A molybdenum-containing dehydrogenase catalyzing an unusual 2-hydroxylation of nicotinic acid.

An enzyme of Ralstonia/ Burkholderia strain DSM 6920 catalyzing the initial hydroxylation of 6-methylnicotinic acid at position 2 was purified to apparent homogeneity. It also catalyzed the unusual conversion of nicotinic acid to 2-hydroxynicotinic acid and was therefore designated as nicotinic acid dehydrogenase (NDH). Native NDH had a molecular mass of 280 kDa and was composed of subunits of 75, 30 and 16 kDa. It contained molybdenum, iron, acid-labile sulfur and FAD in a ratio of 1.6:7.3:8.0:0.6 mol(-1) of native enzyme. The molybdenum cofactor was characterized as molybdopterin cytosine dinucleotide. Zinc was identified as an additional metal ion in a molar ratio of 1.8 mol mol(-1) of native enzyme. Purified NDH exhibited a maximal specific activity of 22.6 micromol nitro blue tetrazoliumchloride reduced min(-1) mg(-1) of protein, using nicotinic acid as electron donor. The apparent K(m) value for nicotinic acid was determined to be 154 microM. Pyridine-3,5-dicarboxylic acid and quinoline-3-carboxylic acid were further substrates, but exhibited significantly different activity pH optima. Several artificial electron acceptors were reduced by NDH, but no activity was detected with NAD or O(2). NDH was inactivated upon incubation with cyanide, but no loss of activity was obtained in the presence of arsenite.

Cys359 of GrdD is the active-site thiol that catalyses the final step of acetyl phosphate formation by glycine reductase from Eubacterium acidaminophilum.

In the amino-acid-fermenting anaerobe Eubacterium acidaminophilum, acetyl phosphate is synthesized by protein C of glycine reductase from a selenoprotein A-bound carboxymethyl-selenoether. We investigated specific thiols present in protein C for responsibility for acetyl phosphate liberation. After cloning of the genes encoding the large and the small subunit (grdC1, grdD1), they were expressed separately in Escherichia coli and purified as Strep-tag proteins. GrdD was the only subunit that catalysed arsenate-dependent hydrolysis of acetyl phosphate (up to 274 U.mg-1), whereas GrdC was completely inactive. GrdD contained two cysteine residues that were exchanged by site-directed mutagenesis. The GrdD(C98S) mutant enzyme still catalysed the hydrolysis of acetyl phosphate, but the GrdD(C359A) mutant enzyme was completely inactive. Next, these thiols were analysed further by chemical modification. After iodoacetate treatment of GrdD, the enzyme activity was lost, but in the presence of acetyl phosphate enzyme activity was protected. Subsequently, the inactivated carboxymethylated enzyme and the protected enzyme were both denatured, and the remaining thiols were pyridylethylated. Peptides generated by proteolytic cleavage were separated and subjected to mass spectrometry. Cys98 was not accessible to carboxymethylation by iodoacetate in the native enzyme in the presence or absence of the substrate, but could be alkylated after denaturation. Cys359, in contrast, was protected from carboxymethylation in the presence of acetyl phosphate, but became accessible to pyridylethylation upon prior denaturation of the protein. This clearly confirmed the catalytic role of Cys359 as the active site thiol of GrdD responsible for liberation of acetyl phosphate.

NAD(P)-dependent aldehyde dehydrogenases induced during growth of Ralstonia eutropha strain Bo on tetrahydrofurfuryl alcohol.

Different aldehyde dehydrogenases (AlDHs) were formed during growth of Ralstonia eutropha Bo on tetrahydrofurfuryl alcohol (THFA). One of these enzymes, AlDH 4, was purified and characterized as a homodimer containing no prosthetic groups, showing a strong substrate inhibition, and having an N-terminal sequence similar to those of various NAD(P)-dependent AlDHs. The conversion rate of THFA by the quinohemoprotein THFA dehydrogenase was increased by AlDH 4.

A cytochrome P450 and a ferredoxin isolated from Mycobacterium sp. strain HE5 after growth on morpholine.

