vis analyses, the λ_max values were 276, 305s, 321, 360, 410, 517, and 547. For CD analyses, the values were 575, 429, 386s, and 236, the λ_min values were 542, 534, 492, 364, 351, 347, 333, 323, 311s, 296, 290, 284, 268, 261, and 248, and the λ₀ values were 563, 459, 384, 238, and 233. For FAB-MS analyses, 1,361.8(8), 1,360.8(22), 1,359.8(50), 1,358.8(65.5), 1,357.8(13), 1,356.8(15), 1,347.8(9), 1,346.8(30), 1,345.8(70), 1,344.8(100), 1,343.8(11), 1,342.8(14), 1,333.8(10), 1,332.8(16), 1,331.8(15), 1,330.8(19), 1,320.8(6), 1,319.8(14), 1,318.8(23), 1,317.8(19), and 1,316.8(25). For NMR analyses (c = 1.5 mM), see Fig. 4 for a 500-MHz ¹H NMR spectrum.

RESULTS

Guided biosynthesis of pseudovitamin B₁₂.

From about 3 kg of wet cells of a 100-liter P. acidi-propionici fermentation, supplemented with approximately 100 mg of adenine/liter of medium, 10 mg of cobinamide/liter, and 17 mg of CoCl₂ · 6H₂O/liter, a sample of about 230 mg (171 μmol, 20% yield relative to cobinamide) of bright-red crystalline pseudovitamin B₁₂ was obtained and subjected to spectral analysis, as described below.

Spectroscopic analysis of pseudovitamin B₁₂ and of factor A.

Aqueous solutions of pseudovitamin B₁₂ and factor A exhibit practically identical UV-Vis and CD spectra. At wavelengths of >300 nm they are similar to the spectrum of vitamin B₁₂ (29, 33). The UV-Vis spectra of both purinylcobamides in aqueous solution display typical maxima at 548, 518, and 360 nm (α, β, and γ bands) (33) and at 276 nm (band of the coordinating nucleotide), as described earlier (see, for example, reference 29). The spectra are consistent with the structures of complete base-on corrinoids, with one cyanide ligand bound to the cobalt center.

The FAB mass spectra of the cyano corrins pseudovitamin B₁₂ and factor A displayed base peaks at m/z = 1,344.8 and 1,358.5, respectively, arising from the intact pseudomolecular ions (MH⁺). In addition prominent fragments (MH⁺-CN) due to loss of the cyanide ligand occurred at m/z = 1,318.8 for pseudovitamin B₁₂ and at m/z = 1,332.6 for factor A. The FAB mass spectra confirmed the molecular formulae C₅₉H₈₃CoN₁₇O₁₄P for pseudovitamin B₁₂ and C₆₀H₈₅CoN₁₇O₁₄P for factor A.

Very specific information on the structure of pseudovitamin B₁₂ and factor A in aqueous solutions was obtained from thorough NMR spectroscopic investigations. Complete assignment of all but three exchange labile hydroxyl and amino protons of the nucleotide moiety and of all carbons was obtained for the spectra of pseudovitamin B₁₂ and factor A. Assignment of ¹H and ¹³C signals was obtained from heteronuclear (¹H,¹³C PFG-HSQC, ¹H,¹³C PFG-HMBC, and ¹H,¹³C PFG-HSQC-TOCSY) and homonuclear correlations (watergate-TOCSY and watergate-ROESY). The ¹³C chemical shift of the axially bound cyanide group of pseudovitamin B₁₂ could be identified by its broad signal in the 1D proton decoupled ¹³C spectrum. Assignments and chemical shifts of the signals in the ¹H, ¹³C, and ¹⁵N spectra are listed in Tables 1 and 2; see Fig. 4 for the 500-MHz ¹H NMR spectra.

