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Jan R. Andreesen - One of the best experts on this subject based on the ideXlab platform.
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Clostridium sticklandii, a specialist in amino acid degradation:revisiting its metabolism through its genome sequence
BMC Genomics, 2010Co-Authors: Nuria Fonknechten, Jan R. Andreesen, Sébastien Chaussonnerie, Sabine Tricot, Aurélie Lajus, Nadia Perchat, Eric Pelletier, Michel Gouyvenoux, Valérie Barbe, Marcel SalanoubatAbstract:Background Clostridium sticklandii belongs to a cluster of non-pathogenic proteolytic clostridia which utilize amino acids as carbon and energy sources. Isolated by T.C. Stadtman in 1954, it has been generally regarded as a "gold mine" for novel biochemical reactions and is used as a model organism for studying metabolic aspects such as the Stickland reaction, coenzyme-B12- and selenium-dependent reactions of amino acids. With the goal of revisiting its carbon, nitrogen, and energy metabolism, and comparing studies with other clostridia, its genome has been sequenced and analyzed. Results C. sticklandii is one of the best biochemically studied proteolytic clostridial species. Useful additional information has been obtained from the sequencing and annotation of its genome, which is presented in this paper. Besides, experimental procedures reveal that C. sticklandii degrades amino acids in a preferential and sequential way. The organism prefers threonine, arginine, serine, cysteine, proline, and Glycine, whereas glutamate, aspartate and alanine are excreted. Energy conservation is primarily obtained by substrate-level phosphorylation in fermentative pathways. The reactions catalyzed by different ferredoxin oxidoReductases and the exergonic NADH-dependent reduction of crotonyl-CoA point to a possible chemiosmotic energy conservation via the Rnf complex. C. sticklandii possesses both the F-type and V-type ATPases. The discovery of an as yet unrecognized selenoprotein in the D-proline Reductase operon suggests a more detailed mechanism for NADH-dependent D-proline reduction. A rather unusual metabolic feature is the presence of genes for all the enzymes involved in two different CO_2-fixation pathways: C. sticklandii harbours both the Glycine synthase/Glycine Reductase and the Wood-Ljungdahl pathways. This unusual pathway combination has retrospectively been observed in only four other sequenced microorganisms. Conclusions Analysis of the C. sticklandii genome and additional experimental procedures have improved our understanding of anaerobic amino acid degradation. Several specific metabolic features have been detected, some of which are very unusual for anaerobic fermenting bacteria. Comparative genomics has provided the opportunity to study the lifestyle of pathogenic and non-pathogenic clostridial species as well as to elucidate the difference in metabolic features between clostridia and other anaerobes.
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Factors and selenocysteine insertion sequence requirements for the synthesis of selenoproteins from a gram-positive anaerobe in Escherichia coli.
Applied and environmental microbiology, 2007Co-Authors: Torsten Gursinsky, Jan R. Andreesen, Angelika Schierhorn, Daniel Gröbe, Jana Jäger, Brigitte SöhlingAbstract:Selenoprotein synthesis in Escherichia coli strictly depends on the presence of a specific selenocysteine insertion sequence (SECIS) following the selenocysteine-encoding UGA codon of the respective mRNA. It is recognized by the selenocysteine-specific elongation factor SelB, leading to cotranslational insertion of selenocysteine into the nascent polypeptide chain. The synthesis of three different selenoproteins from the gram-positive anaerobe Eubacterium acidaminophilum in E. coli was studied. Incorporation of 75Se into Glycine Reductase protein B (GrdB1), the peroxiredoxin PrxU, and selenophosphate synthetase (SelD1) was negligible in an E. coli wild-type strain and was fully absent in an E. coli SelB mutant. Selenoprotein synthesis, however, was strongly increased if selB and selC (tRNASec) from E. acidaminophilum were coexpressed. Putative secondary structures downstream of the UGA codons did not show any sequence similarity to each other or to the E. coli SECIS element. However, mutations in these structures strongly reduced the amount of 75Se-labeled protein, indicating that they indeed act as SECIS elements. UGA readthrough mediated by the three different SECIS elements was further analyzed using gst-lacZ translational fusions. In the presence of selB and selC from E. acidaminophilum, UGA readthrough was 36 to 64% compared to the respective cysteine-encoding UGC variant. UGA readthrough of SECIS elements present in Desulfomicrobium baculatum (hydV), Treponema denticola (selD), and Campylobacter jejuni (selW-like gene) was also considerably enhanced in the presence of E. acidaminophilum selB and selC. This indicates recognition of these SECIS elements and might open new perspectives for heterologous selenoprotein synthesis in E. coli.
