A Gold Mine of Fascinating Enzymes: Those Remarkable, Strictly Anaerobic Bacteria, Methanococcus vannielii andClostridium sticklandii
2002; Elsevier BV; Volume: 277; Issue: 51 Linguagem: Inglês
10.1074/jbc.x200005200
ISSN1083-351X
Autores Tópico(s)Methane Hydrates and Related Phenomena
ResumoI was fortunate to have had Dr. H. A. Barker as a mentor for my graduate studies at Berkeley. He had been trained by famous microbiologists from the Delft School of Microbiology, and he instilled in his students a deep interest in the metabolism of anaerobic microorganisms. Members of the Delft School had found that the microbial decomposition of various compounds under anaerobic conditions sometimes involved unusual chemical reactions that were amenable to detailed study because the responsible catalysts were present in exaggerated amounts or subsequent rate-limiting steps allowed intermediates to accumulate. By studying under Barker, who had worked with C. B. van Niel in Pacific Grove and later with A. J. Kluyver in Delft, I became an indirect descendent of the Delft microbiologists. For my thesis problem, I chose to work on the biosynthesis of methane, an area of research that I knew to be of considerable interest to Barker. Formate, acetate, and various fatty acids were added to simple mineral salts media for selection of organisms able to utilize these substrates for methane production. I used soil samples as inocula that I had dug from San Francisco Bay mud flats. At that time, the bay was heavily contaminated, and the mud flats reeked of hydrogen sulfide at low tide, a clear indication of anaerobic conditions. Microorganisms that grew on acetate, propionate, and short chain fatty acids were obtained, and these were studied using 14C-labeled substrates to determine the source of methane (1Stadtman T.C. Barker H.A. Studies on the methane fermentation. VII. Tracer experiments on the mechanism of methane formation..Arch. Biochem. 1949; 21: 256-264PubMed Google Scholar). Particularly interesting was the unexpected finding that in the fermentation of acetate methane was derived from the methyl carbon and the carboxyl carbon was the source of the carbon dioxide (2Stadtman T.C. Barker H.A. Studies on the methane fermentation. IX. The origin of methane in the acetate and methanol fermentations by Methanosarcina..J. Bacteriol. 1951; 61: 81-86Crossref PubMed Google Scholar). Barker (Fig.1) was particularly excited by these results because they were an exception to an earlier hypothesis of van Niel that methane is derived exclusively from carbon dioxide. However, in the other fatty acid fermentations carbon dioxide did serve as oxidant and was reduced to methane. Formate was actively fermented to a mixture of methane, hydrogen, and carbon dioxide. Two microorganisms were enriched in parallel when formate was supplied as substrate. One proved to be a methane-producing motile coccus that we named Methanococcus vannielii in honor of C. B. van Niel (3Stadtman T.C. Barker H.A. Studies on the methane fermentation X. A new formate-decomposing bacterium, Methanococcus vannielii..J. Bacteriol. 1951; 62: 269-280Crossref PubMed Google Scholar). The other was a rod-shaped organism that I later named Clostridium sticklandii (4Stadtman T.C. On the metabolism of an amino acid fermenting Clostridium..J. Bacteriol. 1954; 67: 314-320Crossref PubMed Google Scholar). Detailed studies on the morphology and biochemical properties ofM. vannielii (3Stadtman T.C. Barker H.A. Studies on the methane fermentation X. A new formate-decomposing bacterium, Methanococcus vannielii..J. Bacteriol. 1951; 62: 269-280Crossref PubMed Google Scholar) constituted a major portion of my Ph.D. thesis, and I was very gratified that the results of all of these studies were published with Barker in a series of papers on methane fermentations. When I first joined the newly formed National Heart Institute at NIH in 1950, I continued working on a project initiated during the year I spent as a postdoctoral fellow at Harvard Medical School. The oxidation of cholesterol using enzymes from an aerobicNocardia species was investigated. At that time, sterols formed by selective oxidation of the cholesterol side chain would have been useful precursors of cortisone and certain hormones such as progesterone. Unfortunately, cholesterol was degraded completely by the organism under a variety of conditions, and we could not detect any of the desired intermediate products. However, an enzyme that oxidized cholesterol at position 3 of the ring to form Δ-4-cholestene-3-one was identified and partially purified. Later, preparations of this cholesterol oxidase were produced by P-L Biochemicals, Inc. and used clinically for determination of cholesterol. After my unsuccessful experience with strictly aerobic bacteria as enzyme sources, I was glad to retreat to the anaerobic world and initiated studies on the fermentation of amino acid substrates by the clostridial species I had isolated along withM. vannielii from San Francisco Bay mud (4Stadtman T.C. On the metabolism of an amino acid fermenting Clostridium..J. Bacteriol. 