With the exception of resonances originating from the constitutionally different nucleotide bases, the ¹H and ¹³C chemical shifts of pseudovitamin B₁₂ and factor A differ only slightly. The largest ¹H shift difference is observed (Δδ = 0.08 ppm) for H_re(C81); the largest ¹³C shift difference is observed (Δδ = 1.2 ppm) for CR3. An unambiguous assignment (pro-R or pro-S) of the signals of all the magnetically nonequivalent methylene hydrogens of pseudovitamin B₁₂ and factor A could be derived from analysis of nuclear Overhauser effects (NOEs) involving diastereotopic acetamide and propionamide side chain methylene protons. The ¹H,¹⁵N PFG-HSQC spectra of pseudovitamin B₁₂ in combination with watergate-ROESY spectra allowed the assignment of all seven side chain amide nitrogens at natural isotopic (¹⁵N) abundance, together with their directly bonded amide protons (Table 2). With the exception of the d-side chain, for each amide group the low-field proton signal was assigned to H_E due to the ROESY cross-peaks displayed between the amide protons and side chain methylene protons, since only H_E can be close in space to the α-methylene protons of the carboxamide functions. Such an inverse assignment concerning the d-side chain amide protons has already been reported, e.g., in NMR studies on methylcobalamin (68). This assignment is compatible with a specific shielding effect of the nearby cobalt-coordinated nucleotide base affecting both amide protons and causing highfield shifts of both amide signals. Analysis of the chemical shifts of the ¹H, ¹⁵N, and ¹³C signals clearly confirmed the constitution and base-on nature of pseudovitamin B₁₂ and factor A.

Notable chemical shift differences between the spectrum of vitamin B₁₂ on one hand and those of pseudovitamin B₁₂ and factor A on the other hand could be observed for signals that were assigned to the constitutionally different nucleotide bases. In the spectra of pseudovitamin B₁₂ and factor A, additional ¹H upfield or downfield shifts with |Δδ| > 0.1 ppm with respect to the signals in the spectrum of vitamin B₁₂ are only observed for protons that experience the different anisotropic effects of the cobalt-coordinating nucleotide bases (Table 1): in the spectra of pseudovitamin B₁₂ and factor A these are the resonances of H(C3), H_re(C31), H_re(C171), H_si(C172), and H(CR1), and in the spectra of pseudovitamin B₁₂ these are the resonances also of H(C51), H(C7A), and H_si(C81). A comparison of the ¹³C NMR data for pseudovitamin B₁₂ and factor A with those for vitamin B₁₂ also show similar chemical shifts (|Δδ| < 2 ppm) of signals due to the constitutionally corresponding carbons, with the exception of the signal for C1R in the spectrum of pseudovitamin B₁₂ and of the signals for C1R and C2R in the spectrum of factor A. The observed NOE intensities were compatible with relevant interproton distances from the crystal structures of vitamin B₁₂ and factor A (41, 42). For the spectra of pseudovitamin B₁₂ and factor A a strong NOE contact was observed between H(C8N) and the methylene protons H(C131) and the weaker ones of H(C8N) to the methyl groups H(C151) and H(C1A) as well as to the methylene proton H_re(C172). Furthermore, in the ROESY spectrum of pseudovitamin B₁₂, weak contacts between H(C2N) and the methyl groups H(C51) and H(C7A), as well as to the methylene protons H_si(C31), H_re(C31), and H_re(C81), exist. Likewise, the spectrum of factor A exhibits the corresponding contacts of H(C21N) and the methyl groups H(C51) and H(C7A), as well as the methylene protons H_si(C31), H_re(C31), and H_re(C81). All of these NOE contacts indicate the nucleotide base to be cobalt coordinating and suggest a “north-south” orientation with respect to the cobalt-corrin portion, constitutional, and conformational properties of pseudovitamin B₁₂ and factor A in aqueous solution, which are also indicated for vitamin B₁₂ and factor A by their crystal structures.

Analysis of the corrinoids isolated from C. cochlearium.

The sample of crystalline cyano-corrinoids obtained from C. cochlearium was analyzed with respect to its composition by HPLC analysis and by UV-Vis, CD, mass, and NMR spectroscopy. These spectra could be examined by comparison with the spectra recorded for pseudovitamin B₁₂ and factor A, two well-characterized, authentic reference compounds.