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Peroxidase activity of selenoprotein GrdB of Glycine Reductase and stabilisation of its integrity by components of proprotein GrdE from Eubacterium acidaminophilum
Archives of Microbiology, 2007Co-Authors: Tina Gröbe, Brigitte Söhling, Michael Reuter, Torsten Gursinsky, Jan R. AndreesenAbstract:The anaerobe Eubacterium acidaminophilum has been shown to contain an uncharacterized peroxidase, which may serve to protect the sensitive selenoproteins in that organism. We purified this peroxidase and found that it was identical with the substrate-specific “protein B”-complex of Glycine Reductase. The “protein B”-complex consists of the selenocysteine-containing GrdB subunit and two subunits, which derive from the GrdE proprotein. The specific peroxidase activity was 1.7 U (mg protein)^−1 with DTT and cumene hydroperoxide as substrates. Immunoprecipitation experiments revealed that GrdB was important for DTT- and NADH-dependent peroxidase activities in crude extracts, whereas the selenoperoxiredoxin PrxU could be depleted without affecting these peroxidase activities. GrdB could be heterologously produced in Escherichia coli with coexpression of selB and selC from E. acidaminophilum for selenocysteine insertion. Although GrdB was sensitive to proteolysis, some full-size protein was present which accounted for a peroxidase activity of about 0.5 U (mg protein)^−1 in these extracts. Mutation of the potentially redox-active UxxCxxC motif in GrdB resulted in still significant, but decreased activity. Heterologous GrdB was protected from degradation by full-length GrdE or by GrdE-domains. The GrdB-GrdE interaction was confirmed by copurification of GrdE with Strep -tagged GrdB. The data suggest that GrdE domains serve to stabilise GrdB.
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Peroxidase activity of selenoprotein GrdB of Glycine Reductase and stabilisation of its integrity by components of proprotein GrdE from Eubacterium acidaminophilum
Archives of Microbiology, 2007Co-Authors: Tina Gröbe, Brigitte Söhling, Michael Reuter, Torsten Gursinsky, Jan R. AndreesenAbstract:The anaerobe Eubacterium acidaminophilum has been shown to contain an uncharacterized peroxidase, which may serve to protect the sensitive selenoproteins in that organism. We purified this peroxidase and found that it was identical with the substrate-specific “protein B”-complex of Glycine Reductase. The “protein B”-complex consists of the selenocysteine-containing GrdB subunit and two subunits, which derive from the GrdE proprotein. The specific peroxidase activity was 1.7 U (mg protein)^−1 with DTT and cumene hydroperoxide as substrates. Immunoprecipitation experiments revealed that GrdB was important for DTT- and NADH-dependent peroxidase activities in crude extracts, whereas the selenoperoxiredoxin PrxU could be depleted without affecting these peroxidase activities. GrdB could be heterologously produced in Escherichia coli with coexpression of selB and selC from E. acidaminophilum for selenocysteine insertion. Although GrdB was sensitive to proteolysis, some full-size protein was present which accounted for a peroxidase activity of about 0.5 U (mg protein)^−1 in these extracts. Mutation of the potentially redox-active UxxCxxC motif in GrdB resulted in still significant, but decreased activity. Heterologous GrdB was protected from degradation by full-length GrdE or by GrdE-domains. The GrdB-GrdE interaction was confirmed by copurification of GrdE with Strep -tagged GrdB. The data suggest that GrdE domains serve to stabilise GrdB.