1954; 67: 314-320Crossref PubMed Google Scholar). Initial studies on the anaerobic metabolism of lysine and ornithine (5Stadtman T.C. Lysine fermentation to fatty acids and ammonia: A cobamide coenzyme-dependent process..J. Biol. Chem. 1962; 237: 2409-2411Abstract Full Text PDF PubMed Google Scholar) and the roles of vitamin B12 in these processes (6Stadtman T.C. B12 coenzyme-dependent amino group migrations..The Enzymes. 1972; 6: 539-563Crossref Scopus (12) Google Scholar) provided examples of interesting new reactions and additional roles of B12coenzyme. The degradation of lysine to acetate, butyrate, and ammonia occurred by two distinct processes. In one, acetate was derived from carbon atoms 1 and 2 of the 6-carbon chain of l-lysine, and the residual 4 carbon atoms were converted to butyrate. In the other, acetate was derived from carbon atoms 5 and 6 ofd-lysine and butyrate from carbon atoms 1–4. These conversions required the participation of an imposing list of cofactors and involved many distinct enzymic steps (7Stadtman T.C. Some vitamin B12- and selenium-dependent enzymes..Ann. N. Y. Acad. Sci. 1983; 41: 233-236Google Scholar). Another amino acid transformation investigated in the early studies was the reductive deamination of glycine by C. sticklandii. Significantly, glycine reduction proved to be an energy-conserving process linked eventually to the formation of ATP (8Stadtman T.C. Elliott P. Tiemann L. Studies on the enzymic reduction of amino acids III. Phosphate esterification coupled with glycine reduction..J. Biol. Chem. 1958; 231: 961-973Abstract Full Text PDF PubMed Google Scholar). Thus, in the presence of orthophosphate and ADP, glycine was reduced to acetate and ammonia and ATP was formed with the stoichiometry shown in Equation1.Glycine+R(SH)2+Pi+ADP→acetate+ammonia+R−SS+ATPEquation 1 Much later the direct products of the glycine reductase complex were shown to be ammonia and acetyl phosphate (9Arkowitz R.A. Abeles R.H. Identification of acetyl phosphate as the product of clostridial glycine reductase: evidence for an acyl enzyme intermediate..Biochemistry. 1989; 28: 4639-4644Crossref PubMed Scopus (28) Google Scholar), and the highly active acetate kinase contaminant in the enzyme preparations converted acetyl phosphate and ADP to acetate and ATP. The key discovery that one of the protein components of the glycine reductase complex is a selenoprotein (10Stadtman T.C. Glycine reduction to acetate and ammonia: identification of ferredoxin and another low molecular weight acidic protein as components of the reductase system..Arch. Biochem. Biophys. 1966; 113: 9-19Crossref PubMed Scopus (25) Google Scholar, 11Turner D.C. Stadtman T.C. Purification of protein components of clostridial glycine reductase system and characterization of protein A as a selenoprotein..Arch. Biochem. Biophys. 1973; 154: 366-381Crossref PubMed Scopus (141) Google Scholar) shifted the emphasis of my later research. Much to my surprise the studies on glycine reductase fromC. sticklandii led us to the discovery early in May 1972 that the “rich culture medium” containing 2% Tryptone, 1% yeast extract, and formate used for routine growth of the organism was selenium-deficient. When supplemented with 1 μm selenite, the cell population exhibited high glycine reductase activity throughout the entire growth phase, whereas in the absence of added selenite glycine reductase was detected only in early log phase cells. A low molecular weight acidic protein component of the glycine reductase complex (10Stadtman T.C. Glycine reduction to acetate and ammonia: identification of ferredoxin and another low molecular weight acidic protein as components of the reductase system..Arch. Biochem. Biophys. 