By a combination of careful HPLC and UV-Vis spectroscopic analysis, the sample was found to contain three corrinoid components, all having UV-Vis spectral features of Co_β-cyano-7"-adeninylcobamides (29, 66). (Fig. 3). The most polar, major fraction (RT = 23 min) and the least polar fraction (RT = 36.1 min) had retention times and UV-Vis spectra identical to those of pseudovitamin B₁₂ and factor A, respectively. The fraction of intermediate polarity (RT = 23.4 min) had similar UV-Vis absorbance characteristics. The mass spectrum of the corrinoid sample from C. cochlearium was consistent with the superposition of the mass spectra of pseudovitamin B₁₂ and factor A. The spectrum of the sample from C. cochlearium exhibits a base peak at m/z = 1,344.8 and the signal at m/z = 1,358.8 with a relative intensity of 65%. These signals are consistent with the pseudomolecular ions (MH⁺) of pseudovitamin B₁₂ and factor A (or an isomer, thereof), respectively. In addition, signals at m/z = 1,318.8 and m/z = 1,332.8, due to the corresponding fragment ions (MH⁺-CN), have the same relative intensity ratio. Comparison of the signal pattern in the FAB mass spectrum with the HPLC trace suggests the sample from C. cochlearium to contain pseudovitamin B₁₂ (representing ca. 60%), factor A, and an isomer of factor A (in total, ca. 40% of the sample).

The 1D ¹H NMR spectrum of the corrinoid sample from C. cochlearium (Fig. 4) also can be deconvoluted mostly as a superposition of spectra of pseudovitamin B₁₂ and of factor A. Table 1 and Fig. 4 show that most of the ¹H (and ¹³C) NMR resonances of pseudovitamin B₁₂ and factor A superimpose completely or in part. Only some signals experience observable shift differences of >0.01 ppm, due to the constitutionally differing nucleotide bases, and are consequently suitable for securing the existence of both corrinoids in aqueous solution. These signals are the intense methyl singlets of H(C51), H(C7A), H(C12A), and H(C1A) and the resonances of H(C8N), H(CR1), H_Z(N34), H_E(N84), H_Z(N84), and H(N174) in the low field region. For all of these signals a doubling is observed, a finding consistent with an intensity ratio of approximately 3:2. In addition, the three protons of the methyl group C21N of the 2-methyladenine moiety of factor A have no counterpart in pseudovitamin B₁₂, and their signal, a singlet at 2.26 ppm, is a feature of the spectrum of factor A. In the ¹H NMR spectrum of the sample from C. cochlearium the corresponding singlet at 2.26 ppm is clearly identifiable. Due to the low total corrinoid concentration (ca. 1.5 mM) of the sample and lower signal resolution, the 2D ¹H,¹³C-PFG-HSQC spectrum and the ¹H,¹³C-PFG-HMBC spectrum of the corrinoid sample of C. cochlearium are less informative, but they are also consistent with the presence of pseudovitamin B₁₂ and of factor A. A cross-peak with the ¹H and ¹³C coordinates (2.26 and 28.4 ppm [HSQC]) and (2.26 and 166.3 ppm [HMBC]), respectively, can specifically be attributed to the methyl group H₃(C21N) of the 2-methyladenine base of factor A. Two further singlets in the ¹H NMR spectrum at chemical shifts of 8.02 and 2.81 ppm could not be explained by the spectrum of either factor A or pseudovitamin B₁₂. Based on their chemical shifts, they were tentatively assigned to H(C2N) of a purinylcobamide and to a methyl group bound to an exocyclic amino function. These tentative assignments of the signals in the ¹H NMR spectrum were supported by corresponding information from ¹H,¹³C-PFG-HSQC and ¹H,¹³C-PFG-HMBC spectra: the signal of 8.02 ppm [assigned to H(C2N)] in the proton dimension correlated with a ¹³C signal of 161.0 ppm (HBMC, assigned to C6N), and the one at 2.81 ppm corresponded with a signal at 31.5 ppm (HSQC, of H₃C-N61N). In addition, the signal at 2.81 ppm in the ¹H NMR spectrum produced an NOE to the one at 6.40 ppm, assigned to H(CR1). The data are consistent with the structure of an N⁶-methyladenine moiety bound via N7N to the ribose unit of the nucleotide function.