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Glycine Reductase mechanism
Current opinion in chemical biology, 2004Co-Authors: Jan R. AndreesenAbstract:The ability of some anaerobic bacteria to conserve energy via a soluble substrate level phosphorylation system by reducing Glycine to acetyl-phosphate has been an intriguing mechanism for about half a century. The genes implicated in this system have been sequenced and form an operon structure with those of the thioredoxin system. The deduced proteins exhibit high degrees of similarity with Glycine Reductase from other bacteria. Faster progress in understanding the exact mechanisms is hampered, for example, by some unique reactions involving selenoethers and redox active selenocysteines, which do not allow an easy heterologous formation in Escherichia coli. Further major obstacles are the processing of a substrate-specific pro-protein to a new carbonyl/pyruvoyl group in one of the two peptides formed that stabilize the substrate-binding selenoprotein, which contains an additional rather unstable carbonyl group.
Thressa C Stadtman - One of the best experts on this subject based on the ideXlab platform.
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selenoproteins tracing the role of a trace element in protein function
PLOS Biology, 2005Co-Authors: Thressa C StadtmanAbstract:In retrospect, the history of selenium biochemistry does not differ greatly from the study of a number of other trace elements in that it occurred over many years, progressing from periods of little general interest to widespread concern regarding toxicity problems and eventually to recognition of selenium as an essential nutrient for many forms of life. My involvement in studies on selenium metabolism is a classic example of serendipity. I was studying an interesting enzyme from an anaerobic bacterium that utilized Glycine as substrate (Glycine Reductase), but the amounts of pure protein I could isolate were very limited because it seemed to be produced only during the very early stages of bacterial cell growth. The rich culture medium supported continued luxuriant growth of the bacterium, but the level of the desired enzyme in the cells merely underwent dilution during the process. Finally, after testing many known growth stimulatory supplements to no avail, my colleagues and I tried two inorganic nutrients, molybdate and selenite. This approach was prompted by the report that addition of these inorganic compounds to a medium used for anaerobic growth of Escherichia coli supported synthesis of the enzyme formate dehydrogenase [1]. Much to my delight, addition of selenite to our growth medium resulted in greatly increased levels of the Glycine Reductase enzyme, and synthesis of the protein continued throughout the entire growth period. Thus, a common belief among microbiologists that a so-called rich culture medium high in tryptone and yeast extract content was nutritionally adequate is incorrect if growth of the organism depends on ability to synthesize a selenium-containing enzyme. In our case other amino acids in the medium could support growth even when the trace of selenium was depleted and synthesis of Glycine Reductase stopped. With this unexpected finding, we had an ideal biological system for unraveling details of the role of selenium in the anaerobic metabolism of Glycine.
