Isolation of an atypically small lipoamide dehydrogenase involved in the glycine decarboxylase complex from Eubacterium acidaminophilum

Freudenberg, W.; Dietrichs, D; Lebertz, H.; Andreesen, Jan R.

doi:10.1128/jb.171.3.1346-1354.1989

Cited by 33 publications

(35 citation statements)

References 58 publications

(80 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…NADPH-dependent glycine reduction was 0.1 U/mg of protein in glycine-grown cells and 0.06 U/mg of protein in betaine-grown cells. Thioredoxin of E. acidaminophilum does not bind to DEAE (26), in contrast to the thioredoxin reductase-like flavoprotein (16), protein PA (6; this study), protein PB (1,25,42), and protein Pc (31,42) of E. acidaminophilum or C. sticklandii. After chromatography of cell extract on DEAE at pH 7.8, both the nonbinding and the binding protein fractions were tested for glycine reductase activity.…”

Section: I L Q 0 K K V I a I G D R D G I P G P A I E E C V K S A G mentioning

confidence: 94%

“…Enzymes, coenzymes, and a glycan detection kit were obtained from Boehringer Mannheim (Mannheim, Germany); molecular weight marker kit SDS-70L and alkaline phosphatase-conjugated goat-anti-rabbit immunoglobulin G (IgG) were from Sigma GmbH (Deisenhofen, Germany); molecular weight marker kit HMW and LMW, Sephacryl S-100 HR, DEAE-Sephacel, Sephadex G-100, Sepharose Q, and protein A-and protein G-Sepharose CL-6B were from Pharmacia (Freiburg, Germany); DLlipoamide was from Serva (Heidelberg, Germany); DEAEcellulose (Whatman DE-52) was from Whatman Ltd. (Maidstone, England); hydroxylapatite Bio-Gel-HTP was from Bio-Rad Laboratories (Munchen, Germany); and [75Se]selenite was from Amersham Buchler (Braunschweig, Germany). All other chemicals were of the highest purity available from commercial sources or prepared as described previously (16). Antibodies raised against the thioredoxin reductase-like flavoprotein and thioredoxin were prepared as described previously (16,17).…”

mentioning

confidence: 99%

“…All other chemicals were of the highest purity available from commercial sources or prepared as described previously (16). Antibodies raised against the thioredoxin reductase-like flavoprotein and thioredoxin were prepared as described previously (16,17). Thioredoxin was prepared as described before (26).…”

mentioning

confidence: 99%

“…The assay is based on the dihydrolipoamide dehydrogenase activity of the system thioredoxin reductase-like flavoprotein plus thioredoxin that was further stimulated by protein PA (further-stimulation assay). Protein PA was monitored by using an assay that contained, in a final volume of 1 ml; 50 mm potassium phosphate (pH 7.8), 1 mM Na2EDTA, 5 ,ug of thioredoxin reductase-like flavoprotein and 10 ,ug of thioredoxin (purified from E. acidaminophilum and C. litoralis, respectively [16,26]), and 2 mM dihydrolipoamide as an electron donor. After preincubation for 5 to 15 min at 30°C, the reaction was started by adding 0.5 mM NADP+, and the increase of NADPH was monitored at 340 nm.…”

mentioning

confidence: 99%

See 3 more Smart Citations

Interaction of selenoprotein PA and the thioredoxin system, components of the NADPH-dependent reduction of glycine in Eubacterium acidaminophilum and Clostridium litorale [corrected]

et al. 1991

Self Cite

View full text Add to dashboard Cite

Purification of protein PA of the glycine reductase complex from Eubacterium acidaminophUum and Clostridium litoralis was monitored by a new spectrophotometric assay. The procedure depended on a specific two-to threefold stimulation of a dihydrolipoamide dehydrogenase activity that is elicited by the interaction of a thioredoxin reductase-like flavoprotein and thioredoxin from both organisms. Protein PA isolated from E.acidaminophilum by 75 Se iabeling and monitoring of the dithioerythritol-dependent glycine reductase activity was identical in its biochemical, structural, and immunological properties to the protein isolated by using the stimulation assay. Proteins PA from both organisms were glycoproteins of Mr about 18,500 and exhibited very similar N-terminal amino acid sequences. Depletion of thioredoxin from crude extracts of E. acidaminophilum totally diminished the NADPH-dependent but not the dithioerythritol-dependent glycine reduction. The former activity could be fully restored by adding thioredoxin. Antibodies raised against the thioredoxin reductase-like flavoprotein or thioredoxin inhibited to a high extent NADPH-dependent but not dithioerythritol-dependent glycine reductase activity. These results indicate the involvement of the thioredoxin system in the electron flow from reduced pyridine nucleotides to glycine reductase.

