Identification and Characterization of a Novel Vitamin B12 (Cobalamin) Biosynthetic Enzyme (CobZ) from Rhodobacter capsulatus, Containing Flavin, Heme, and Fe-S Cofactors

Helen M Mcgoldrick,Charles A Roessner,Evelyne Raux,Andrew D Lawrence,Kirsty J Mclean,Andrew W Munro,Stefano Santabarbara,Stephen E.J Rigby,Peter Heathcote,A Ian Scott,Martin J Warren

doi:10.1074/jbc.m411884200

Helen M Mcgoldrick, Charles A Roessner + Show 9 more

Open Access

https://doi.org/10.1074/jbc.m411884200

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Abstract

One of the most intriguing steps during cobalamin (vitamin B12) biosynthesis is the ring contraction process that leads to the extrusion of one of the integral macrocyclic carbon atoms from the tetrapyrrole-derived framework. The aerobic cobalamin pathway requires the action of a monooxygenase called CobG (precorrin-3B synthase), which generates a hydroxylactone intermediate that is subsequently ring-contracted by CobJ. However, in the photosynthetic bacterium Rhodobacter capsulatus, which harbors an aerobic-like pathway, there is no cobG in the main cobalamin biosynthetic operon although it does contain an additional uncharacterized gene called orf663. To demonstrate the involvement of Orf663 in cobalamin synthesis, the first dedicated 10 genes of the B12 pathway (including orf663), encoding enzymes for the transformation of uroporphyrinogen III into hydrogenobyrinic acid (HBA), were sequentially cloned into a plasmid to generate an artificial operon, which, when transformed into Escherichia coli, endowed the host with the ability to make HBA. Deletion of orf663 from this operon prevented HBA synthesis, demonstrating that it was essential for corrin construction. HBA synthesis was restored to this recombinant strain either by returning orf663 or by substituting it with cobG. Recombinant overproduction of Orf663, now renamed CobZ, allowed the characterization of a novel cofactor-rich protein, housing two Fe-S centers, a flavin, and a heme group, which like B12 itself is a modified tetrapyrrole. A mechanism for Orf663 (CobZ) in cobalamin biosynthesis is proposed.

Highlights

One of the most intriguing steps during cobalamin biosynthesis is the ring contraction process that leads to the extrusion of one of the integral macrocyclic carbon atoms from the tetrapyrrole-derived framework
After growing R. capsulatus under both chemoheterotrophic and phototrophic conditions we observed that cobalamin accumulated to similar levels within the cells under both sets of conditions (11 pmol per A600 unit), indicating that this organism can make cobalamin even in the presence of low concentrations of molecular oxygen
This result coupled to the observation that R. capsulatus does not contain an orthologue of cobG [9], utilized by other bacteria operating an aerobic cobalamin biosynthetic pathway [16], suggests that it may employ an alternative system to help promote ring contraction

Summary

The abbreviations used are

CobG, precorrin-3B synthase; CobZ, gene product of orf663 and an enzyme isofunctional protein to CobG, but containing flavin and heme group as well as Fe-S centers; HBA, hydrogenobyrinic acid. Presence of cobalamin pathways that appear aerobic in character, but are missing an orthologue of cobG. In this project we sought to try and overproduce the enzymes found in the main R. capsulatus cobalamin operon [9] in Escherichia coli to genetically engineer a strain with a capacity to synthesize hydrogenobyrinic acid (HBA) in vivo and thereby identify the protein required for initiating the ring contraction process

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION