1991/07/01 by H Priefert, Horst Priefert, S Hein +7 · 70 citations
Biochemistry, Genetics and Molecular Biology · Engineering · #Acetoin #Alcaligenes #Amino acid #Bacteria #Bacterial Genetics and Biotechnology #Biochemistry #Biofuel production and bioconversion #Biology #Consensus sequence #EcoRI #Escherichia coli #Gene #Genetics #Homology (biology) #Microbial Metabolic Engineering and Bioproduction #Molecular biology #Nucleic acid sequence #Open reading frame #Operon #Peptide sequence #Plasmid #Pseudomonas #Structural gene #lac operon
paper · open access · doi:10.1128/jb.173.13.4056-4071.1991
published in Journal of Bacteriology 173(13), 4056-4071 (American Society for Microbiology)
openalex publication_date 1991/07/01 · openalex created_date 2025/10/10 · openalex updated_date 2026/07/28
Acetoin:dichlorophenolindophenol oxidoreductase (Ao:DCPIP OR) and the fast-migrating protein (FMP) were purified to homogeneity from crude extracts of acetoin-grown cells of Alcaligenes eutrophus. Ao:DCPIP OR consisted of alpha and beta subunits (Mrs, 35,500 and 36,000, respectively), and a tetrameric alpha 2 beta 2 structure was most likely for the native protein. The molecular weight of FMP subunits was 39,000. The N-terminal amino acid sequences of the three proteins were determined, and oligonucleotides were synthesized on the basis of the codon usage of A. eutrophus. With these, the structural genes for the alpha and beta subunits of Ao:DCPIP OR and FMP, which were referred to as acoA, acoB, and acoC, respectively, were localized on one single EcoRI restriction fragment which has been cloned recently (C. Fründ, H. Priefert, A. Steinbüchel, and H. G. Schlegel, J. Bacteriol. 171:6539-6548, 1989). The nucleotide sequences of a 5.3-kbp region of this fragment and one adjacent fragment were determined, and the structural genes for acoA (1,002 bp), acoB (1,017 bp), and acoC (1,125 bp) were identified. Together with the gene acoX, whose function is still unknown and which is represented by a 1,080-bp open reading frame, these genes are probably organized in one single operon (acoXABC). The transcription start site was identified 27 bp upstream of acoX; this site was preceded by a region which exhibited complete homology to the enterobacterial sigma 54-dependent promoter consensus sequence. The amino acid sequences deduced from acoA and acoB for the alpha subunit (Mr, 35,243) and the beta subunit (Mr, 35,788) exhibited significant homologies to the primary structures of the dehydrogenase components of various 2-oxo acid dehydrogenase complexes, whereas those deduced from acoC for FMP (Mr, 38,941) revealed homology to the dihydrolipoamide acetyltransferase of Escherichia coli. The occurrence of a new enzyme type for the degradation of acetoin is discussed.