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.

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.

Synthesis of substrate analogs of methyltransferases in the vitamin B(12) biosynthetic pathway and characterization of their enzymatic products.

The specificity toward substrate analogs of the first two methyltransferases in the vitamin B(12) biosynthetic pathway was probed with 15 synthetic porphyrinogens. Several novel methylated chlorins and isobacteriochlorins were isolated and characterized, suggesting the same methylation sequence C-2>C-7>C-20 as for the natural substrate, uro'gen III. The results allow us to narrow down possible structural requirements concerning substrate recognition by the methyltransferase enzymes.

Structural characterization of novel cobalt corrinoids synthesized by enzymes of the vitamin B12 anaerobic pathway.

Investigation on the use of the oxidized form (factor 3 (3a)) of the trimethylated intermediate (precorrin 3 (2)) as a substrate for the enzymes of the anaerobic pathway to vitamin B12 led to the synthesis of three pairs of novel cobalt corrinoids. The products were made with the aid of the Salmonella typhimurium enzymes CbiH, CbiF, CbiG, and CbiT, were synthesized in several 13C labeled versions, and were isolated as methylesters after esterification. Structures were determined by detailed NMR and MS analyses. Each set of products was obtained in the decarboxylated (RMe) and non-decarboxylated (R=CH2COOCH3) forms (at the C-12 position of the porphyrinoid).

Identification and functional analysis of enzymes required for precorrin-2 dehydrogenation and metal ion insertion in the biosynthesis of sirohaem and cobalamin in Bacillus megaterium.

In Bacillus megaterium, the hemAXBCDL genes were isolated and were found to be highly similar to the genes from Bacillus subtilis that are required for the conversion of glutamyl-tRNA into uroporphyrinogen III. Overproduction and purification of HemC (porphobilinogen deaminase) and -D (uroporphyrinogen III synthase) allowed these enzymes to be used for the in vitro synthesis of uroporphyrinogen III from porphobilinogen. A second smaller cluster of three genes (termed sirABC) was also isolated and found to encode the enzymes that catalyse the transformation of uroporphyrinogen III into sirohaem on the basis of their ability to complement a defined Escherichia coli (cysG) mutant. The functions of SirC and -B were investigated by direct enzyme assay, where SirC was found to act as a precorrin-2 dehydrogenase, generating sirohydrochlorin, and SirB was found to act as a ferrochelatase responsible for the final step in sirohaem synthesis. CbiX, a protein found encoded within the main B. megaterium cobalamin biosynthetic operon, shares a high degree of similarity with SirB and acts as the cobaltochelatase associated with cobalamin biosynthesis by inserting cobalt into sirohydrochlorin. CbiX contains an unusual histidine-rich region in the C-terminal portion of the protein, which was not found to be essential in the chelation process. Sequence alignments suggest that SirB and CbiX share a similar active site to the cobaltochelatase, CbiK, from Salmonella enterica.