show abstract

Section: I L Q 0 K K V I a I G D R D G I P G P A I E E C V K S A G mentioning

confidence: 94%

mentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

See 2 more Smart Citations

Interaction of selenoprotein PA and the thioredoxin system, components of the NADPH-dependent reduction of glycine in Eubacterium acidaminophilum and Clostridium litorale [corrected]

et al. 1991

Self Cite

View full text Add to dashboard Cite

show abstract

“…An affinity column was prepared by linking purified polyclonal antibodies against D-amino acid oxidase to activated CNBr-Sepharose 4B according to [25]. The crude extract was applied to it after the column had been equilibrated with buffer B.…”

Section: Methodsmentioning

confidence: 99%

Evidence for the functional importance of Cys298 in D‐amino acid oxidase from Trigonopsis variabilis

Schräder

Andreesen

1993

European Journal of Biochemistry

View full text Add to dashboard Cite

D-Amino acid oxidase from Trigonopsis variabilis was purified to homogeneity by a combination of freezekhawing, isoelectric precipitation and chromatography on Mono Q. This purification procedure required very little working effort. The homogeneous enzyme exhibited a ratio AZXJA45U of about 6.5 and was obtained in high yield (63%) and a good stability. Using D-methionine as a substrate, a specific activity of 120 U/mg was determined colorimetrically at 26 "C, corresponding to 185 U/mg polarographically at 37°C. Polyclonal antibodies were raised against the homogeneous protein and Western immunoblot analysis showed that the 39-kDa subunit can undergo defined cleavages at the carboxy terminus of amino acid positions 104, 106 and 108, leading to 27-kDa and 12-kDa fragments as revealed by SDSPAGE, which are still enzymically active in their native form. The enzyme was inactivated by all sulfhydryl-modifying reagents tested. Inactivation by 5,5'-dithiobis(-2-nitrobenzoate) was correlated with a modification of up to 2 mollmol protein of the six cysteine residues present in the monomer. Identification of the most reactive cysteine was achieved by inactivation of the enzyme with the fluorescent, sulfhydryl-modifying reagent monobromobimane. In the presence of a substrate amino acid, under anaerobic conditions, the protein could be protected from modification and, thus, inactivation by this reagent. Peptide mapping by reversephase chromatography of endoproteinase Glu-C-digested monobromobimane-labeled enzyme revealed one major fluorescence peak which was not obtained when the protein was modified in the presence of a substrate amino acid under anaerobic conditions. Isolation and sequencing of the labeled peptide led to the identification of Cys298 as the reactive cysteine residue. D-Amino acid oxidase catalyzes the oxidation of D-amino acids to their corresponding 2-imino acids which are subsequently hydrolysed non-enzymically to 2-0x0 acids and ammonia. Reoxidation of the enzyme by molecular oxygen is accompanied by the release of H,O,. The enzyme contains one FADlsubunit and belongs to the class of dehydrogenases/ oxidases [l]. This protein is of special importance in research and biotechnology 12-51 and was, therefore, subject to a diversity of investigations (for review see [2]). For a long time, the enzyme from pig kidney has been the only source of homogeneous D-amino acid oxidase 161. Recently, the enzyme from the yeast Rhodotorulu gracilis was purified to homogeneity and its properties were determined [7 -101. D-Amino acid oxidase activity was also detected in the yeast Trigonopsis vuriubilis, and the enzyme was purified to near homogeneity, but in contrast to R. gracilis the enzyme preparation showed a very low stability [ll]. Although the amino acid sequence of the enzyme is published [12] Chemical modification of D-amino acid oxidase from pig kidney by amino-acid-specific reagents led to the identification of histidine [15, 161, arginine [17], tyrosine [16, 181, lysine [18], methionine [19] and cysteine [20...

show abstract

Various functions of selenols and thiols in anaerobic Gram‐positive, amino acids‐utilizing bacteria

et al. 1999

View full text Add to dashboard Cite

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.

show abstract

Isolation of an atypically small lipoamide dehydrogenase involved in the glycine decarboxylase complex from Eubacterium acidaminophilum

Cited by 33 publications

References 58 publications

Interaction of selenoprotein PA and the thioredoxin system, components of the NADPH-dependent reduction of glycine in Eubacterium acidaminophilum and Clostridium litorale [corrected]

Interaction of selenoprotein PA and the thioredoxin system, components of the NADPH-dependent reduction of glycine in Eubacterium acidaminophilum and Clostridium litorale [corrected]

Evidence for the functional importance of Cys298 in D‐amino acid oxidase from Trigonopsis variabilis

Various functions of selenols and thiols in anaerobic Gram‐positive, amino acids‐utilizing bacteria

Contact Info

Product

Resources

About