DISCUSSION

Authentic samples of pseudovitamin B₁₂ (29), prepared by guided biosynthesis as described here, and of factor A were subjected to an extensive spectroscopic analysis. The most detailed spectroscopic and structural information was obtained from 1D and 2D homo- and heteronuclear NMR spectra in aqueous solution (40). From experiments with B₁₂ derivatives (in aqueous medium with a mean deuterium content of 10% or less) signals for nearly all ¹H, ¹³C, and ¹⁵N atoms were detected and could be assigned (a complete listing of assigned ¹H, ¹³C, and ¹⁵N signals is given in Tables 1 and 2; see also Fig. 4 for 500 MHz ¹H NMR spectra of pseudovitamin B₁₂ and factor A). The close correspondence of the chemical shifts of most ¹H, ¹⁵N, and ¹³C NMR signals in the spectra of the purinylcobamides pseudovitamin B₁₂ and factor A on the one hand and of the DMB-cobamide vitamin B₁₂ on the other (16) supports a constitutionally and configurationally identical buildup of their cyano-Co(III)-corrin segments. The spectra also indicate attachment at the ribose moiety of the purine base of the cobalt coordinating nucleotide via its N7 in the unique α-configuration. Notable chemical shift differences can be observed only for signals that have been assigned either to the constitutionally different nucleotide bases or to protons that experience the different anisotropic effects of the cobalt-coordinating nucleotide bases. Detailed information on the conformational properties of pseudovitamin B₁₂ and factor A in aqueous solution was obtained from the ROESY data. By comparing the observed NOE intensities with the interproton distances from the crystal structures of vitamin B₁₂ (45) and of factor A (41), the base-on nature of pseudovitamin B₁₂ and factor A in aqueous solution and at pH 5.2 was confirmed, as was the north-south orientation of the cobalt-coordinating purine bases in pseudovitamin B₁₂ and factor A.

The corrinoid sample from C. cochlearium thus represents a cocrystallisate of pseudovitamin B₁₂, factor A, and a third cyanocobamide. The latter is indicated by the mass spectrum to have the same molecular mass as factor A and is tentatively assigned the structure of a Co_β-cyano-7"-[N⁶-methyl]adeninylcobamide, a previously unknown purinylcobamide. In view of the earlier isolation of pseudovitamin B₁₂ and pseudocoenzyme B₁₂ from C. tetanomorphum (2), the isolation of purinylcobamides from the related C. cochlearium is not unexpected. The corrinoids were isolated in their cyano-Co(III) forms, which are unlikely to have a direct cofactor function (23, 43). The presence of purinylcobamides in C. cochlearium (and the absence of benzimidazolylcobamides, when grown without the supplement of the corresponding base) can be explained primarily by the difficulty (or even incapacity) of this anaerobe to biosynthesize benzimidazoles. Such a situation is not untypical of anaerobes (see, for example, references 39, 60, and 66). The appearance, besides pseudovitamin B₁₂, of its homologue factor A (and of 7"-[N⁶-methyl]adeninylcobamide), is in line with frequent observations on the coexistence of purinylcobamides in anaerobes (61). While the direct sources of the adeninyl bases are not established, their origin from degradation of oligo(deoxy)nucleotides has been suggested tentatively (28, 61). The appearance of 7"-[N⁶-methyl]adeninylcobamides would lend further support to this suggestion, since derivatives of N⁶-methyladenine are ubiquitous natural products of oligonucleotides (50). Clearly, additional methyl groups increase the hydrophobicity of the nucleotide appendage. A related situation is encountered with the naturally occurring benzimidazolylcobamides, which may also differ by the degree of their biosynthetic methylation at the nucleoside base (60).