A cytochrome P450 and an iron-sulfur protein, whose expression was specifically induced during growth of Mycobacterium sp. strain HE5 on morpholine as the sole source of carbon, nitrogen, and energy were purified to apparent homogeneity. Due to the lack of enzymatic activity, carbon monoxide difference spectra and determination of the acid-labile sulfur, respectively, were used to detect the proteins during purification. The cytochrome P450, designated P450mor, was characterized as a monomer with an apparent molecular mass of 44.7 kDa. The amino acid sequence of an internal peptide comprising 19 amino acids was identical to the sequence derived from a gene encoding a cytochrome P450 from Mycobacterium smegmatis mc(2)155 suggested to be involved in the utilization of piperidine and pyrrolidine. The iron-sulfur protein was characterized as a ferredoxin exhibiting a molecular mass of 6.8 kDa and named Fdmor. An identity of 48-77% was obtained for the 30 N-terminal amino acids of Fdmor and the corresponding sequences of different 3Fe-4S-ferredoxins known to be involved in P450-dependent reactions. From these data we concluded that growth of Mycobacterium sp. strain HE5 on morpholine led to the expression of a cytochrome P450-dependent monooxygenase system composed of at least two different proteins.

A selenocysteine-containing peroxiredoxin from the strictly anaerobic organism Eubacterium acidaminophilum.

A strongly 75Se-labeled 22 kDa protein detected previously showed in its N-terminal sequence the highest similarity to the family of thiol-dependent peroxidases, now called peroxiredoxins. The respective gene prxU was cloned and analyzed. prxU encodes a protein of 203 amino acids (22,470 Da) and contains an in-frame UGA codon (selenocysteine) at the position of the so far strictly conserved and catalytically active Cys47. The second conserved cysteine present in 2-Cys peroxiredoxins was replaced by alanine. Heterologous expression of the Eubacterium acid-aminophilum PrxU as a recombinant selenoprotein in Escherichia coli was not possible. A cysteine-encoding mutant gene, prxU47C, containing UGC instead of UGA was strongly expressed. This recombinant PrxU47C mutant protein was purified to homogeneity by its affinity tag, but was not active as a thiol-dependent peroxidase. The identification of prxU reveals that the limited class of natural selenoproteins may in certain organisms also include isoenzymes of peroxiredoxins, previously only known as non-selenoproteins containing catalytic cysteine residues.

The NADH-dependent reductase of a putative multicomponent tetrahydrofuran mono-oxygenase contains a covalently bound FAD.

NADH-cytochrome c oxidoreductase activity specifically expressed during growth on tetrahydrofuran was detected in cell extracts of Pseudonocardia sp. strain K1. The enzyme catalyzing this reaction was purified to apparent homogeneity by a three-step purification procedure. It was characterized as a monomer of apparent molecular mass 40 kDa. Spectroscopic studies indicated that it contains an iron-sulfur cluster and a flavin cofactor. An amount of 1 mol of flavin and 1 mol of iron was determined per mol of homogeneous protein. The N-terminal amino-acid sequence exhibited great similarity to the reductase component of various oxygenases. Cloning and sequencing of the corresponding gene designated as thmD revealed an ORF encoding a protein of 360 amino acids. An overall similarity of up to 38% was obtained to the NAD(P)H-acceptor reductase of several binuclear iron-containing mono-oxygenases. Conserved sequence motifs were identified that were similar to the chloroplast-type ferredoxin 2Fe-2S centre and to nucleotide-binding domains. Studies on the flavin cofactor showed that it could not be removed from the protein by denaturation, indicating a covalent attachment. Spectroscopic studies revealed that the flavin is at the FAD level and covalently bound to the protein via the flavin 8alpha-methyl group. Thus, the isolated reductase component is the first enzyme of this type for which a covalent attachment of the flavin has been observed.

In vitro processing of the proproteins GrdE of protein B of glycine reductase and PrdA of D-proline reductase from Clostridium sticklandii: formation of a pyruvoyl group from a cysteine residue.