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Some Functions of the Essential Trace Element, Selenium
Trace Elements in Man and Animals 10, 2002Co-Authors: Thressa C StadtmanAbstract:In recent years there have been impressive increases in information concerning selenium biochemistry. After the initial discoveries that bacteria (Pinsent, 1954) and animals (Patterson et al., 1957; Schwarz and Foltz, 1957) require this trace element, almost 20 years elapsed before selenium was demonstrated to be an essential component of a mammalian enzyme, glutathione peroxidase,(Flohe et al., 1973; Rotruck et al., 1973) and a protein component of the Clostridial Glycine Reductase complex (Turner and Stadtman, 1973). Selenocysteine was determined to be the chemical form of selenium present in Glycine Reductase (Cone et al., 1976). Other prokaryotic selenoenzymes, several formate dehydrogenases (Stadtman, 1979, 1980a, 1980b, 1990 and 1996), a hydrogenase of Methanococcus vannielii (Yamazaki, 1982) and a clostridial nicotinic acid hydroxylase (Dilworth, 1982) were discovered. Mammalian glutathione peroxidase isoenzymes (Ursini et al., 1995) and selenoprotein P, a glycoprotein of unknown function, were isolated (Hill et al., 1991). Selenoprotein P is unusual in that it contains 10 selenocysteine residues in the polypeptide chain. Selenoprotein W, a 10 kDa muscle protein of unknown function was purified from normal muscle and partially characterized (Vendeland et al., 1993; Vendeland et al., 1995). The function of selenoprotein W is unknown but the occurrence of the selenocysteine residue in a Cys-x-x-Secys motif is suggestive of a redox role. The importance of selenium in mammalian physiology was magnified by the discovery of its occurrence in a 5' deiodinase that converts the prothyroid hormone, tetraiodothyronine, to the active hormone, 3,5,3'-tri-iodothyronine (Berry et al., 1991) and this established a direct role of selenium in eukaryotic developmental processes. Several years after the identification of selenocysteine in selenoproteins this unusual amino acid was shown to be inserted cotranslationally. An in-frame TGA codon was
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Bacterial selenoenzymes and mechanisms of action
Selenium, 2001Co-Authors: Thressa C StadtmanAbstract:There are two types of bacterial selenoenzymes known at present: those that contain selenium in the form of selenocysteine residues in the polypeptide chains and those that instead contain a catalytically essential selenium in a dissociable cofactor form. Among the latter are the molybdopterin containing enzymes, nicotinic acid hydroxylase, xanthine dehydrogenase and the recently discovered purine hydroxylase. The crystal structure of a related enzyme, carbon monooxide dehydrogenase, has been determined. This enzyme, unlike the other three, is present in various aerobic bacteria. Escherichia coli formate dehydrogenase is a selenium-dependent enzyme that contains selenocysteine, molybdenum, a molybdopterin-guanine dinucleotide and an iron-sulfur center. The crystal structures of the oxidized and reduced enzymes have been reported. This enzyme differs from classical molybdenum hydroxylases in that oxygen from water is not incorporated in the product. Clostridial Glycine Reductase is a selenocysteine-dependent enzyme that reduces Glycine to acetylphosphate and ammonia. The reaction mechanism of this enzyme involves the cleavage of a carbon-nitrogen bond of the Schiff base derivative of the Glycine substrate and represents a new role of the ionized selenol group of the selenocysteine residue in metabolism. Selenium-carboxymethylselenocysteine is formed as the enzyme-bound intermediate on the selenoprotein A subunit of the enzyme complex in this reaction. Reductive cleavage of the selenoether, transfer to a cysteine residue of another subunit forming an acetylthioester and reaction with phosphate results in the formation of acetylphosphate.
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Chapter 10. Bacterial selenoenzymes and mechanisms of action
2001Co-Authors: Thressa C StadtmanAbstract:Summary: There are two types of bacterial selenoenzymes known at present: those that contain selenium in the form of selenocysteine residues in the polypeptide chains and those that instead contain a catalytically essential selenium in a dissociable cofactor form. Among the latter are the molybdopterin containing enzymes, nicotinic acid hydroxylase, xanthine dehydrogenase and the recently discovered purine hydroxylase. The crystal structure of a related enzyme, carbon monooxide dehydrogenase, has been determined. This enzyme, unlike the other three, is present in various aerobic bacteria. Escherichia coli formate dehydrogenase is a selenium-dependent enzyme that contains selenocysteine, molybdenum, a molybdopterin-guanine dinucleotide and an iron-sulfur center. The crystal structures of the oxidized and reduced enzymes have been reported. This enzyme differs from classical molybdenum hydroxy lases in that oxygen from water is not incorporated in the product. Clostridial Glycine Reductase is a selenocysteine-dependent enzyme that reduces Glycine to acetylphosphate and ammonia. The reaction mechanism of this enzyme involves the cleavage of a carbon-nitrogen bond of the Schiff base derivative of the Glycine substrate and represents a new role ofthe ionized selenol group of the selenocysteine residue in metabolism. Selenium carboxymethylselenocysteine is formed as the enzyme-bound intermediate on the selenoprotein A subunit of the enzyme complex in this reaction. Reductive cleavage of the selenoether, transfer to a cysteine residue of another subunit forming an acetylthioester and reaction with phosphate results in the formation of acetylphosphate.
