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PMID: 8655474 Published · ppublish English Journal Article Research Support, U.S. Gov't, Non-P.H.S.

Cloning, sequencing, and expression of clustered genes encoding beta-hydroxybutyryl-coenzyme A (CoA) dehydrogenase, crotonase, and butyryl-CoA dehydrogenase from Clostridium acetobutylicum ATCC 824.

Journal of bacteriology ·Vol. 178 ·No. 11 ·1996-06-00 ·Pages 3015-24

Boynton ZL, Bennet GN, Rudolph FB

Abstract

The enzymes beta-hydroxybutyryl-coenzyme A (CoA) dehydrogenase (BHBD), crotonase, and butyryl-CoA dehydrogenase (BCD) from Clostridium acetobutylicum are responsible for the formation of butyryl-CoA from acetoacetyl-CoA. These enzymes are essential to both acid formation and solvent formation by clostridia. Clustered genes encoding BHBD, crotonase, BCD, and putative electron transfer flavoprotein alpha and beta subunits have been cloned and sequenced. The nucleotide sequence of the crt gene indicates that it encodes crotonase, a protein with 261 amino acid residues and a calculated molecular mass of 28.2 kDa; the hbd gene encodes BHBD, with 282 residues and a molecular mass of 30.5 kDa. Three open reading frames (bcd, etfB, and etfA) are located between crt and hbd. The nucleotide sequence of bcd indicates that it encodes BCD, which consists of 379 amino acid residues and has high levels of homology with various acyl-CoA dehydrogenases. Open reading frames etfB and etfA, located downstream of bcd, encode 27.2- and 36.1-kDa proteins, respectively, and show homology with the fixAB genes and the alpha and beta subunits of the electron transfer flavoprotein. These findings suggest that BCD in clostridia might interact with the electron transfer flavoprotein in its redox function. Primer extension analysis identified a promoter consensus sequence upstream of the crt gene, suggesting that the clustered genes are transcribed as a transcriptional unit and form a BCS (butyryl-CoA synthesis) operon. A DNA fragment containing the entire BCS operon was subcloned into an Escherichia coli-C. acetobutylicum shuttle vector. Enzyme activity assays showed that crotonase and BHBD were highly overproduced in cell extracts from E. coli harboring the subclone. In C. acetobutylicum harboring the subclone, the activities of the enzymes crotonase, BHBD, and BCD were elevated.

MeSH Terms
3-Hydroxyacyl CoA Dehydrogenases/chemistry,genetics Amino Acid Sequence Base Sequence Butyryl-CoA Dehydrogenase Cloning, Molecular Clostridium/enzymology,genetics Enoyl-CoA Hydratase/chemistry,genetics Fatty Acid Desaturases/chemistry,genetics Genes, Bacterial Molecular Sequence Data Multigene Family
Chemicals
3-Hydroxyacyl CoA Dehydrogenases 3-hydroxybutyryl-CoA dehydrogenase Fatty Acid Desaturases Butyryl-CoA Dehydrogenase Enoyl-CoA Hydratase
Authors & Affiliations
3 authors, click to expand affiliations / ORCID
Boynton Z L
Department of Biochemistry and Cell Biology, The Institute of Biosciences and Bioengineering, Rice University, Houston, Texas 77005-1892, USA.
Bennet G N
Rudolph F B
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Article Info
Journal
Journal of bacteriology
Abbr.
J Bacteriol
ISSN
0021-9193
DOI
10.1128/jb.178.11.3015-3024.1996
Published
1996-06-00
Pages
3015-24
Language
English
Region
United States
NLM ID
2985120R
PMCID
PMC178046
Subset
IM
Databases
GENBANK
U17110
External Links
PubMed source DOI: 10.1128/jb.178.11.3015-3024.1996 PMC Full Text View journal details
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