Biosynthesis of 5,6-dimethylbenzimidazole from 6,7-dimethyl-¹⁴C-8-ribityllumazine

Biosythesis of corrins. I. Experiments with carbon-14-labeled porphobilinogen and carbon-14-labeled uroporphyrinogens

“…[14CH3]-L-Methionine (10)(11)(12)(13)(14)(15) ^Ci/mmol) was used as received (New England Nuclear, crystalline solid) or after dilution as necessary with cold material (Sigma, sigma grade).…”

“…(ii) Incubation of Uroporphyrinogen I Synthetase with [8-l4C]Porphobilinogen. Incubations were customarily conducted with 12 screw-cap test tubes, each containing 0.5 ml of 0.1 M Tris buffer (pH 8.2), 0.1 ml of 0.1 M ethylenediamine tetraacetate (EDTA, pH 8.2), glutathione (reduced form, 1 mg), 1.1 ml of distilled water, 1.0 ml of [8-14C]porphobilinogen (10) solution (ca. 0.5 mg/ml; e.g., preparation 4: 0.512 mg/ml, specific activity 602,500 dpm/µ ), and 0.3 ml of the partially purified and dialyzed uroporphyrinogen I synthetase solution (see above).…”

“…The incubations were stored in the dark at 29°, except for periodic additions of glucose and adjustments of pH under nitrogen (glove bag) (see Table IV). After 70 hr, both fermentations were harvested (cell wt, 8.7 g and 9.3 g, respectively), and the vitamin Bi2 was extracted and purified as outlined in sections C and D. Cyanocobalamin Expt 12 Expt 13 Spec act., dpm/gmol The spinach synthetase incubation was conducted for 12 hr, and the resulting uro'gen was oxidized and diluted with radioinactive, chemically synthesized uroporphyrins I-IV (total 25.3 mg, 142,500 dpm/µ ). The feeding experiment was conducted as previously described with the addition of 50% glucose solution (1 ml) and the adjustment of pH at 19.5 and 45 hr (pH 5.5-7.0 and 6.0-7.0, respectively).…”

“…dence concerned the report18 that a 14C-labeled specimen of PBG (10) was incorporated into vitamin Bn, although the location of the label in the vitamin was not determined. From the beginning of our studies, we were intrigued, as others have been by the structural similarity of uroporphyrinogen III (uro'gen III) (12) and cobyrinic acid (4) and we were attracted by the economy of a biosynthetic scheme wherein the corrin and porphyrin pathways diverge after the formation of uro'gen III.10 The fact that the latter reduced porphyrin system is an authenticated metabolite of P. shermanii taken together with work19•20 on Clostridium tetanomorphum (a B]2-producing organism) where relatively large amounts of uro'gen III were formed at the expense of copro'gen III (13) or proto'gen IX (14) (Figure 6) constitutes some indirect evidence for the intermediacy of the type III uro'gen (12). Our early simplistic notion21 for the uro'gen -* corrin conversion involving cyclopropane chemistry is illustrated in Scheme I.…”

“…In view of subsequent experimentation described in the succeeding papers of this series, it has become necessary to devise a completely new concept for corrin biosynthesis. The formation of uro'gen III (12) from PBG (10) and the further transformation of the former to corrins involve perhaps the most exciting unsolved challenges in porphyrincorrin biochemistry, but before we reexamined the many experiments and explanations that have been made for these processes, the key experiment of testing the possible intermediacy of uro'gen III for vitamin B j2 in P. shermanii became our first objective. Thus, regardless of the controversial and at times puzzling aspects of type III uroporphyrin biosynthesis in various organisms (a topic which forms the basis of a later paper of this series), clear and unambiguous proof for the intermediacy of uro'gen III constituted the first requirement in establishing a recognizable intermediate between PBG (10) and cobyrinic acid (4).…”