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Glycine Reductase selenoprotein A is not a glycoprotein: the positive periodic acid-Schiff reagent test is the result of peptide bond cleavage and carbonyl group generation.
Proceedings of the National Academy of Sciences of the United States of America, 1995Co-Authors: Yoshinobu Kimura, Thressa C StadtmanAbstract:Abstract The complete amino acid sequence of Clostridium sticklandii selenoprotein A, a selenocysteine-containing protein component of the Glycine Reductase complex, has been established. Both the intact protein and peptide fragments produced by Staphylococcus aureus V8 protease or trypsin were purified by reversed-phase high-performance liquid chromatography and subjected to electrospray ionization mass spectrometric analysis and standard Edman degradation. Selenoprotein A consists of 157 amino acids with a chemical molecular weight of 17,011, in reasonable agreement with the observed molecular weight (17,022.7) determined from its ionization mass spectrum. The sequence of the amino-terminal region of the isolated native protein is Ser-Arg-Phe-Thr-Gly-Lys- Lys-Ile-Val-Ile-Ile-Gly-Asp-Arg-Asp-. An N-terminal methionine residue deduced from the gene sequence was not present. Although selenoprotein A reacted positively in a glycoprotein stain when using either the periodic acid-Schiff reagent procedure or a commercial glycan detection kit, no saccharide was detected by carbohydrate analyses after acid hydrolysis or methanolysis. Identity of the amino acid sequence determined by analysis with that deduced from the gene sequence is further evidence of the absence of bound carbohydrate.
Brigitte Söhling - One of the best experts on this subject based on the ideXlab platform.
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Factors and selenocysteine insertion sequence requirements for the synthesis of selenoproteins from a gram-positive anaerobe in Escherichia coli.
Applied and environmental microbiology, 2007Co-Authors: Torsten Gursinsky, Jan R. Andreesen, Angelika Schierhorn, Daniel Gröbe, Jana Jäger, Brigitte SöhlingAbstract:Selenoprotein synthesis in Escherichia coli strictly depends on the presence of a specific selenocysteine insertion sequence (SECIS) following the selenocysteine-encoding UGA codon of the respective mRNA. It is recognized by the selenocysteine-specific elongation factor SelB, leading to cotranslational insertion of selenocysteine into the nascent polypeptide chain. The synthesis of three different selenoproteins from the gram-positive anaerobe Eubacterium acidaminophilum in E. coli was studied. Incorporation of 75Se into Glycine Reductase protein B (GrdB1), the peroxiredoxin PrxU, and selenophosphate synthetase (SelD1) was negligible in an E. coli wild-type strain and was fully absent in an E. coli SelB mutant. Selenoprotein synthesis, however, was strongly increased if selB and selC (tRNASec) from E. acidaminophilum were coexpressed. Putative secondary structures downstream of the UGA codons did not show any sequence similarity to each other or to the E. coli SECIS element. However, mutations in these structures strongly reduced the amount of 75Se-labeled protein, indicating that they indeed act as SECIS elements. UGA readthrough mediated by the three different SECIS elements was further analyzed using gst-lacZ translational fusions. In the presence of selB and selC from E. acidaminophilum, UGA readthrough was 36 to 64% compared to the respective cysteine-encoding UGC variant. UGA readthrough of SECIS elements present in Desulfomicrobium baculatum (hydV), Treponema denticola (selD), and Campylobacter jejuni (selW-like gene) was also considerably enhanced in the presence of E. acidaminophilum selB and selC. This indicates recognition of these SECIS elements and might open new perspectives for heterologous selenoprotein synthesis in E. coli.