The determination of two of the cobamides in C. cochlearium as the 7"-purinylcobamides pseudovitamin B₁₂ and factor A points to the functional relevance in this anaerobe of the corresponding coenzyme forms, pseudocoenzyme B₁₂ (Co_β-5′-deoxy-5′-adenosyl-7"-adeninylcobamide) and adenosyl factor A (Co_β-5′-deoxy-5′-adenosyl-7"-[2-methyl]adeninylcobamide). These two adenosylcobamides are known to exist in their base-off constitution mainly in neutral aqueous solution, in contrast to coenzyme B₁₂ (47; W. Fieber, B. Hoffmann, H. Bothe, W. Buckel, R. Konrat, and B. Kräutler, unpublished data). It is also likely that the corresponding methyl-Co(III)-corrinoids (Co_β-methyl-7"- adeninylcobamide and Co_β-methyl-7"-[2-methyl]adeninylcobamide) have a functional role in biosynthetic methyl group transfer reactions (e.g., in some methanogens, such as in a variety of Methanococcales spp. [66]).

The different constitution of the nucleotide bases of coenzyme B₁₂ and of the purinylcobamides could be considered to be of functional relevance in view of the recently established base-off–His-on mode of binding of the corrinoid cofactor to some adenosyl-dependent mutases (55, 58) (see above). The predominance of the base-off form of the purinylcobamides might therefore be considered to be a means in support of a proper preorganization of these “complete” corrinoids and to assist their incorporation in the base-off–His-on mode into the apo form of glutamate mutase. To test this notion, preliminary comparisons were carried out with the adenosylcobamides coenzyme B₁₂, pseudocoenzymec B₁₂, and adenosyl factor A as cofactors of glutamate mutase from C. cochlearium. In contrast to the simple expectations, Michaelis-Menten studies of glutamate mutase revealed coenzyme B₁₂ to bind better than its base-off analogues pseudocoenzyme B₁₂ and adenosyl factor A and the catalytic activity of the mutase to be similar, irrespective of the adenosyl-corrinoid cofactor used (Fieber et al., unpublished). Accordingly, a direct catalytic advantage for the adenosylcobamide-dependent glutamate mutase from C. cochlearium by the use of the purinylcobamides pseudocoenzyme B₁₂ and adenosyl-factor A instead of the benzimidazolycobamide coenzyme B₁₂ can be excluded.

The purinylcobamides carry a nucleotide base that is able to undergo H bonding with H donors and acceptors and that can become protonated (47), even when coordinated to the corrin-bound cobalt center. This situation is drastically different from that in the benzimidazolylcobamides, where H bonding and protonation of the nucleotide base cannot directly occur in the cobalt-coordinated state. A mechanism for the control of the organometallic reactivity of protein-bound corrinoid cofactors involves weakening of the cobalt coordination of the axial transligand, conveyed (in part) by H bonding it to other protein residues in “regulatory” diads or triads (21, 43, 51, 52). Only in their base-off–His-on form can bound benzimidazolylcobamides be set to participate directly in such H-bonded regulatory units via a coordinated histidine. A possible functional advantage of the presence of a purinyl base in complete corrinoid cofactors in their H-bonding base-on constitution may therefore not be dismissed. Indeed, diol-dehydratase and cobamide-dependent ribonucleotide reductase are two enzymes that depend upon adenosylcobamides and which have been discovered lately to carry their corrinoid cofactors in the base-on form (1, 48, 63).

While two of the native corrinoids of the obligate anaerobes C. tetanomorphum and C. cochlearium are widely occurring purinylcobamides, C. tetanomorphum has been shown to produce benzimidazolylcobamides when supplied with benzimidazoles (70), and coenzyme B₁₂ can act as functional corrinoid cofactor of glutamate mutase in both of these clostridia (9, 37, 60). The incapacity of C. tetanomorphum and C. cochlearium to biosynthesize benzimidazoles parallels the situation encountered in several other anaerobes (29, 39, 66). The native cobamides from C. cochlearium are indicative of the biosynthesis of three relevant adeninylcobamides by this anaerobe. Two of these homologous corrinoids (pseudovitamin B₁₂ and factor A) are known from other anaerobes also (61). It will be of interest to definitively clarify the structure of the third cobamide of C. cochlearium and to examine its occurrence in other anaerobic microorganisms.