Components of glycine reductase from Eubacterium acidaminophilum

Abstract

The genes encoding thioredoxin reductase (trxB), thioredoxin (trxA), protein PA of glycine reductase (&A) and the first 23 amino acids of the large subunit of protein P, of glycine reductase ( g r d o belonging to the reductive deamination systems present in Eubacterium acidaminophilum were cloned and sequenced. The proteins were products of closely linked genes with 314 codons (thioredoxin reductase), 110 codons (thioredoxin), and 158 codons (protein PA). The protein previously called 'atypically small lipoamide dehydrogenase' or 'electron transferring flavoprotein' could now conclusively be identified as a thioredoxin reductase (subunit mass of 34781 Da) by the alignment with the enzyme of Escherichia coli showing the same typical order of the corresponding domains. The thioredoxin (molecular mass of 11 742 Da) deviated considerably from the known consensus sequence, even in the most strongly conserved redox-active segment WCGPC that was now GCVPC. The selenocysteine of protein PA (molecular mass of 16609 Da) was encoded by TGA. The protein was highly similar to those of Clostridium purinolyticum and Clostridium sticklundii involved in glycine reductase. Thioredoxin reductase and thioredoxin of E. acidaminophilum could be successfully expressed in E. coli.Eubacterium acidaminophilum utilizes glycine as both electron donor and electron acceptor, whereas sarcosine and betaine are exclusively used as electron acceptor [l]. Due to this fact, this organism is well suited to compare the biochemistry of the unusual reductive deaminations and the energy conservation associated with it. Glycine reductase, sarcosine reductase, and betaine reductase seem to be generally composed of three proteins: selenoprotein PA, protein Ps, and protein Pc, of which only P, is substrate-specific [2-101 (Fig. 1). In the first step the substrate is bound to protein P, that is specific for glycine, sarcosine, or betaine (M. Meyer and J. R. Andreesen, unpublished results). In all three reductases, the P,-activated substrate is proposed to be nucleophilically attacked by the selenol anion of protein PA to transform it to a carboxymethylated selenoether [3] and the respective amine [4]. By action of protein Pc, acetyl phosphate is formed in all cases leaving the selenoprotein PA in its oxidized state [3, 9, lo]. Finally the selenoprotein PA becomes reduced by the thioredoxin system to start a new catalytic cycle of reductive deamination [7, 81 (Fig. 1). In E. aciduminophilum the NADPH-derived electrons are transferred to protein PA by two proteins, a thioredoxin-reductase-like flavoprotein and an atypical thioredoxin [7,

The Heterodisulfide Reductase from Methanobacterium Thermoautotrophicum Contains Sequence Motifs Characteristic of pyridine‐Nucleotide‐Dependent Thioredoxin Reductases

“…4. The sequence region containing the FAD-binding motif has highest similarity to the FAD-binding domain of thioredoxin reductase (TrxB) from Eubacterium acidaminophitum (Lubbers and Andreesen, 1993) and of lipoamide dehydrogenase (LPD) from Pisum sativum (Bourguignon et al, 1992). Within a stretch of 43 amino acids, HdrA was found to be 33% identical and 79% similar to the corresponding sequence of TrxB (Fig.…”

Thioredoxins: structure and function in plant cells

“…In addition, E. co/z thioredoxin is also essential for the assembly of filamentous phages (Russel & Model, 1985), and can participate in the reduction of selenium and hydroperoxides (Bjornstedt, Kumar & Holmgren, 1995 ;Cha, Kim & Kim, 1995). In other prokaryotic cells, thioredoxin has more specific functions such as being a component of the glycine and arsenate reduction systems and of the penicillin biosynthesis pathway (Lubbers & Andreesen, 1993;Cohen et al, 1993Cohen et al, , 1994Ji et al, 1994). Thioredoxin also has a general 'non specific' disulphide reductase function, reducing any accessible disulphide bridge on a given protein (Holmgren, 1989).…”

Identification of d-Proline Reductase fromClostridium sticklandiias a Selenoenzyme and Indications for a Catalytically Active Pyruvoyl Group Derived from a Cysteine Residue by Cleavage of a Proprotein

“…To start a new reaction cycle, protein A is reduced by the thioredoxin system that obtains its electrons from NADPH (14). The genes encoding protein A, protein B, protein C, thioredoxin, and thioredoxin reductase are organized in an operon-like structure (11,15). For D-proline reductase, the existence of an analogous high energy acyl-enzyme intermediate has been excluded, indicating that proline reduction is not coupled to energy conservation by substrate level phosphorylation (10).…”