GrdE and PrdA of Clostridium sticklandii are subunits of glycine reductase and D-proline reductase, respectively, that are processed post-translationally to form a catalytic active pyruvoyl group. The cleavage occurred on the N-terminal side of a cysteine residue, which is thus the precursor of a pyruvoyl moiety. Both proproteins could be over-expressed in Escherichia coli and conditions were developed for in vitro processing. GrdE could be expressed as full-size protein, whereas PrdA had to be truncated N-terminally to achieve successful over-expression. Both proproteins were cleaved at the in vivo observed cleavage site after addition of 200 mM NaBH4 in Tris buffer (pH 7.6) at room temperature as analysed by SDS/PAGE and MS. Cleavage of GrdE was observed with a half-time of approximately 30 min. Cys242, as the precursor of the pyruvoyl group in GrdE, was changed to alanine, serine, or threonine by site-directed mutagenesis. The Cys242-->Ser and Cys242-->Thr mutant proteins were also cleaved under similar conditions with extended half-times. However, the Cys242-->Ala mutant protein was not cleaved indicating a pivotal role of the thiol group of cysteine or hydroxyl group of serine and threonine during the processing of pyruvoyl group-dependent reductases.

Tungstate Uptake by a highly specific ABC transporter in Eubacterium acidaminophilum.

The Gram-positive anaerobe Eubacterium acidaminophilum contains at least two tungsten-dependent enzymes: viologen-dependent formate dehydrogenase and aldehyde dehydrogenase. (185)W-Labeled tungstate was taken up by this organism with a maximum rate of 0.53 pmol min(-)1 mg(-)1 of protein at 36 degrees C. The uptake was not affected by equimolar amounts of molybdate. The genes tupABC coding for an ABC transporter specific for tungstate were cloned in the downstream region of genes encoding a tungsten-containing formate dehydrogenase. The substrate-binding protein, TupA, of this putative transporter was overexpressed in Escherichia coli, and its binding properties toward oxyanions were determined by a native polyacrylamide gel retardation assay. Only tungstate induced a shift of TupA mobility, suggesting that only this anion was specifically bound by TupA. If molybdate and sulfate were added in high molar excess (>1000-fold), they were also slightly bound by TupA. The K(d) value for tungstate was determined to be 0.5 microm. The genes encoding the tungstate-specific ABC transporter exhibited highest similarities to putative transporters from Methanobacterium thermoautotrophicum, Haloferax volcanii, Vibrio cholerae, and Campylobacter jejuni. These five transporters represent a separate phylogenetic group of oxyanion ABC transporters as evident from analysis of the deduced amino acid sequences of the binding proteins. Downstream of the tupABC genes, the genes moeA, moeA-1, moaA, and a truncated moaC have been identified by sequence comparison of the deduced amino acid sequences. They should participate in the biosynthesis of the pterin cofactor that is present in molybdenum- and tungsten-containing enzymes except nitrogenase.

Analysis of a 2,4,6-trichlorophenol-dehalogenating enrichment culture and isolation of the dehalogenating member Desulfitobacterium frappieri strain TCP-A.

An anaerobic, 2,4,6-trichlorophenol ortho-dehalogenating mixed culture was enriched from sediment of the river Saale (Germany). Two isolated dechlorinating colonies (MK1 and MK2) consisted of rods of different lengths and thicknesses, indicating heterogeneity. Following subcultivation with thiosulfate as alternative electron acceptor and cocultivation with Clostridium celerecrescensT, the 2,4,6-trichlorophenol-dehalogenating bacterium Desulfitobacterium frappieri strain TCP-A was isolated and characterized regarding its taxonomic properties and the spectrum of chlorophenols that it dehalogenated. Four other bacterial strains were coenriched and identified as organisms with closest phylogenetic relatedness to the Clostridium type strains C. indolis, C. glycolicum, C. hydroxybenzoicum and C. sporosphaeroides (16S rDNA sequence identities of 99.5, 99.2, 94.4, and 93.5%, respectively). Amplified ribosomal DNA restriction analysis of the original dehalogenating cultures MK1 and MK2 (when not exposed to thiosulfate) confirmed the microbial heterogeneity and revealed the presence of two additional species related to the type strains of C. celerecrescens and Clostridium propionicum. Only one copy of the 16S rRNA genes of Desulfitobacterium frappieri in each of the clone libraries of MK1 and MK2 (containing 136 and 56 clones, respectively) was found by dot-blot hybridization, suggesting a relatively low number of the dehalogenating bacterium within the enrichment culture.

