The central metabolic pathway from acetyl-CoA to butyryl-CoA inClostridium acetobutylicum

A sensitive assay for medium chain acyl-CoA dehydrogenase has been developed by substituting ferricenium hexafluorophosphate for the physiological acceptor, electron transferring flavoprotein. The ferricenium ion is a facile oxidant of the octanoyl-CoA-reduced enzyme with a Vmax of 1400 min-1 and a KM of 55 microM at pH 7.6. The ferricenium assay does not require additional mediator dyes, exhibits low background rates, and avoids the necessity of purifying substantial amounts of electron transferring flavoprotein. Unlike the fluorescence-based electron transferring flavoprotein assay, this new procedure can be performed aerobically. Both assays give comparable results when tested with crude fibroblast homogenates from normal and medium chain acyl-CoA dehydrogenase deficient patients. The convenience of the ferricenium method suggests it may be generally useful as a screening assay for a number of acyl-CoA dehydrogenases.

The fixA, fixB, fixC, and fixX genes of Rhizobium meliloti 1021 constitute an operon and are required for nitrogen fixation in alfalfa nodules. DNA homologous to the R. meliloti fixABC genes is present in all other Rhizobium and Bradyrhizobium species examined, but fixABC-homologous sequences were found in only one free-living diazotroph, Azotobacter vinelandii. To determine whether the fixABCX genes share sequence homology with any of the 17 Klebsiella pneumoniae nif genes, we determined the entire nucleotide sequence of the fixA, fixB, fixC, and fixX genes and defined four open reading frames that code for polypeptides of molecular weights 31,146, 37,786, 47,288, and 10,937, respectively. Neither DNA nor amino acid sequence homology to the R. meliloti fixA, -B, -C, and -X genes was found in the K. pneumoniae nif operon. The fixX gene contains a cluster of cysteine residues characteristic of ferredoxins and is highly homologous to an Azotobacter ferredoxin which has been shown to donate electrons to nitrogenase. The fixABC operon contains a promoter region that is highly homologous to other nifA-activated promoters. We also found a duplication of the 5' end of the fixABCX operon; a 250-bp region located 520 bp upstream of the fixABCX promoter bears more than 65% homology to the 5' end of the transcribed region, including the first 32 codons of fixA.