The AAA(+) motor complex of subunits CobS and CobT of cobaltochelatase visualized by single particle electron microscopy.
Cobalamins belong to the tetrapyrrole family of prosthetic groups. The presence of a metal ion is a key feature of these compounds. In the oxygen-dependent (aerobic) cobalamin biosynthetic pathway, cobalt is inserted into a ring-contracted tetrapyrrole called hydrogenobyrinic acid a,c-diamide (HBAD) by a cobaltochelatase that is constituted by three subunits, CobN, CobS and CobT, with molecular masses of 137, 37 and 71kDa, respectively. Based on the similarities with magnesium chelatase, cobaltochelatase has been suggested to belong to the AAA(+) superfamily of proteins. In this paper we present the cloning of the Brucella melitensis cobN, cobS and cobT, the purification of the encoded protein products, and a single-particle reconstruction of the macromolecular assembly formed between CobS and CobT from negatively stained electron microscopy images of the complex. The results show for the first time that subunits CobS and CobT form a chaperone-like complex, characteristic for the AAA(+) class of proteins. The molecules are arranged in a two-tiered ring structure with the six subunits in each ring organized as a trimer of dimers. The similarity between this structure and that of magnesium chelatase, as well as analysis of the amino acid sequences confirms the suggested evolutionary relationship between the two enzymes.
Lundqvist
Joakim
J
Department of Molecular Biophysics, Lund University, SE-22100 Lund, Sweden.
Elmlund
Dominika
D
Heldt
Dana
D
Deery
Evelyne
E
Söderberg
Christopher A G
CA
Hansson
Mats
M
Warren
Martin
M
Al-Karadaghi
Salam
S
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics; D017347:Brucella melitensis / Q000201:enzymology*; D003001:Cloning, Molecular; D008190:Lyases / Q000737:chemistry* / Q000235:genetics; D008854:Microscopy, Electron / Q000379:methods; D018832:Molecular Chaperones / Q000737:chemistry* / Q000235:genetics; D008969:Molecular Sequence Data; D011487:Protein Conformation; D021122:Protein Subunits / Q000737:chemistry / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics
D001426:Bacterial Proteins; D018832:Molecular Chaperones; D021122:Protein Subunits; D011994:Recombinant Proteins; D008190:Lyases; C033211:cobaltochelatase