Molecular analysis of the grd operon coding for genes of the glycine reductase and of the thioredoxin system from Clostridium sticklandii.

A probe based on the sequence of the gene encoding selenoprotein A of glycine reductase of Clostridium sticklandii was used to obtain clones of adjacent DNA that encoded the other components of glycine reductase, proteins B and C, in addition to thioredoxin and thioredoxin reductase. The genes of the thioredoxin system and the glycine reductase were shown to be transcribed together, confirming an operon structure. In addition, a gene (grdX) encoding a 13.7-kDa protein of unknown function seemed to be associated with the reductase genes. Four potential promoters were identified by mapping the 5'-end of the mRNAs. The sequence of promoter P1 was shown to be similar to the sigma70 promoter consensus sequence. The other three promoters were similar to each other, but not to known promoter consensus sequences. The transcripts starting at each of the four promoters were terminated to about 80% at a predicted loop structure downstream of grdB; the remaining transcripts continued through this structure and covered the genes encoding both subunits of protein C and bmpA, a gene that was also expressed monocistronically.

Catalytic and molecular properties of the quinohemoprotein tetrahydrofurfuryl alcohol dehydrogenase from Ralstonia eutropha strain Bo.

The quinohemoprotein tetrahydrofurfuryl alcohol dehydrogenase (THFA-DH) from Ralstonia eutropha strain Bo was investigated for its catalytic properties. The apparent k(cat)/K(m) and K(i) values for several substrates were determined using ferricyanide as an artificial electron acceptor. The highest catalytic efficiency was obtained with n-pentanol exhibiting a k(cat)/K(m) value of 788 x 10(4) M(-1) s(-1). The enzyme showed substrate inhibition kinetics for most of the alcohols and aldehydes investigated. A stereoselective oxidation of chiral alcohols with a varying enantiomeric preference was observed. Initial rate studies using ethanol and acetaldehyde as substrates revealed that a ping-pong mechanism can be assumed for in vitro catalysis of THFA-DH. The gene encoding THFA-DH from R. eutropha strain Bo (tfaA) has been cloned and sequenced. The derived amino acid sequence showed an identity of up to 67% to the sequence of various quinoprotein and quinohemoprotein dehydrogenases. A comparison of the deduced sequence with the N-terminal amino acid sequence previously determined by Edman degradation analysis suggested the presence of a signal sequence of 27 residues. The primary structure of TfaA indicated that the protein has a tertiary structure quite similar to those of other quinoprotein dehydrogenases.

A selDABC cluster for selenocysteine incorporation in Eubacterium acidaminophilum.

The four genes required for selenocysteine incorporation were isolated from the gram-positive, amino acid-fermenting anaerobe Eubacterium acidaminophilum, which expresses various selenoproteins of different functions. The sel genes were located in an unique organization on a continuous fragment of genomic DNA in the order selD1 (selenophosphate synthetase 1), selA (selenocysteine synthase), selB (selenocysteine-specific elongation factor), and selC (selenocysteine-specific tRNA). A second gene copy, encoding selenophosphate synthetase 2 (selD2), was present on a separate fragment of genomic DNA. SelD1 and SelD2 were only 62.9% identical, but the two encoding genes, selD1 and selD2, contained an in-frame UGA codon encoding selenocysteine, which corresponds to Cys-17 of Escherichia coli SelD. The function of selA, selB, and selC from E. acidaminophilum was investigated by complementation of the respective E. coli deletion mutant strains and determined as the benzyl viologen-dependent formate dehydrogenase activity in these strains after anaerobic growth in the presence of formate. selA and selC from E. acidaminophilum were functional and complemented the respective mutant strains to 83% (selA) and 57% (selC) compared to a wild-type strain harboring the same plasmid. Complementation of the E. coli selB mutant was only observed when both selB and selC from E. acidaminophilum were present. Under these conditions, the specific activity of formate dehydrogenase was 55% of that of the wild type. Transformation of this selB mutant with selB alone was not sufficient to restore formate dehydrogenase activity.

