Cloning and expression of the gene cluster encoding key proteins involved in acetyl-CoA synthesis in Clostridium thermoaceticum: CO dehydrogenase, the corrinoid/Fe-S protein, and methyltransferase.
Acetogenic bacteria fix CO or CO2 by a pathway of autotrophic growth called the acetyl-CoA (or Wood) pathway. Key enzymes in the pathway are a methyltransferase, a corrinoid/Fe-S protein, a disulfide reductase, and a carbon monoxide dehydrogenase. This manuscript describes the isolation of the genes that code for the methyltransferase, the two subunits of the corrinoid/Fe-S protein, and the two subunits of carbon monoxide dehydrogenase. These five genes were found to be clustered within an approximately 10-kilobase segment on the Clostridium thermoaceticum genome. The proteins were expressed at up to 5-10% of Escherichia coli cell protein, and isopropyl beta-D-thiogalactopyranoside had no effect on the levels of expression, implying that the C. thermoaceticum inserts contained transcriptional and translational signals that were recognized by E. coli. The methyltransferase is expressed in E. coli in a fully active dimeric form with a specific activity and heat stability similar to the enzyme expressed in C. thermoaceticum. However, both the corrinoid/Fe-S protein and carbon dioxide dehydrogenase, although expressed in high amounts and with identical subunit molecular weights in E. coli, are inactive and less heat stable than are the native enzymes from C. thermoaceticum.
Roberts
D L
DL
Department of Chemistry, University of Wisconsin-Milwaukee 53201.
James-Hagstrom
J E
JE
Garvin
D K
DK
Gorst
C M
CM
Runquist
J A
JA
Baur
J R
JR
Haase
F C
FC
Ragsdale
S W
SW
D000105:Acetyl Coenzyme A / Q000096:biosynthesis*; D000445:Aldehyde Oxidoreductases / Q000235:genetics*; D000595:Amino Acid Sequence; D003013:Clostridium / Q000201:enzymology / Q000235:genetics*; D045728:Corrinoids; D004269:DNA, Bacterial / Q000096:biosynthesis; D005796:Genes; D005798:Genes, Bacterial; D007506:Iron-Sulfur Proteins / Q000235:genetics*; D008667:Metalloproteins / Q000235:genetics*; D008780:Methyltransferases / Q000235:genetics*; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes; D005810:Multigene Family; D009693:Nucleic Acid Hybridization; D010957:Plasmids; D015183:Restriction Mapping; D014805:Vitamin B 12 / Q000378:metabolism*
D045728:Corrinoids; D004269:DNA, Bacterial; D007506:Iron-Sulfur Proteins; D008667:Metalloproteins; D009097:Multienzyme Complexes; D000105:Acetyl Coenzyme A; D000445:Aldehyde Oxidoreductases; C019823:carbon monoxide dehydrogenase; D008780:Methyltransferases; D014805:Vitamin B 12