Biosynthesis of vitamin B12: identity of fragment extruded during ring contraction to the corrin macrocycle.

Abstract

Incorporation experiments with labeled sirohydrochlorin and trimethylisobacteriochlorin demonstrate that ring contraction in vivo to the corrin macrocycle of vitamin B12 liberates acetic acid. The C-20 atom of the precursors becomes the acetate carboxyl carbon.The trimethylisobacteriochlorin isolated (1, 2) from the vitamin B12 producer Propionibacterium shermanii was proved (3,4) to have structure 11 in which, surprisingly, the third methyl group to be inserted is located at C-20 (Fig. 1). The surprise stems from the fact that the carbon atom at C-20 of structure 11 has to be extruded at some stage during the demonstrated conversion of the trimethyl system (structure 11)* into cobyrinic acid (structure 13) by using cell-free enzyme preparations from P. shermanii (ref. 7 and refs. therein) or Clostridium tetanomorphum (1). Double-labeling experiments further proved (4, 7) that the C-20 methyl group is also lost during the conversion of 11 to 13 and, hence, study of the nature of the extruded fragment is of considerable interest; the necessary experiments are outlined here.There are, in principle, several approaches to this problem based on different known precursors of cobyrinic acid (structure 13). [20-14C, 2,7-methyl-3H]Sirohydrochlorin (structure 8), [20-14C, 2,7-methyl-'4C]sirohydrochlorin (structure 9), and [2,7,20-methyl-14C]trimethylisobacteriochlorin (structure 12) were selected to avoid formation of formaldehyde, which can arise chemically from uroporphyrinogen-III (structure 2). Incubation of the former precursor (structure 8) with a cobaltcontaining cell-free enzyme system (8) from P. shermanii (Table 1, Exps. 1 and 2) yielded first the trimethylisobacteriochlorin (structure 10) in dihydro form,* followed by further biological transformation into cobyrinic acid (structure 13). From the incubation mixtures were isolated cobyrinic acid (structure 13) as its ester (structure 14), acetic and formic acids as their p-bromophenacyl esters, and acetaldehyde and formaldehyde as their dimedone derivatives. In each case, radioinactive carrier material was added to assist the isolation. The results in Table 1 (Exps. 1 and 2) demonstrate reproducible incorporation of sirohydrochlorin (structure 8)* into cobyrinic acid (structure 13) with formation of labeled acetic acid. The isolated formic acid (Exp. 2) and formaldehyde and acetaldehyde (Exps. 1 and 2) carried negligible activity (<2 X 10-3 equiv.), and this trace also appeared in the corresponding materials from the blank run (Exp. 3). This parallel blank also showed clearly that the production of acetic acid in Exps. 1 and 2 is an enzymic process.These results were confirmed with triply 14C-labeled sirohydrochlorin (structure 9; Exp. 4), in which a remarkably high conversion of sirohydrochlorin (structure 9)* into cobyrinic acid (structure 13) was achieved. The acetic acid isolated from Exps.

Algae Need Their Vitamins

“…There are essentially two alternative routes, comprising up to 20 enzymatic steps from the tetrapyrrole primogenitor uroporphyrinogen III (66). The first to be characterized was the so-called late-insertion pathway (4,63), which has an absolute requirement for molecular oxygen (58) and in which the cobalt ion is inserted into the tetrapyrrole macrocycle after ring contraction. The second route is called the early-insertion pathway (54), where the cobalt ion is chelated before ring contraction and which can operate under anaerobic conditions.…”

Biosynthesis of vitamin B12: isolation of precorrin-6x, a metal-free precursor of the corrin macrocycle retaining five S-adenosylmethionine-derived peripheral methyl groups.

“…Pulse-labeling experiments (3) revealed that the fourth methyl is introduced at C-17 and extension of this approach showed that C-12 is methylated next followed by C-1 with C-5 and C-15 last (4-7). Finally, the ring contraction steps leading to the corrin macrocycle were found to involve extrusion of C-20 and its attached methyl group, with these appearing, respectively, as the carboxyl and methyl groups of acetic acid (8,9).…”

Biosynthesis of the corrin macrocycle of coenzyme B12 in Pseudomonas denitrificans

“…The entire ring contraction process seems to be initiated by the methylation at C-17. It leads to precorrin-4 with an acetyl group at C-1 (42) and not at C-19 as previously assumed (2,20,27). Precorrin-4, which has been isolated in its oxidized form only, must have the structure given in Fig.…”

“…After precorrin-2, the last precursor common to siroheme, coenzyme F430, heme dl, and coenzyme B12 (21,26) ( Fig. 1), the pathway specifically dedicated to corrinoids is initiated by the introduction of the C-20 methyl group which is expelled later along with the C-20 bridge carbon in the form of acetic acid, as a result of the ring contraction process (2,27). The C-20 methylation reaction is catalyzed by CobI and yields precorrin-3A (39,46).…”