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

Abstract

The enzymes ␤-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 ␣ and ␤ 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 ␣ and ␤ 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.

Dissection of the Caffeate Respiratory Chain in the Acetogen Acetobacterium woodii : Identification of an Rnf-Type NADH Dehydrogenase as a Potential Coupling Site

“…The reductase and Etf constitute an enzyme complex. A very similar reaction is catalyzed by butyrylCoA dehydrogenases involved in butyrate fermentation that also receive electrons from NADH via Etf (1,6,37). These enzymes belong to the class of acyl-CoA dehydrogenases.…”

“…Next, we tested the hypothesis that the activation of caffeate reduction by ATP is not due to the (re)activation of some enzyme involved in the process but, analogous to similar reductive reactions (1,6,14,22,37), is due to the activation of caffeate and subsequent formation of caffeyl-CoA prior to reduction. Addition of acetyl-CoA or CoA plus ATP resulted in an eightfold increase in the initial caffeate reduction rate, similar to ATP-dependent stimulation, indicating that there was formation of caffeylCoA.…”

“…Stock cultures were grown in Hungate tubes. The medium was prepared using the anaerobic techniques described previously (8,23 4 Cl, 0.5 g of cysteine hydrochloride, 0.33 g of MgSO 4 ⅐ 6H 2 O, 2.9 g of NaCl, 6.0 g of KHCO 3 , 2.0 g of yeast extract, 1.0 ml of trace element solution SL 9, 1.0 ml of a selenite-tungstate solution, and 2.0 ml of vitamin solution DSMZ 141. The pH was adjusted to 7.1 to 7.2 with HCl.…”

A Polyketide Synthase in Glycopeptide Biosynthesis

“…1). The alignment of the DpgB sequence with protein sequence data bases showed similarity (24% ID/43% SM) to an enoyl-CoA hydratase with 3-hydroxybutryl-CoA dehydratase activity from Clostridium acetobutylicum (33).…”

Restricted Distribution of the Butyrate Kinase Pathway among Butyrate-Producing Bacteria from the Human Colon

“…Two molecules of acetyl coenzyme A (acetyl-CoA) are condensed and subsequently reduced to butyryl-CoA in a sequence of reactions that resembles the reverse ␤-oxidation of fatty acids (7,11,32). For the final step of butyrate synthesis from butyryl-CoA, two alternative pathways are known: the enzymes phosphotransbutyrylase and butyrate kinase convert butyryl-CoA to butyrate with the intermediate formation of butyryl-phosphate.…”