TY - JOUR
T1 - Identification and Characterization of a Novel Vitamin B12 (Cobalamin) Biosynthetic Enzyme (CobZ) from Rhodobacter capsulatus, Containing Flavin, Heme, and Fe-S Cofactors
AU - McGoldrick, Helen M.
AU - Roessner, Charles A.
AU - Raux, Evelyne
AU - Lawrence, Andrew D.
AU - McLean, Kirsty J.
AU - Munro, Andrew W.
AU - Santabarbara, Stefano
AU - Rigby, Stephen E.J.
AU - Heathcote, Peter
AU - Scott, A. Ian
AU - Warren, Martin J.
PY - 2005/1/14
Y1 - 2005/1/14
N2 - 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 uncharaeterized 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.
AB - 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 uncharaeterized 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.
KW - Biochemistry
KW - Biosynthesis
KW - Carbon
KW - Cloning
KW - Cytology
KW - Enzymes
KW - Escherichia coli
KW - Genes
KW - Photosynthesis
KW - Vitamins
UR - http://www.scopus.com/inward/record.url?scp=19944434206&partnerID=8YFLogxK
U2 - 10.1074/jbc.M411884200
DO - 10.1074/jbc.M411884200
M3 - Article
C2 - 15525640
AN - SCOPUS:19944434206
VL - 280
SP - 1086
EP - 1094
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
SN - 0021-9258
IS - 2
ER -