Long-chain acyl-CoA ester intermediates of beta-oxidation of mono- and di-carboxylic fatty acids by extracts of Corynebacterium sp. strain 7E1C.
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.
Broadway
N M
NM
Department of Applied Biology, University of Hull, U.K.
Dickinson
F M
FM
Ratledge
C
C
D015094:3-Hydroxyacyl CoA Dehydrogenases / Q000037:antagonists & inhibitors; D000105:Acetyl Coenzyme A / Q000096:biosynthesis; D000214:Acyl Coenzyme A / Q000096:biosynthesis / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D003352:Corynebacterium / Q000378:metabolism*; D004951:Esterification; D004952:Esters / Q000378:metabolism; D005227:Fatty Acids / Q000378:metabolism*; D007700:Kinetics; D009243:NAD / Q000378:metabolism; D010084:Oxidation-Reduction
D000214:Acyl Coenzyme A; D004952:Esters; D005227:Fatty Acids; D009243:NAD; D000105:Acetyl Coenzyme A; D015094:3-Hydroxyacyl CoA Dehydrogenases