Identification and characterization of a novel vitamin B12 (cobalamin) biosynthetic enzyme (CobZ) from Rhodobacter capsulatus, containing flavin, heme, and Fe-S cofactors.
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.
McGoldrick
Helen M
HM
School of Biological Sciences, Queen Mary, University of London, Mile End Road, London E1 4NS, United Kingdom.
Roessner
Charles A
CA
Raux
Evelyne
E
Lawrence
Andrew D
AD
McLean
Kirsty J
KJ
Munro
Andrew W
AW
Santabarbara
Stefano
S
Rigby
Stephen E J
SE
Heathcote
Peter
P
Scott
A Ian
AI
Warren
Martin J
MJ
D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D003067:Coenzymes / Q000737:chemistry / Q000378:metabolism*; D004578:Electron Spin Resonance Spectroscopy; D004926:Escherichia coli / Q000235:genetics; D005415:Flavins / Q000378:metabolism*; D006418:Heme / Q000378:metabolism*; D007506:Iron-Sulfur Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015394:Molecular Structure; D010084:Oxidation-Reduction; D010957:Plasmids / Q000235:genetics; D011199:Potentiometry; D016354:Rhodobacter capsulatus / Q000201:enzymology* / Q000235:genetics; D017186:Titrimetry; D014805:Vitamin B 12 / Q000096:biosynthesis*
D001426:Bacterial Proteins; D003067:Coenzymes; D005415:Flavins; D007506:Iron-Sulfur Proteins; D006418:Heme; D014805:Vitamin B 12