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Peroxidase activity of selenoprotein GrdB of Glycine Reductase and stabilisation of its integrity by components of proprotein GrdE from Eubacterium acidaminophilum
Archives of Microbiology, 2007Co-Authors: Tina Gröbe, Brigitte Söhling, Michael Reuter, Torsten Gursinsky, Jan R. AndreesenAbstract:The anaerobe Eubacterium acidaminophilum has been shown to contain an uncharacterized peroxidase, which may serve to protect the sensitive selenoproteins in that organism. We purified this peroxidase and found that it was identical with the substrate-specific “protein B”-complex of Glycine Reductase. The “protein B”-complex consists of the selenocysteine-containing GrdB subunit and two subunits, which derive from the GrdE proprotein. The specific peroxidase activity was 1.7 U (mg protein)^−1 with DTT and cumene hydroperoxide as substrates. Immunoprecipitation experiments revealed that GrdB was important for DTT- and NADH-dependent peroxidase activities in crude extracts, whereas the selenoperoxiredoxin PrxU could be depleted without affecting these peroxidase activities. GrdB could be heterologously produced in Escherichia coli with coexpression of selB and selC from E. acidaminophilum for selenocysteine insertion. Although GrdB was sensitive to proteolysis, some full-size protein was present which accounted for a peroxidase activity of about 0.5 U (mg protein)^−1 in these extracts. Mutation of the potentially redox-active UxxCxxC motif in GrdB resulted in still significant, but decreased activity. Heterologous GrdB was protected from degradation by full-length GrdE or by GrdE-domains. The GrdB-GrdE interaction was confirmed by copurification of GrdE with Strep -tagged GrdB. The data suggest that GrdE domains serve to stabilise GrdB.
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Peroxidase activity of selenoprotein GrdB of Glycine Reductase and stabilisation of its integrity by components of proprotein GrdE from Eubacterium acidaminophilum
Archives of Microbiology, 2007Co-Authors: Tina Gröbe, Brigitte Söhling, Michael Reuter, Torsten Gursinsky, Jan R. AndreesenAbstract:The anaerobe Eubacterium acidaminophilum has been shown to contain an uncharacterized peroxidase, which may serve to protect the sensitive selenoproteins in that organism. We purified this peroxidase and found that it was identical with the substrate-specific “protein B”-complex of Glycine Reductase. The “protein B”-complex consists of the selenocysteine-containing GrdB subunit and two subunits, which derive from the GrdE proprotein. The specific peroxidase activity was 1.7 U (mg protein)^−1 with DTT and cumene hydroperoxide as substrates. Immunoprecipitation experiments revealed that GrdB was important for DTT- and NADH-dependent peroxidase activities in crude extracts, whereas the selenoperoxiredoxin PrxU could be depleted without affecting these peroxidase activities. GrdB could be heterologously produced in Escherichia coli with coexpression of selB and selC from E. acidaminophilum for selenocysteine insertion. Although GrdB was sensitive to proteolysis, some full-size protein was present which accounted for a peroxidase activity of about 0.5 U (mg protein)^−1 in these extracts. Mutation of the potentially redox-active UxxCxxC motif in GrdB resulted in still significant, but decreased activity. Heterologous GrdB was protected from degradation by full-length GrdE or by GrdE-domains. The GrdB-GrdE interaction was confirmed by copurification of GrdE with Strep -tagged GrdB. The data suggest that GrdE domains serve to stabilise GrdB.
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Cys359 of GrdD is the active-site thiol that catalyses the final step of acetyl phosphate formation by Glycine Reductase from Eubacterium acidaminophilum.
European journal of biochemistry, 2001Co-Authors: Ulf-martin Kohlstock, Jan R. Andreesen, Michael Reuter, Karl Peter Rücknagel, Angelika Schierhorn, Brigitte SöhlingAbstract: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.
