The substrate-specific selenoprotein B of glycine reductase (PB glycine ) from Eubacterium acidaminophilum was purified and characterized. The enzyme consisted of three different subunits with molecular masses of about 22 (a), 25 (b) and 47 kDa (g), probably in an a 2 b 2 g 2 composition. PB glycine purified from cells grown in the presence of [ 75 Se]selenite was labeled in the 47-kDa subunit. The 22-kDa and 47-kDa subunits both reacted with fluorescein thiosemicarbazide, indicating the presence of a carbonyl compound. This carbonyl residue prevented N-terminal sequencing of the 22-kDa (a) subunit, but it could be removed for Edman degradation by incubation with o-phenylenediamine.A DNA fragment was isolated and sequenced which encoded b and a subunits of PB glycine (grdE), followed by a gene encoding selenoprotein A (grdA2) and the g subunit of PB glycine (grdB2). The cloned DNA fragment represented a second GrdB-encoding gene slightly different from a previously identified partial grdB1-containing fragment. Both grdB genes contained an in-frame UGA codon which confirmed the observed selenium content of the 47-kDa (g) subunit. Peptide sequence analyses suggest that grdE encodes a proprotein which is cleaved into the previously sequenced N-terminal 25-kDa (b) subunit and a 22-kDa (a) subunit of PB glycine . Cleavage most probably occurred at an -Asn-Cys-site concomitantly with the generation of the blocking carbonyl moiety from cysteine at the a subunit.

The gram-positive anaerobic bacterium Clostridium kluyveri (3) ferments ethanol and acetate to butyrate, caproate, and molecular hydrogen (7). ATP, required for growth, is gained by substrate-level phosphorylation from acetyl phosphate, and the quantity is proportional to the amount of hydrogen produced (46, 50). Investigations of additional metabolic abilities revealed that this organism can utilize crotonate, vinylacetate, and 4-hydroxybutyrate as substrates (4, 5) and is able to ferment the unusual substrate combination of succinate plus ethanol (27). A pathway was proposed, one in which succinate is first activated and then reduced by a two-step reaction to give 4-hydroxybutyrate, which is then further metabolized to crotonyl-coenzyme A (CoA) ( Fig. 1) (27). In a previous study, we discussed enzymes involved in the anaerobic breakdown of succinate by C. kluyveri, specifically, a succinyl-CoA:CoA transferase, a CoA-and NADP ϩ -dependent succinate-semialdehyde dehydrogenase, and a 4-hydroxybutyrate dehydrogenase (49). Wolff et al. (55) independently confirmed these data by 13 C-nuclear magnetic resonance studies as well as enzymatic investigations on the dehydrogenases. 4-Hydroxybutyryl-CoA dehydratase, which catalyzes the last step of the succinatespecific pathway, the dehydration and isomerization of 4-hydroxybutyryl-CoA to crotonyl-CoA, was recently identified, purified, and characterized (45). We present here some molecular aspects of this pathway, including the cloning, sequencing, and heterologous expression of a C. kluyveri DNA region which encodes a succinyl-CoA:CoA transferase, the succinatesemialdehyde dehydrogenase, and the 4-hydroxybutyrate dehydrogenase. MATERIALS AND METHODSBacterial strains, plasmids, media, and growth conditions. C. kluyveri (DSM 555) was obtained from the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Braunschweig, Germany. Escherichia coli JM109 (58) and the pBluescript SK vector (Stratagene, San Diego, Calif.) were from the laboratory collection. C. kluyveri cells used for DNA isolation were grown at 37ЊC under strictly anaerobic conditions on ethanol (300 mM) and succinate (100 mM) as previously described (49). Cells for RNA preparation were cultured in the same medium except that succinate was replaced by acetate (100 mM). E. coli cultures were routinely grown at 30ЊC in Luria-Bertani (LB) medium (42) on a rotary shaker. Tetrazolium indicator plates (6) containing 4 g of 4-hydroxybutyrate per liter were employed for the screening procedure (oxidation of 4-hydroxybutyrate). Utilization of 4-hydroxybutyrate (4 g/liter) as a carbon source for recombinant E. coli clones was investigated in M9 medium (42), supplemented with a small amount of yeast extract (0.2 g/liter), MgSO 4 (2 mM), and CaCl 2 (0.1 mM). Ampicillin (75 mg/liter) was added to the media for E. coli as a selection marker, when needed.Nucleic acids isolation and recombinant DNA techniques. Chromosomal DNA from C. kluyveri was isolated by the method of Saito and Miura (41). Total RNA from C. kluyveri...

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...

Electron transfer reactions for the reduction of glycine in Eubacterium acidaminophilum involve many selenocysteine (U)- and thiol-containing proteins, as shown by biochemical and molecular analysis. These include an unusual thioredoxin system (-CXXC-), protein A (-CXXU-) and the substrate-specific protein B of glycine reductase (-UXXCXXC-). Most probably a selenoether is formed at protein B by splitting the C-N-bond after binding of the substrate. The carboxymethyl group is then transferred to the selenocysteine of protein A containing a conserved motif. The latter protein acts as a carbon and electron donor by giving rise to a protein C-bound acetyl-thioester and a mixed selenide-sulfide bond at protein A that will be reduced by the thioredoxin system. The dithiothreitol-dependent D-proline reductase of Clostridium sticklandii exhibits many similarities to protein B of glycine reductase including the motif containing selenocysteine. In both cases proprotein processing at a cysteine residue gives rise to a blocked N-terminus, most probably a pyruvoyl group. Formate dehydrogenase and some other proteins from E. acidaminophilum contain selenocysteine, e.g., a 22 kDa protein showing an extensive homology to peroxiredoxins involved in the detoxification of peroxides.

Anaerobically prepared cell extracts of Clostridium kluyveri grown on succinate plus ethanol contained high amounts of 4-hydroxybutyryl-CoA dehydratase, which catalyzes the reversible dehydration of 4-hydroxybutyryl-CoA to crotonyl-CoA. The enzyme was purified 12-fold under strictly anaerobic conditions to over 95% homogeneity and had a specific activity of 123 nkat mg-1. The finding of this dehydratase means that all of the enzymes necessary for fermentation of succinate plus ethanol by C. kluyveri have now been demonstrated to exist in this organism and confirms the proposed pathway involving a reduction of succinate via 4-hydroxybutyrate to butyrate. Interestingly, the enzyme is almost identical to the previously isolated 4-hydroxybutyryl-CoA dehydratase from Clostridium aminobutyricum. The dehydratase was revealed as being a homotetramer (m = 59 kDa/subunit), containing 2 +/- 0.2 mol FAD, 13.6 +/- 0.8 mol Fe and 10.8 +/- 1.2 mol inorganic sulfur. The enzyme was irreversibly inactivated after exposure to air. Reduction by sodium dithionite also yielded an inactive enzyme which could be reactivated, however, up to 84% by oxidation with potassium hexacyanoferrate(III). The enzyme possesses an intrinsic vinylacetyl-CoA isomerase activity which was also found in 4-hydroxybutyryl-CoA dehydratase from C. aminobutyricum. Moreover, the N-terminal sequences of the dehydratases from both organisms were found to be 63% identical.