Vitamin Bn, biosynthesis

Roper, J.M., Raux, E., Brindley, A.A., Schubert, H.L., Gharbia, S.E., Shah, H.N., and Warren, M.J., 2000. The enigma of cobalamin (vitamin Bn) biosynthesis in Porphyromonas gingivalis. Identification and characterization of a functional corrin pathway. The Journal of Biological Chemistry. 275 40316-40323. 

Illumination of suspensions of P. shermanii by light (2000-2500 lux) for 48-72 h sharply decreases the production of vitamin B and its corrinoid precursors, cobyrinic acid and its amides, and is accompanied by a considerable increase in porphyrin synthesis (mainly coproporphyrins III) (Yeliseev and Bykhovsky, 1990). The amidation of cobyrinic acids is an important regulatory step of the vitamin Bn biosynthesis (Yeliseev et al., 1988). When methylation is impaired, polycarboxylic corrinoids accumulate, inhibiting steps that precede the synthesis of corrinoids, including the methylation of UPB III (Bykhovsky and Zaitseva, 1989). 

Eh and pH, An important condition for the synthesis and activity of enzymes involved in vitamin Bn biosynthesis is maintaining low Eh values. Vitamin Bn synthesis de novo begins at lower Eh values than cell division (Vorobjeva, 1976). 

The beneficial effect of mycobacterial culture liquid was due to its content of amino acids, vitamins, peptides, and possibly of specific stimulators of vitamin synthesis, since the vitamin content of the cells was increased to a greater extent than their biomass. Although amino acids exert a significant effect on the synthesis of vitamin Bn, the complete culture liquid of mycobacteria was more effective, which can be explained by the complex influence of many metabolites released by mycobacteria in the exponential phase of growth. Of special interest are polypeptides secreted by mycobacteria into the medium, which may have a specific stimulatory effect on vitamin Bn biosynthesis. Chromatographic separation of mycobacterial polypeptides is shown in Fig. 4.23B. Specific stimulatory effects were displayed by fractions 10, 11 and 12. 

Normally, the strains of P. pentosaceum, P. freudenreichii and P. shermanii studied produced 10-11 mmoles of propionic acid per 100 ml, although the vitamin Bn levels in these strains varied greatly, being 56, 1650 and 1070 pg per g biomass, respectively. Inhibition of vitamin Bn biosynthesis resulted in a reduced propionic acid production, with acetic, or acetic and formic acids, becoming the main end products of fermentation. As a result, the ratio of propionic/acetic acid in the cells that produced insignificant quantities of vitamin Bn (growing in cobalt-free medium with methionine) was low, 1 4, as compared with the normal ratio of 2 1 (Vorobjeva, 1976). 

The similarity between the structures of the corrinoids and the porphyrins becomes evident from comparison of cobyrinic acid (75) (the simplest of the corronoids so far isolated) with uroporphyrinogen III (70). The possibility of a biosynthetic relationship between these structures was suggested by Shemin, who reported the incorporation of [14C]ALA into vitamin Bn and confirmed by the subsequent demonstration that PBG was also incorporated. The ubiquitous precursorial role of uroporphyrinogen III in heme, chlorophyll and corrinoid biosynthesis proposed by Porra (65BBA(107)176) was, however, not substantiated by experimental evidence until much later, when under carefully controlled conditions cells of Propionibacterium shermanii were shown to incorporate radioactivity from [14C]uroporphyrinogen III into vitamin Bn (72JA8269). 

The unmatched complex structure of the low-molecular weight natural product vitamin Bn posed a challenge to synthetic chemistry, which was met in the early 1970s by Eschemnoser and by Woodward and their coworkers. The biosynthesis of the structure of natural cobalt-corrins is no less intricate and facets of its pathways still remain to be uncovered (see section 2.3). 

Deficiency of folate or vitamin Bn can cause hematological changes similar to hereditary orotic aciduria. Folate is directly involved in thymidylic acid synthesis and indirectly involved in vitamin Bn synthesis. Orotic aciduria without the characteristic hematological abnormalities occurs in disorders of the urea cycle that lead to accumulation of carbamoyl phosphate in mitochondria (e.g., ornithine transcarbamoylase deficiency see Chapter 17). The carbamoyl phosphate exits from the mitochondria and augments cytosolic pyrimidine biosynthesis. Treatment with allopurinol or 6-azauridine also produces orotic aciduria as a result of inhibition of orotidine-5 phosphate decarboxylase by their metabolic products. 

The third stage in studying propionic acid bacteria is closely associated with the discovery of vitamin B12, since these bacteria proved to be the record producers of vitamin B12 in nature. This stage is characterized by studying conditions and pathways of the biosynthesis of corrinoids, their metabolic functions, and by starting industrial vitamin Bn production based on the vital functions of propionibacteria. The results of these investigations have convinced us that the metabolism of propionic acid bacteria is tuned to the high level of corrinoids in the cell. 

ALA, the common precursor in the biosynthesis of heme and vitamin Bn, is synthesized from L-glutamate in archaebacteria, anaerobic bacteria and... 

Early stages of the biosynthesis of tetrapyrrole compounds are inhibited by hemin and by the complete form of vitamin B12 (Lascels and Hatch, 1969 Vitamin B represses specifically its own synthesis (Bykhovsky et al., 1968, 1975a). The inhibiting effect of oxygen on vitamin Bn synthesis was attributed to the repression of ALA dehydratase and ALA synthase activities (Menon and Shemin, 1967), but now it is believed (Oh-hama et al., 1993) that in propionic acid bacteria ALA is formed in the C5 pathway. Bykhovsky... 

The effect of pH is a composite of separate effects on synthesis, on enzyme activities, and on vitamin Bn storage in the cell (below pH 5.5 the vitamin is washed out of the cell). In general, the process of biosynthesis is more sensitive to changes in pH than bacterial growth. This fact has a special... 

Biosynthesis of vitamin Bn by propionic acid bacteria, as we have seen (Section 4.4.5), almost parallels growth under anaerobic conditions. Vitamin Bn accumulates in the cell (the above-mentioned case of vitamin Bn secretion apparently is peculiar to a single mutant strain) mainly as coenzyme forms of incomplete corrinoids. These features are taken into account in the industrial production of vitamin Bn. The culture is grown anaerobically, in a medium containing glucose, corn-steep liquor, ammonium sulfate, and a cobaltous salt, since Co is part of the vitamin Bn molecule. Acids formed by the culture are continuously neutralized with a solution of NaOH or (NH4)20H. 

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