Selenium, the element of the moon, in life on earth.

The present status of selenium biochemistry is reviewed with particular emphasis on biomedical problems related to the selenium status of humans and experimental animals. Historical milestones of selenium biochemistry starting from the identification of the first selenoenzymes up to the elucidation of prokaryotic and eukaryotic selenoprotein biosynthesis are compiled. Topical hypotheses on the biological role of selenium in general and of individual selenoproteins in respect to antioxidant defense, redox regulation of metabolic processes, thyroid function, spermatogenesis, oncogenesis, and atherogenesis are critically evaluated.

Tetrahydrofuran degradation by a newly isolated culture of Pseudonocardia sp. strain K1.

An organism capable to grow aerobically on tetrahydrofuran as sole source of carbon and energy was isolated from a waste water treatment plant. The organism designated as strain K1 was identified as Pseudonocardia sp. by chemotaxonomic and morphological characteristics as well as analysis of the gene encoding the 16S rRNA. The highest binary sequence similarity value of 99.0% was obtained to Pseudonocardia sulfidoxydans and Pseudonocardia hydrocarbonoxydans. Optimal growth with a doubling time of 14 h was observed at a tetrahydrofuran concentration of 20 mM and pH 7.0 at 28 degrees C. Under these conditions the substrate was completely degraded within 72 h. In situ concentrations of up to 60 mM were tolerated by the organism without a significantly increased doubling time. The strain also grew on diethyl ether, polyethylene glycol and on gamma-butyrolactone and 4-hydroxybutyrate - two potential intermediates in tetrahydrofuran degradation - as sole carbon and energy source.

Various functions of selenols and thiols in anaerobic gram-positive, amino acids-utilizing bacteria.

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.

Selenium-containing xanthine dehydrogenase from Eubacterium barkeri.

A specific dehydrogenase, different from nicotinic acid hydroxylase, was induced during growth of Eubacterium barkeri on xanthine. The protein designated as xanthine dehydrogenase was enriched 39-fold to apparent homogeneity using a three-step purification scheme. It exhibited an NADP-dependent specific activity of 164 micromol xanthine oxidized per min and per mg of protein. In addition it showed an NADPH-dependent oxidase and diaphorase activity. A molecular mass of 530 kDa was determined for the native enzyme and SDS/PAGE revealed three types of subunits with molecular masses of 17.5, 30 and 81 kDa indicating a dodecameric native structure. Molybdopterin was identified as the molybdenum-complexing cofactor using activity reconstitution experiments and fluorescence measurements after KI/I2 oxidation. The molecular mass of the cofactor indicated that it is of the dinucleotide type. The enzyme contained iron, acid-labile sulfur, molybdenum, tungsten, selenium and FAD at molar ratios of 17.5, 18.4, 2.3, 1.1, 0.95 and 2.8 per mol of native enzyme. Xanthine dehydrogenase was inactivated upon incubation with arsenite, cyanide and different purine analogs. Reconstitution experiments of xanthine dehydrogenase activity by addition of selenide and selenite performed with cyanide-inactivated enzyme and with chloramphenicol-treated cells, respectively, indicated that selenium is not attached to the protein in a covalently bound form such as selenocysteine.

Defluvibacter lusatiae gen. nov., sp. nov., a new chlorohenol-degrading member of the alpha-2 subgroup of proteobacteria.