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Substrate-specific selenoprotein B of Glycine Reductase from Eubacterium acidaminophilum. Biochemical and molecular analysis.
European journal of biochemistry, 1999Co-Authors: Matthias Wagner, Andreas Pich, Denise Sonntag, Rudolf Grimm, Christoph Eckerskorn, Brigitte Söhling, Jan R. AndreesenAbstract: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.
Laurent Bouillaut - One of the best experts on this subject based on the ideXlab platform.
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Role of the global regulator Rex in control of NAD + ‐regeneration in Clostridioides (Clostridium) difficile
Molecular Microbiology, 2019Co-Authors: Laurent Bouillaut, Thomas Dubois, Michael Francis, Nadine Daou, Marc Monot, Joseph Sorg, Abraham Sonenshein, Bruno DupuyAbstract:For the human pathogen Clostridioides (also known as Clostridium) difficile, the ability to adapt to nutrient availability is critical for its proliferation and production of toxins during infection. Synthesis of the toxins is regulated by the availability of certain carbon sources, fermentation products and amino acids (e.g. proline, cysteine, isoleucine, leucine and valine). The effect of proline is attributable at least in part to its role as an inducer and substrate of D-proline Reductase (PR), a Stickland reaction that regenerates NAD+ from NADH. Many Clostridium spp. use Stickland metabolism (co-fermentation of pairs of amino acids) to generate ATP and NAD+ . Synthesis of PR is activated by PrdR, a proline-responsive regulatory protein. Here we report that PrdR, in the presence of proline, represses other NAD+ -generating pathways, such as the Glycine Reductase and succinate-acetyl CoA utilization pathways leading to butyrate production, but does so indirectly by affecting the activity of Rex, a global redox-sensing regulator that responds to the NAD+ /NADH ratio. Our results indicate that PR activity is the favored mechanism for NAD+ regeneration and that both Rex and PrdR influence toxin production. Using the hamster model of C. difficile infection, we revealed the importance of PrdR-regulated Stickland metabolism in the virulence of C. difficile.
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Role of the global regulator Rex in control of NAD+ -regeneration in Clostridioides (Clostridium) difficile.
Molecular microbiology, 2019Co-Authors: Laurent Bouillaut, Abraham L. Sonenshein, Thomas Dubois, Nadine Daou, Marc Monot, Joseph Sorg, Michael B. Francis, Bruno DupuyAbstract:For the human pathogen Clostridioides (also known as Clostridium) difficile, the ability to adapt to nutrient availability is critical for its proliferation and production of toxins during infection. Synthesis of the toxins is regulated by the availability of certain carbon sources, fermentation products and amino acids (e.g. proline, cysteine, isoleucine, leucine and valine). The effect of proline is attributable at least in part to its role as an inducer and substrate of D-proline Reductase (PR), a Stickland reaction that regenerates NAD+ from NADH. Many Clostridium spp. use Stickland metabolism (co-fermentation of pairs of amino acids) to generate ATP and NAD+ . Synthesis of PR is activated by PrdR, a proline-responsive regulatory protein. Here we report that PrdR, in the presence of proline, represses other NAD+ -generating pathways, such as the Glycine Reductase and succinate-acetyl CoA utilization pathways leading to butyrate production, but does so indirectly by affecting the activity of Rex, a global redox-sensing regulator that responds to the NAD+ /NADH ratio. Our results indicate that PR activity is the favored mechanism for NAD+ regeneration and that both Rex and PrdR influence toxin production. Using the hamster model of C. difficile infection, we revealed the importance of PrdR-regulated Stickland metabolism in the virulence of C. difficile.
