The sodium pump glutaconyl-CoA decarboxylase from Acidaminococcus fermentans. Specific cleavage by n-alkanols

Abstract

1. Glutaconyl-CoA decarboxylase from Acidaminococcus Jermentans was inactivated by incubation with n-alkanols at 37 "C. The concentration of the alcohol required for complete inactivation decreased with increasing chain length; e.g. 2 M ethanol was as potent as 2 mM hexanol or 0.5 mM decanol. The data indicate a binding of the alcohol to the enzyme with an energy of about 4 kJ/methylene group.2. Sodium ions prevented the inactivation (50% at 30 mM NaC1). K ' , NHZ, Cs' and Mg2+ had no influence, whereas Lit was ten times less effective than Na' .3. The enzyme was cleaved during the inactivation into a soluble part, consisting of the 01 ( M , 120000) and p polypeptide chains (60000), whereas the hydrophobic y chain (30000) precipitated.4. The soluble part catalysed the sodium-ion-independent but avidin-sensitive glutaconyl-CoA/crotonyl-CoA exchange as measured with the substrates [3-'H]crotonyl-CoA and unlabelled glutaconate and with glutaconate CoA-transferase as auxiliary enzyme.5. In the presence of free biotin or its methyl ester the soluble part catalysed the formation of crotonyl-CoA from glutaconyl-CoA (apparent K, for biotin 40 mM, V,,, 1 YO of the native decarboxylation reaction). This apparent reactivation was most likely caused by the carboxylation of free biotin. 6 . Based on these and other observations the following functions may be assigned to the different polypeptide chains of glutaconyl-CoA decarboxylase : biotin carrier (a), carboxytransferase (p) and carboxylase, the actual sodium pump (7).

Identification of Additional Players in the Alternative Biosynthesis Pathway to Isovaleryl‐CoA in the Myxobacterium Myxococcus xanthus

“…[29] The resulting glutaconyl-CoA is then decarboxylated to crotonylCoA, which is subsequently converted to two acetyl-CoA units as shown in Acidaminococcus fermentans. [30,31] The glutaconylCoA decarboxylase involved in this process was shown to be a membrane bound sodium pump that consists of four subunits of which no homologues can be found in M. xanthus, except for the carboxylase a subunit, which shows highest identity/ similarity to LiuB (27 %/49 %) but also to AccB (acetyl-CoA carboxylase, 24 %/43 %) and PccB (propionyl-CoA carboxylase, 24 %/41 %). Although glutaconyl-CoA and 3-methylglutaconlyCoA-believed to be an intermediate in the alternative pathway to IV-CoA-differ only in one methyl group, all intermediates in the alternative pathway seem to be CoA-bound and thus no activation of a free acid seems to be required.…”

Fermentative Cyclohexane Carboxylate Formation in <b><i>Syntrophus aciditrophicus</i></b>

“…The specific activity, measured by the formation of crotonyl-CoA from glutaconyl-CoA (see above), was very low (2 mU/mg protein in the presence of biotin and 0.6 mU/mg in its absence). For comparison, with GdcA from A. fermentans a specific activity of 40 mU/mg was achieved: 1% of that of the holoenzyme [Buckel and Liedtke, 1986]. The GcdB and GcdC components could not be produced in E. coli .…”

Substrate-Induced Radical Formation in 4-Hydroxybutyryl Coenzyme A Dehydratase from Clostridium aminobutyricum

“…The specific activity of 4HBD (1,31,32) was measured anaerobically in a coupled assay based on determining the amount of crotonyl-CoA formed by ␤-oxidation to acetyl-CoA. The cuvette (light path [d] ϭ 1 cm) contained 100 mM potassium phosphate, pH 7.4, 2 mM EDTA, 2 mM dithiothreitol, 2 mM NAD ϩ , 1 mM sodium 4-hydroxybutyrate or vinylacetate, 0.1 mM CoA, 0.1 mM acetyl-phosphate, 1 mM acetyl-CoA, 1.0 U 4-hydroxybutyrate CoA-transferase (24), and a mixture of auxiliary enzymes from Acidaminococcus fermentans (0.3 mg/ml) (33). After 3 min of incubation at room temperature, the reaction was started by adding 4HBD.…”