Hydrogenobyrinic acid

Introduction of the cobalt atom into the corrin ring is preceeded by conversion of hydrogenobyrinic acid to the diamide (34). The resultant cobalt(II) complex (35) is reduced to the cobalt(I) complex (36) prior to adenosylation to adenosylcobyrinic acid i7,i -diamide (37). Four of the six remaining carboxyhc acids are converted to primary amides (adenosylcobyric acid) (38) and the other amidated with (R)-l-amino-2-propanol to provide adenosylcobinamide (39). Completion of the nucleotide loop involves conversion to the monophosphate followed by reaction with guanosyl triphosphate to give diphosphate (40). Reaction with a-ribazole 5 -phosphate, derived biosyntheticaHy in several steps from riboflavin, and dephosphorylation completes the synthesis. 

Naturally, the biosynthesis of cobalamins themselves require delivery of Co ions at a particular point in the reaction scheme. Cobaltochelatase catalyzes the ATP-dependent insertion of Co11 into the corrin ring during the biosynthesis of coenzyme B12 in Pseudomonas denitrifleans. Cobaltochelatase is a heterodimeric enzyme (140 KDA and 450 KDA subunits each inactive in isolation), and the two components have been isolated and purified to homogeneity.1119 The reaction product is divalent cobyrinic acid, demonstrating that hydrogenobyrinic acid and its diamide (255) are precursors of AdoCbl. 

Gene transfer from the angiosperm Catharanthus roseus, and over-expression in the bacterium Escherichia coli, yielded the synthase for strictosidine, a known alkaloid of the tryptophan-secologanin class (Scott 1992). A similar strategy has clarifted the biosynthesis of hydrogenobyrinic acid, an advanced precursor of vitamin B,2 (Scott 1994). 

Alkal. indole strictosidine (Catharanthus roseus Ang. Escherichia coli Bact. Scott 1992) corrins hydrogenobyrinic acid (Pseudomonas denitrificans Bact. E. coli. Scott 1994). 

Scheme 19. Biosynthesis of hydrogenobyrinic acid from precorrin-3A...

Two cofactors were found to be essential for the production of hydrogenobyrinic acid 60 from precorrin-3A 55, namely SAM, as would be expected, but also reduced nicotinamide adenine dinucleotide phosphate (NADPH, partial structure 59) which was surprising. Scheme 19. Omission of NADPH from the incubation gave a critically important result no 60 was formed but a new pale-yellow product appeared in its place. When a labelled form of this new pigment was incubated with the enzyme system, now with NADPH included, it was specifically converted into hydrogenobyrinic acid 60 in high yield. Clearly, a new intermediate for Bi2-biosynthesis had been found which opened the door to dramatic progress [89]. 

The structure 61 for precorrin-6A forced a complete change in the way the biosynthesis of vitamin B12 was viewed. This structure was full of surprises (a) the ring contraction step does not occur near the end of the pathway but has been completed at precorrin-6A 61 and is possibly carried out even earlier (b) oxidation has occurred prior to the formation of precorrin-6A and reduction is needed after it so the oxidation level along the pathway is not constant (c) decarboxylation of the C-12 acetate group is not an early step it occurs on the pathway beyond precorrin-6A (d) the 12a-methyl group of hydrogenobyrinic acid 60 is introduced initially at C-11 with subsequent migration to C-12. 

Enzymic catalysis of a rearrangement reaction has special mechanistic fascination and, for vitamin B12, a methyl group at C-11 of 66 shifts to C-12 of 60, Scheme 25. This step clears the blockage to movement of the double bonds so tautomerisation can now occur, with thermodynamic gain, to form the extended conjugated system of hydrogenobyrinic acid 60. The enzyme responsible for... 

It should be noted that the C-labelling in the above studies was not, unlike earlier, for NMR analysis but simply to increase the mass so that the biosynthesised hydrogenobyrinic acid would be distinguished in the mass spectrum from the huge peak due to endogenous material appearing 8 mass units lower. 

The position had now been reached where the biosynthetic route to hydrogenobyrinic acid 60, a precursor of vitamin B12, had been mapped out from ALA through several steps to uro gen III 10, before going forward along the methylation pathway to precorrin-2 12 and precorrin-3A 55. Then there was a gap to be filled before precorrin-6A 61 and subsequent intermediates. We will now... 

Scheme 25. Methyl migration from C-11 to C-12 in the formation of hydrogenobyrinic acid...

The researches reviewed so far had revealed the functions of five of the eight enzymes overproduced by the special strain of Ps. denitrificans described earlier. These five were Cobl, M, K, L and H, set here in the order in which they act on the biosynthetic pathway for hydrogenobyrinic acid 60 en route to vitamin B12 3. Three enzymes were still available, CobG, CobF and CobJ, that together with the 11-methyltransferase, CobM, carry out the conversion of precorrin-3A 55 into precorrin-6 A 61 via precorrin-4 69. So the enzymes for the two methylations at C-17 and at C-1 remained to be identified. 

All the Bi2-intermediates discovered as a result of the experiments outlined in the main part of Sect. 3 have been assembled in Scheme 30 to show the entire biosynthetic pathway from precorrin-3A 55 through to hydrogenobyrinic acid 60. By any measure, it is a remarkable sequence rich in surprises and unexpected chemistry. The early biosynthetic route from ALA 15 to precorrin-3A 55 is shown in Scheme 29, so Schemes 29 and 30 in combination show the complete story. Space only allows discussion of some of the more intriguing mechanistic aspects of Scheme 30 a fuller account is available [76]. 

Scheme 30. Complete pathway from precorrin-3A through to hydrogenobyrinic acid...

Scheme 33 shows the steps elucidated [76] for the biosynthesis of coenzyme Bi2 4 from hydrogenobyrinic acid 60 in Ps. denitrificans. This collects together the knowledge generated for this organism by the French teams of Blanche and Crouzet. A more complete review of this work and that of others is available [76]. 

The substrate for cobalt insertion was found to be the fl,c-diamide 78 generated from hydrogenobyrinic acid 60 by CobB. Remarkably, the superficially simple step of cobalt insertion requires the cooperation of three proteins, CobN, Cobs and CobT, to form a cobalt cheletase system which is ATP-dependent. The product, cob(II)yrinic acid fl,c-diamide, is reduced to the Co(l) state by a flavin dependent enzyme (gene as yet unknown), which was isolated as pure protein. The highly nucleophilic Co(I)-complex was thus ready for adenosylation at... 

Scheme 33. Complete pathway from hydrogenobyrinic acid to coenzyme Bi...

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