Genetically Engineered Synthesis and Structural Characterization of Cobalt−Precorrin 5A and −5B, Two New Intermediates on the Anaerobic Pathway to Vitamin B₁₂:  Definition of the Roles of the CbiF and CbiG Enzymes

Abstract

Two new cobalt corrinoid intermediates, cobalt-precorrin 5A and cobalt-precorrin 5B, have been synthesized with the aid of overexpressed enzymes of the vitamin B(12) pathway of Salmonella entericaserovar typhimurium. These compounds were made in several regioselectively (13)C-labeled forms, and their structures have been established by multidimensional NMR spectroscopy. The addition of CbiF to the enzymes known to synthesize cobalt-precorrin 4 resulted in the formation of cobalt-precorrin 5A, and the inclusion of CbiG with CbiF produced cobalt-precorrin 5B, which has allowed us to define the role of these enzymes in the anaerobic biosynthetic pathway. CbiF is the C-11 methylase, and CbiG, an enzyme which shows homology with CobE of the aerobic pathway, is the gene product responsible for the opening of the ring A delta-lactone and extrusion of the "C(2)" unit. The discovery of these long-sought intermediates paves the way for defining the final stages of the anaerobic pathway. It is of considerable evolutionary interest that nature uses two distinct pathways to vitamin B(12), both conserved over several billion years and featuring completely different mechanisms for ring-contraction of the porphyrinoid to the corrinoid ring system. Thus the aerobic pathway utilizes molecular oxygen to trigger the events at C-20 leading to contraction and expulsion of the "C(2)" unit as acetic acid from a metal-free intermediate, whereas the anaerobic route features internal delivery of oxygen from a carboxylic acid terminus to C-20 followed by extrusion of the "C(2)" unit as acetaldehyde, using cobalt complexes as substrates.

Elucidation of the anaerobic pathway for the corrin component of cobalamin (vitamin B 12 )

“…The starting point was the production of both cobalt-precorrin-5A and -5B from cobalt-precorrin-4. Although these syntheses validated the activities of CbiF and CbiG, the yields were low (10-20%) as previously noted (15). In contrast, the direct synthesis of cobalt-precorrin-5B from cobalt-factor III with an enzyme mixture incubation results in yields greater than 80%.…”

“…Cobalt-precorrin-4 can be separated from this species on a DEAE-Sephadex column. The UV-visible spectrum of this product has a broad absorbance peak at 340 nm and a minor peak at 440 nm ( (16), generating the intermediate cobalt-precorrin-5B (15). This reaction is a prerequisite to the C-1 methylation step (8).…”

“…It has also been shown that cobalt-precorrin-4 can be converted into cobaltprecorrin-5A by Escherichia coli extracts that contain overproduced Salmonella enterica CbiF in the presence of SAM in a yield of ∼5-10% (15). Similarly, cobalt-precorrin-4 can be transformed into cobalt-precorrin-5B by incubation with cell lysates containing CbiF and CbiG, indicating that CbiG catalyses the opening of the δ-lactone ring and the subsequent deacylation (15) to release the C2 fragment as acetalydehyde (16). However, the poor conversion rates of these reactions have prevented a detailed study of the mechanisms underpinning these transformations, and all of the intermediates have been characterized only as their derivatized methylester form rather than as free acids.…”

Fine-Tuning Our Knowledge of the Anaerobic Route to Cobalamin (Vitamin B 12 )

“…However, the mismethylation activity of CbiF and the extraordinary sensitivity of the CbiF methylation products to oxidation (14) greatly complicated the isolation of intermediates subsequent to cobalt-precorrin-4. Kajiwara et al (5) were able to synthesize, purify, and characterize cobaltprecorrin-5A ( Fig. 1), the product resulting from the CbiFcatalyzed methylation of cobalt-precorrin-4 at C-11, by the careful elimination of oxygen not only from the in vitro incubations containing cobalt, precorrin-3, CbiH, and CbiF but also during the isolation procedures.…”

“…Three recent papers (by Roessner et al [12], Kajiwara et al [5], and Santander et al [14]) report the use of a combination of genetic engineering, enzymology, bio-organic chemistry, and high-resolution NMR spectroscopy to help fine-tune what is known of the anaerobic pathway to cobalamin. They present new experiments that firmly establish the structures of two intermediates of the pathway and confirm the functions of 5 more of the 13 cbi gene-encoded enzymes that contribute to the conversion of precorrin-2 to adenosyl-cobinamide ( Fig.…”