Sequence and expression of the gene encoding the corrinoid/iron-sulfur protein from Clostridium thermoaceticum and reconstitution of the recombinant protein to full activity.
The corrinoid/iron-sulfur protein (C/Fe-SP) from Clostridium thermoaceticum acts as a methyl group carrier in the anaerobic acetyl-CoA pathway of CO and CO2 fixation. Consisting of a small (approximately 33 kDa) and a large (approximately 55 kDa) subunit, the C/Fe-SP contains 1 mol of cobalt in a corrinoid cofactor and 1 mol of [4Fe-4S]2+/1+ cluster/mol of alpha beta dimer. Cobalt is the site of methylation, and the [4Fe-4S] center appears to serve an electron transfer function. The genes encoding both subunits have been cloned previously and are located within a gene cluster that includes other genes required for CO2 fixation by anaerobic bacteria. When the genes encoding the C/Fe-SP were expressed in Escherichia coli, the protein was found to be inactive. We report the amino acid sequences of the large and small subunits of the C/Fe-SP based on the DNA sequences of the cloned genes. The [4Fe-4S] cluster was found to be located in the large subunit. Although the primary structural lattice for cobamide binding resides in the small subunit, both subunits are required for formation of a stable cobamide-binding protein. Based on sequence comparisons with other [4Fe-4S]-containing proteins, 3 of the 4 cysteine residues that serve as ligands to the iron sites in the cluster have been located. The two subunits were independently overexpressed in E. coli to a level of 30-50% of cell protein; however, the resulting protein was inactive, lacked stoichiometric amounts of Fe-S cluster, and lacked cobamide. By combining the recombinant subunits, unfolding them with urea, and refolding in the presence of cobamide, iron, and inorganic sulfide, the resulting C/Fe-SP was found to contain stoichiometric amounts of cobamide and [4Fe-4S] cluster and had spectroscopic and enzymatic properties similar to those of the native protein. We expect that the methods developed here may be used for heterologous overexpression and reconstitution of other complex metalloenzymes. The C/Fe-SP was found to utilize with equal efficiency either vitamin B12 or the natural cofactor 5-methoxybenzimidazolylcobamide as a methyl carrier.
Lu
W P
WP
Department of Biochemistry, University of Nebraska, Lincoln 68583-0718.
Schiau
I
I
Cunningham
J R
JR
Ragsdale
S W
SW
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites; D003001:Cloning, Molecular; D003013:Clostridium / Q000201:enzymology / Q000235:genetics*; D003038:Cobamides / Q000378:metabolism; D004269:DNA, Bacterial; D004926:Escherichia coli; D007506:Iron-Sulfur Proteins / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D010957:Plasmids; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism
D001426:Bacterial Proteins; C080269:C-Fe-SP protein, Moorella thermoacetica; D003038:Cobamides; D004269:DNA, Bacterial; D007506:Iron-Sulfur Proteins; D011994:Recombinant Proteins