The two Gram-negative bacterial strains S1 and S4 were isolated from activated sludge of an industrial waste water treatment plant and exhibited a stable capability to degrade 2,4-dichlorophenol, 4-chloro-2-methylphenol, 4-chlorophenol and phenol. The cells were short rods with a polar flagellum, being mesophilic, strictly aerobic, oxidase-positive, and chemoorganotrophic. They utilized a range of amino acids, but only a restricted number of carbohydrates. Reassociation experiments with DNA from strains S1 and S4 revealed high interstrain similarity, indicating, that both strains belong to the same species. The phylogenetic position was determined by comparison of the almost complete 16S rDNA sequence of strain S1 with sequences of related bacteria. Strain S1 clustered with members of the alpha-2 subgroup of the Proteobacteria by forming a separate lineage within the radiation of Mesorhizobium, Phyllobacterium and Sinorhizobium. Both strains can be differentiated from members of related taxa by a set of physiological and chemotaxonomic properties including the ability to grow with norvaline, L-tryptophan, putrescine, glutarate and malonate, and by the presence of spermidine as major polyamine and of 12:0 3OH as fatty acid. Strain S1 is described as type strain of a new species and assigned to a new genus with the proposed name Defluvibacter lusatiae.

Morpholine-induced formation of L-alanine dehydrogenase activity in Mycobacterium strain HE5.

An NAD-dependent, morpholine-stimulated L-alanine dehydrogenase activity was detected in crude extracts from morpholine-, pyrrolidine-, and piperidine-grown cells of Mycobacterium strain HE5. Addition of morpholine to the assay mixture resulted in an up to 4. 6-fold increase of L-alanine dehydrogenase activity when L-alanine was supplied at suboptimal concentration. L-alanine dehydrogenase was purified to near homogeneity using a four-step purification procedure. The native enzyme had a molecular mass of 160 kDa and contained one type of subunit with a molecular mass of 41 kDa, indicating a tetrameric structure. The sequence of 30 N-terminal amino acids was determined and showed a similarity of up to 81% to that of various alanine dehydrogenases. The pH optimum for the oxidative deamination of L-alanine, the only amino acid converted by the enzyme, was determined to be pH 10.1, and apparent Km values for L-alanine and NAD were 1.0 and 0.2 mM, respectively. Km values of 0. 6, 0.02, and 72 mM for pyruvate, NADH, and NH4+, respectively, were estimated at pH 8.7 for the reductive amination reaction.

Identification of D-proline reductase from Clostridium sticklandii as a selenoenzyme and indications for a catalytically active pyruvoyl group derived from a cysteine residue by cleavage of a proprotein.

Highly active D-proline reductase was obtained from Clostridium sticklandii by a modified purification scheme. The cytoplasmic enzyme had a molecular mass of about 870 kDa and was composed of three subunits with molecular masses of 23, 26, and 45 kDa. The 23-kDa subunit contained a carbonyl group at its N terminus, which could either be labeled with fluorescein thiosemicarbazide or removed by o-phenylenediamine; thus, N-terminal sequencing became feasible for this subunit. L-[14C]proline was covalently bound to the 23-kDa subunit if proline racemase and NaBH4 were added. Selenocysteine was detected in the 26-kDa subunit, which correlated with an observed selenium content of 10.6 g-atoms in D-proline reductase. No other non-proteinaceous cofactor was identified in the enzyme. A 4.8-kilobase pair (kb) EcoRI fragment was isolated and sequenced containing the two genes prdA and prdB. prdA coding for a 68-kDa protein was most likely translated as a proprotein that was posttranslationally cleaved at a threonine-cysteine site to give the 45-kDa subunit and most probably a pyruvoyl-containing 23-kDa subunit. The gene prdB encoded the 26-kDa subunit and contained an in frame UGA codon for selenocysteine insertion. prdA and prdB were transcribed together on a transcript of 4.5 kb; prdB was additionally transcribed as indicated by a 0.8-kb mRNA species.

Substrate-specific selenoprotein B of glycine reductase from Eubacterium acidaminophilum. Biochemical and molecular analysis.

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