Resolution of the fatty acid reductase from Photobacterium phosphoreum into acyl protein synthetase and acyl-CoA reductase activities. Evidence for an enzyme complex.
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Acyltransferases↗
Biomedical subjects
D Riendeau
Publications and source records attributed to D Riendeau.
Fatty acid reductase in bioluminescent bacteria. Resolution from aldehyde reductases and characterization of the aldehyde product.
Fatty acid reductase from the bioluminescent bacterium Photobacterium phosphoreum, has been partially purified free of aldehyde reductase activity and with a low endogenous fatty acid content permitting the characterization of the aldehyde product of the reaction. Two aldehyde reductases, both dependent on NADH, were separated by anion-exchange chromatography from the fatty acid reductase activity. The partially purified fatty acid reductase catalyzed the synthesis exclusively of long chain aldehydes from fatty acids in the presence of ATP and NADPH as demonstrated by the conversion of [3H]tetradecanoic acid to [3H]aldehyde. Comparison of the amount of [3H]aldehyde produced with the bioluminescence responses of luciferase to the aldehyde product and standard aldehydes, both with respect to maximum light intensity and luminescent decay, established that tetradecanoic acid has been converted to tetradecanal, the aldehyde of the same chain length. These results are consistent with a mechanism involving activation of the fatty acid with ATP followed by reduction of a fatty acyl intermediate to the corresponding aldehyde.
Aldehyde Oxidoreductases↗
Co-induction of fatty acid reductase and luciferase during development of bacterial bioluminescence.
The luminescent bacterium Photobacterium phosphoreum has been shown to possess a fatty acid reductase based on the stimulation of the aldehyde-dependent luminescent reaction on incubation of the enzyme with ATP, NADPH, and tetradecanoic acid (Riendeau, D., and Meighen, E. (1979) J. Biol. Chem. 254, 7488-7490). A direct, luciferase-independent assay for the fatty acid reductase has now been developed using [3H]tetradecanoic acid as substrate and thin layer chromatography to separate and identify the products of the reaction. Tritiated aldehyde was the only product of the reaction at early times of assay, and the amount produced was linearly dependent on time and extract concentration. The labeled aldehyde was further reduced to alcohol after prolonged incubation, indicating that long chain aldehyde reductase(s) are also present in bioluminescet bacteria. Measurement of the fatty acid reductase activity in extracts during growth and development of the bioluminescent bacteria showed that the fatty acid reductase activity is co-induced with luciferase, suggesting that these enzymes are coordinately regulated and directly implicating the fatty acid reductase in aldehyde biosynthesis in the bacterial bioluminescent system.
Aldehyde Oxidoreductases↗
Evidence for a fatty acid reductase catalyzing the synthesis of aldehydes for the bacterial bioluminescent reaction. Resolution from luciferase and dependence on fatty acids.
The enzyme responsible for the stimulation by ATP AND NADPH of light emission catalyzed by bacterial luciferase has been partially purified from extracts of the luminescent bacterium, Photobacterium phosphoreum. The stimulatory activity was found to be stabilized by high concentrations of mercaptoethanol, permitting it to be separated from luciferase into an active and stable form and enabling further characterization of its functional properties. The activity of the enzyme was shown to be dependent not only on ATP and NADPH but also on the presence of a long chain fatty acid, and was inhibited by the addition of NADH and horse liver alcohol dehydrogenase. The specificity for fatty acids, as measured by the stimulation of luciferase activity, had a very limited range, with maximal luminescence being obtained with myristic acid and lower responses being observed only with tridecanoic and pentadecanoic acid. These results provide evidence in vitro for an enzyme in bioluminescent bacteria that functions as a fatty acid reductase converting fatty acids to aldehydes which in turn can be utilized by luciferase in the light-emitting reaction.
Alcohol Oxidoreductases↗
N-(4-chlorophenyl)-N-hydroxy-N'-(3-chlorophenyl)urea, a general reducing agent for 5-, 12-, and 15-lipoxygenases and a substrate for their pseudoperoxidase activities.
Lipoxygenases contain a nonheme iron that undergoes oxidation and reduction during the catalytic cycle. The conversion from the Fe3+ enzyme form to the Fe2+ form can be achieved using reducing inhibitors, a reaction that can be reversed with lipid hydroperoxides. The present study describes the properties of N-(4-chlorophenyl)-N-hydroxy-N'-(3-chlorophenyl)urea (CPHU), which functions as a reducing agent for various lipoxygenases and stimulates the degradation of lipid hydroperoxide catalyzed by these enzymes (pseudoperoxidase activity). CPHU was a substrate for the pseudoperoxidase reaction of purified soybean lipoxygenase-1 with apparent Km values for CPHU and 13-hydroperoxy-9,11-octadecadienoic acid (13-HpODE) of 14 and 15 microM, respectively. CPHU was converted during the pseudoperoxidase reaction to a mixture of products that can be resolved by reverse-phase high pressure liquid chromatography. By comparison with the chemical reaction of CPHU and potassium nitrosodisulfonate, the major enzymatic reaction product was tentatively identified as a one-electron oxidation product of CPHU. At low concentrations (50 microM), dithiothreitol completely protected against the degradation of hydroxyurea without inhibiting the pseudoperoxidase reaction. Under these conditions, the rate of the pseudoperoxidase reaction with CPHU as a substrate can be quantitated by the change in absorbance at 234 nm owing to the consumption of 13-HpODE. In addition to soybean lipoxygenase-1, CPHU was found to be a substrate for the pseudoperoxidase activities of purified recombinant human 5-lipoxygenase and porcine leukocyte 12-lipoxygenase. The results are consistent with CPHU reacting with lipoxygenase by a one-electron oxidation to generate the ferrous enzyme form and the nitroxide radical, which could be reduced back to CPHU by DTT.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
Interfacial catalysis and production of a high ratio of leukotriene A4 to 5-HPETE by 5-lipoxygenase in a coupled assay with phospholipase A2.
The activity of purified 5-lipoxygenases (5-LO) shows a requirement for the presence of phosphatidylcholine or other lipids, in addition to Ca2+ and ATP. The enzymatic activity of purified human 5-lipoxygenase was dependent on the ratio of arachidonic acid to phospholipids rather than on the bulk arachidonic acid concentration, suggesting that the concentration of substrate at the lipid-water interface is important for the rate of the 5-LO reaction. Enzyme activity was also examined using vesicles of dimyristoylphosphatidylmethanol/1-palmitoyl-2-arachidonoylphosp hat idylcholine. Using PLA2 to release arachidonic acid from phospholipid, the ratio of leukotriene A4 (detected as trans-LTB4 isomers) to 5-hydroperoxyeicosatetraenoic acid (5-HPETE) accumulated depended on the 5-LO concentration and was relatively independent of the amount of PLA2. The ratio of leukotriene A4 to 5-HPETE production increased with the amount of 5-LO (1-15 micrograms/ml) to reach values (> 10) similar to those observed with ionophore-challenged human leukocytes. The data are consistent with the catalysis of 5-LO occurring at the surface of phospholipid vesicles with the 5-HPETE product being re-utilized by the LTA4 synthase reaction of 5-lipoxygenase under conditions of limiting arachidonic acid availability.
Arachidonate 5-Lipoxygenase↗