Long-chain acyl-CoA ester intermediates of β-oxidation of mono- and di-carboxylic fatty acids by extracts of Corynebacterium sp. strain 7E1C

Abstract

beta-Oxidation of palmitate and tetradecanedioic acid was studied in cell-free extracts of the Gram-positive bacterium Corynebacterium sp. strain 7E1C, and the acyl-CoA ester intermediates formed were analysed by h.p.l.c. beta-Oxidation assays displayed a lag phase before a constant rate of NAD+ reduction was obtained. The length of the lag phase was inversely proportional to the number of units of activity added to assays. This is a characteristic feature of a system of consecutive reactions proceeding via free intermediates. During beta-oxidation of palmitate all the saturated acyl-CoAs from C16 to C8 were detected together with trace amounts of unsaturated and 3-hydroxy-intermediates. The time-course of intermediate formation again indicated a precursor-product relationship indicative of free intermediates being formed. When 3-hydroxyacyl-CoA dehydrogenase was inhibited by completely removing NAD+ from assays, the major acyl-CoAs, detected during palmitate beta-oxidation were palmitoyl-CoA, hexadeca-2-enoyl-CoA and 3-hydroxypalmitoyl-CoA. These compounds also displayed a precursor-product relationship. Under normal assay conditions the acyl-CoA dehydrogenase(s) are the probable rate-limiting enzyme(s) of the beta-oxidation spiral. These results indicate that in cell-free extracts of Corynebacterium sp. strain 7E1C, beta-oxidation proceeds via free acyl-CoA intermediates and is at variance with the concept of substrate channelling or of a 'leaky hose pipe' model as proposed for mitochondrial beta-oxidation in eukaryotic cells. The significant accumulation of chain-shortened acyl-CoA esters is similar to the situation observed for mammalian peroxisomal beta-oxidation.

The enzymology of dicarboxylic acid formation by Corynebacterium sp. strain 7E1C grown on n-alkanes

“…Corynebacterium sp. strain 7E1C (ATCC 19067) was grown for 24 h on nutrient broth and then transferred to Jay's/succinate medium in a vortex-stirred bottle as described earlier (Broadway et al, 1992). Cells were harvested at the mid-exponential phase of growth.…”

“…Cells were harvested at the mid-exponential phase of growth. Cell-free extracts were prepared as described previously (Broadway et al, 1992). A mutant of this organism, Corynebacterium ATCC 2 1747, which produces increased amounts of dicarboxylic acids (Du Pont, 1973, 1974 was also examined.…”

“…P-Oxidation was measured in 'linked ' and 'direct' assays as described previously (Broadway et al, 1992).…”

“…The other was a direct method and used chemically synthesized acyl-CoAs. The linked assay could be used only with extracts from succinate-grown cells, whereas the direct assay could be used with extracts from cells grown on various carbon sources (Broadway et al, 1992). For both assays, activity was measured by either NAD+ reduction or acetyl-CoA formation.…”

“…The acyl-CoA esters of o-hydroxyfatty acids and of dicarboxylic acids were synthesized and purified as described previously (Broadway et al, 1992).…”