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Proline-Dependent Regulation of Clostridium difficile Stickland Metabolism
Journal of bacteriology, 2012Co-Authors: Laurent Bouillaut, William T. Self, Abraham L. SonensheinAbstract:Clostridium difficile, a proteolytic Gram-positive anaerobe, has emerged as a significant nosocomial pathogen. Stickland fermentation reactions are thought to be important for growth of C. difficile and appear to influence toxin production. In Stickland reactions, pairs of amino acids donate and accept electrons, generating ATP and reducing power in the process. Reduction of the electron acceptors proline and Glycine requires the d-proline Reductase (PR) and the Glycine Reductase (GR) enzyme complexes, respectively. Addition of proline in the medium increases the level of PR protein but decreases the level of GR. We report the identification of PrdR, a protein that activates transcription of the PR-encoding genes in the presence of proline and negatively regulates the GR-encoding genes. The results suggest that PrdR is a central metabolism regulator that controls preferential utilization of proline and Glycine to produce energy via the Stickland reactions.
Bruno Dupuy - One of the best experts on this subject based on the ideXlab platform.
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Role of the global regulator Rex in control of NAD + ‐regeneration in Clostridioides (Clostridium) difficile
Molecular Microbiology, 2019Co-Authors: Laurent Bouillaut, Thomas Dubois, Michael Francis, Nadine Daou, Marc Monot, Joseph Sorg, Abraham Sonenshein, Bruno DupuyAbstract:For the human pathogen Clostridioides (also known as Clostridium) difficile, the ability to adapt to nutrient availability is critical for its proliferation and production of toxins during infection. Synthesis of the toxins is regulated by the availability of certain carbon sources, fermentation products and amino acids (e.g. proline, cysteine, isoleucine, leucine and valine). The effect of proline is attributable at least in part to its role as an inducer and substrate of D-proline Reductase (PR), a Stickland reaction that regenerates NAD+ from NADH. Many Clostridium spp. use Stickland metabolism (co-fermentation of pairs of amino acids) to generate ATP and NAD+ . Synthesis of PR is activated by PrdR, a proline-responsive regulatory protein. Here we report that PrdR, in the presence of proline, represses other NAD+ -generating pathways, such as the Glycine Reductase and succinate-acetyl CoA utilization pathways leading to butyrate production, but does so indirectly by affecting the activity of Rex, a global redox-sensing regulator that responds to the NAD+ /NADH ratio. Our results indicate that PR activity is the favored mechanism for NAD+ regeneration and that both Rex and PrdR influence toxin production. Using the hamster model of C. difficile infection, we revealed the importance of PrdR-regulated Stickland metabolism in the virulence of C. difficile.
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Role of the global regulator Rex in control of NAD+ -regeneration in Clostridioides (Clostridium) difficile.
Molecular microbiology, 2019Co-Authors: Laurent Bouillaut, Abraham L. Sonenshein, Thomas Dubois, Nadine Daou, Marc Monot, Joseph Sorg, Michael B. Francis, Bruno DupuyAbstract:For the human pathogen Clostridioides (also known as Clostridium) difficile, the ability to adapt to nutrient availability is critical for its proliferation and production of toxins during infection. Synthesis of the toxins is regulated by the availability of certain carbon sources, fermentation products and amino acids (e.g. proline, cysteine, isoleucine, leucine and valine). The effect of proline is attributable at least in part to its role as an inducer and substrate of D-proline Reductase (PR), a Stickland reaction that regenerates NAD+ from NADH. Many Clostridium spp. use Stickland metabolism (co-fermentation of pairs of amino acids) to generate ATP and NAD+ . Synthesis of PR is activated by PrdR, a proline-responsive regulatory protein. Here we report that PrdR, in the presence of proline, represses other NAD+ -generating pathways, such as the Glycine Reductase and succinate-acetyl CoA utilization pathways leading to butyrate production, but does so indirectly by affecting the activity of Rex, a global redox-sensing regulator that responds to the NAD+ /NADH ratio. Our results indicate that PR activity is the favored mechanism for NAD+ regeneration and that both Rex and PrdR influence toxin production. Using the hamster model of C. difficile infection, we revealed the importance of PrdR-regulated Stickland metabolism in the virulence of C. difficile.