Biosynthetic studies on vitamin B₁₂

Abstract

The role of folates in the enzymatic formation of the porphyrin macrocycles was investigated. Uroporphyrinogen III cosynthetase-free hydroxymethylbilane synthase from Rhodopseudomonas spheroides was incubated with the substrate PBG. In contrast to earlier findings with enzyme from rat liver, the presence of tetrahydrofolic acid, 5-methyl-tetrahydrofolic acid or N⁵,N¹⁰-methenyl-tetrahydrofolic acid showed no effect on the ratio of uro'gen III/uro'gen I produced. Several precursors of cobyrinic acid including ALA, PBG, uro'gen III and reduced factor III were used in cobalt-free feeding experiments with whole cells and cell-free preparations from Propionibacterium shermanii. In the absence of cobalt, no intermediates beyond factor III could be isolated. Sufficient factor II and factor III octamethylesters were isolated to allow acquisition of their natural abundance ¹³C NMR spectra and tentative assignments are presented. Reduction of these compounds afforded the corresponding dipyrrocorphins which were characterized both by ¹H and ¹³C NMR. The synthesis of [3-¹³C]ALA is described. High-field ¹³C NMR analyses of three isotopomers (derived from [3-¹³C]ALA, [4-¹³C]ALA and [5-¹³C]ALA) of two new tetramethylated tetrapyrroles permitted their structural characterization. Both compounds are derived from the symmetric uro'gen I and represent the first case of natural methylations on the type I isomer. Factor S₁ is methylated at the four acetate termini and Factor S₃ is methylated at three acetate termini and one α-pyrrolic position. Pulse labeling experiments with whole cells and cell-free preparations from P. shermanii using [¹³CH₃]methionine and [¹³CH₃]S-adenosylmethionine led to the definition of the sequence of methylations beyond the known trimethylated isobacteriochlorin, factor III. The fourth methylation site is C-17, followed by C-12, C-1 and finally C-15/C-5. ¹³C NMR analyses of the incubation products of [4-¹³C]ALA and [¹³CH₃]methionine with P. shermanii showed no formation of C-17 or C-1 methylated species, suggesting that cobalt may be inserted prior the formation of the C-17 methylated pyrrocorphin. A mechanistic scheme based on these results is proposed. [4-¹³C]ALA and [3-¹³C]ALA were fed to P. shermanii in a medium containing 50% D₂O. Analyses of the ¹³C NMR spectra of the cyanocobalamin specimens indicated that both C-18 and C-19 were deuterated. This finding ruled out the intermediacy of 18,19 unsaturated corrins setting further constraints on the possible mechanisms of the last steps of vitamin B₁₂ biosynthesis.