Purification and characterization of a coenzyme‐A‐dependent succinate‐semialdehyde dehydrogenase from Clostridium kluyveri

Abstract

Cell extracts of Clostridium kluyveri, grown on ethanol plus succinate contained a succinylCoA : CoA transferase (0.28 U/mg), a coenzyme-A-dependent succinate-semialdehyde dehydrogenase (0.73 U/mg> and a NAD+-dependent 4-hydroxybutyrate dehydrogenase (0.25 U/mg). The semialdehyde dehydrogenase, which catalyzed the NADPH-dependent reduction of succinyl-CoA to succinate semialdehyde, was purified 59-fold to homogeneity. A molecular mass of 115000 Da was determined for the native enzyme; SDSRAGE revealed one protein band at 55000, indicating that the active form is a dimer. The enzyme was highly specific for succinyl-CoA and succinate semialdehyde. The pH optimum was 7.0 for the reduction of succinyl-CoA, and 8.5 for the reverse reaction. K,,, values were determined for both the forward and. reverse directions. The kinetic data suggest a ping-pong mechanism.Clostridium kluyveri is an organism with an unusual fermentative metabolism; ethanol and acetate are oxidized to butyrate and caproate plus molecular hydrogen [l]. The amount of H, produced is related to the ATP which becomes available to the organism for growth and other expenditures 12, 31. C, kluyveri is also able to grow on crotonate, vinylacetate and 4-hydroxybutyrate as sole carbon and energy sources [4, 51. More recently it was shown that C. kluyveri can ferment a substrate combination of ethanol and succinate, producing acetate, butyrate and caproate [6]. A pathway was proposed, in which succinate is activated to succinyl-CoA, then metabolized to 4-hydroxybutyrate via two reduction steps analogous to acetaldehyde and alcohol dehydrogenase /6]. Experimental evidence of the enzymes required has not been presented. We report here on the detection of a succinyl-CoA : CoA transferase, a 4-hydroxybutyrate dehydrogenase [7] and a succinate-semialdehyde dehydrogenase in ethanolhccinate-grown cells of C. kluyveri and on the punfication and characterization of succinate-semialdehyde dehydrogenase. (0.05 %), para-amino benzoic acid (0.05 mg/l), biotin (0.01 mgA) and with the trace element solution (10 mVI) described by Kenealy and Zeikus [9]. The medium was prepared strictly anaerobically using an atmosphere of N,/CO, (80 : 20). After sterilization, the medium was reduced with cysteine/HCl and Na,S 3 9 H,O (0.025% of each). The cells (450 g) were harvested anaerobically, frozen in liquid nitrogen and stored at -2OOC until used. MATERIALS AND METHODS Organism and growth conditions MaterialsQ-Sepharose fast flow was obtained from Pharmacia. Reactive-Red-120-agarose (type 3000), succinate semialdehyde and the coenzyme-A derivatives were obtained from Sigma. 4-Hydroxybutyrate (sodium salt) and 2-amino-2-methyl-l,3-propanediol were from Aldrich, coenzyme A and other biochemicals from Boehringer.Succinyl-CoA was prepared from succinate anhydride and coenzyme A according to Simon and Shemin [lo]. The reaction was followed by the increasing absorbance at 235 nm, due to the formation of the thioester bond [ll]. To remove the remaining coenzyme A, a few crystals of 53'-dithi...

Adenine Binding Mode Is a Key Factor in Triggering the Early Release of NADH in Coenzyme A-dependent Methylmalonate Semialdehyde Dehydrogenase

“…A possible correlation between organic acidemia and MSDH deficiency has been explored (26,27), and very recently, Sass et al (28) proposed that polymorphism in the human ALDH6A1 gene encoding MSDH is directly correlated with 3-hydroxybutyric aciduria, a severe metabolic disease. Detailed kinetic studies of the MSDH-catalyzed reaction have shown that (i) the rate constant associated with the acylation step is high (k ac Ͼ 1000 s Ϫ1 ), indicating that the position of the nicotinamide ring relative to the hemithioacetal allows efficient hydride transfer, and (ii) that NADH release occurs before the rate-limiting ␤-decarboxylation and CoA attack on the thioacylenzyme intermediate (22), thus supporting the ping-pong kinetic mechanism that has previously been reported for other CoA-dependent ALDHs (29,30).…”

Computational and Experimental Analysis of Redundancy in the Central Metabolism of Geobacter sulfurreducens

“…Acetate-succinate CoA transferase (Ato) activity was determined as described by Sohling and Gottschalk [55], via a coupled assay in which the product of the Ato reaction, acetyl-CoA, is condensed with oxaloacetate by the enzyme citrate synthase and the liberation of CoASH is monitored by measuring the reduction of 5,59-dithio-bis(2-nitrobenzoic acid) (DTNB) at 412 nm (E 412 ¼ 13.6 mM À1 cm À1 ). The reaction mixture contained 100 mM potassium phosphate buffer, pH 7.0, 200 mM, 1 mM oxaloacetate, 1 mM DTNB, 0.1 mM succinyl-CoA, and 3 U of porcine citrate synthase (Sigma-Aldrich).…”

Methylmalonate-semialdehyde Dehydrogenase from Bacillus subtilis

“…The kinetic mechanism of the CoA-dependent ALDH family to which MSDHs belong was shown to be of ping-pong type with NADH release preceding transthioesterification (5,6). However, the MSDH-catalyzed reaction also includes a supplementary ␤-decarboxylation step that occurs before transthioesterification.…”