1966; 113: 9-19Crossref PubMed Scopus (25) Google Scholar), termed protein A, that we had isolated previously from early log phase cells proved to be the missing factor in cell-free extracts prepared from end of log phase cell populations that were not supplemented with selenium. A typical dilution curve was exhibited for protein A levels in extracts as a function of growth ofC. sticklandii in the non-selenite-supplemented medium. In retrospect, I realized that in the 1950s, when the Bethesda tap water could be used for culture of various anaerobic bacteria, the levels of glycine reductase in C. sticklandii were considerably higher than they were later when we were forced to use distilled water because of high levels of neutral detergents in the water supply. It is evident that many of the so-called “rich culture media” used by microbiologists are selenium-deficient, and this is true also for various serum-supplemented media used for culture of mammalian cells. To determine whether selenium was an actual component of protein A,C. sticklandii was grown in media containing [75Se]selenite. This resulted in the incorporation of radioactivity in protein A, and the 75Se content of the protein was enriched in parallel with enzyme activity during isolation of the protein in near homogeneous form (11Turner D.C. Stadtman T.C. Purification of protein components of clostridial glycine reductase system and characterization of protein A as a selenoprotein..Arch. Biochem. Biophys. 1973; 154: 366-381Crossref PubMed Scopus (141) Google Scholar). Thus, by the end of June 1972 we had evidence of the existence of an essential selenium-containing protein, the protein A component of glycine reductase. There followed a “learning period” for me concerning the chemistry of selenium and its relative, sulfur, to determine the identity of the selenium compound in the labeled protein A. I obtained several organoselenium compounds from the National Cancer Institute library that originally had been collected as potential carcinogens. However, the chemists who had synthesized these compounds had introduced phenyl groups for stability purposes, thus limiting their use as possible model compounds for our studies. Before the identification in 1957 of selenium as an essential nutrient for rats (12Schwarz K. Foltz C.M. Selenium as an integral part of Factor 3 against dietary necrotic liver degeneration..J. Am. Chem. Soc. 1957; 79: 3292-3293Crossref Scopus (1291) Google Scholar) and birds (13Patterson E.L. Milstrey R. Stokstad E.L.R. Effect of selenium in preventing exudative diathesis in chicks..Proc. Soc. Exp. Biol. Med. 1957; 95: 617-620Crossref PubMed Scopus (114) Google Scholar), it was known in biology mainly for its toxic properties. I had determined previously (10Stadtman T.C. Glycine reduction to acetate and ammonia: identification of ferredoxin and another low molecular weight acidic protein as components of the reductase system..Arch. Biochem. Biophys. 1966; 113: 9-19Crossref PubMed Scopus (25) Google Scholar) that reaction of the reduced form of protein A with iodoacetamide inhibited its biological activity as an essential component of the glycine reductase complex and had assumed that one or more essential SH groups had been alkylated. When we treated the 75Se-labeled protein with iodoacetamide or iodoacetate, the biological activity likewise was destroyed, but elimination of radioactive selenium as inorganic forms previously observed during acid hydrolysis was prevented almost completely. Instead, we could recover the radiolabel from the acid hydrolysates in a compound containing an alkyl group attached to the selenium. This derivative was identified as Se-carboxymethyl-selenocysteine by comparison with the corresponding alkyl derivative of authentic selenocysteine (14Cone J.E. Martin del Rio R. Davis J.N. Stadtman T.C. Chemical characterization of the selenoprotein component of clostridial glycine reductase: identification of selenocysteine as the organoselenium moiety..Proc. Natl. Acad. Sci. U. S. A. 1976; 73: 2659-2663Crossref PubMed Scopus (226) Google Scholar). We made several other alkyl derivatives of the selenoprotein for further identification and established that the Se-carboxymethyl, Se-carboxyethyl, and Se-aminoethyl forms were the most satisfactory from the standpoint of stability during acid hydrolysis and subsequent chromatographic separation on an amino acid analyzer column. Throughout these studies, my able assistant, Joe Nathan Davis, provided invaluable expertise and together with two postdoctoral fellows, Joyce Cone and Raphael Martin del Rio, we could establish that protein A contains 1 gram atom of selenium per mol and the selenium is present in the form of a selenocysteine residue in the polypeptide (14Cone J.E. Martin del Rio R. Davis J.N. Stadtman T.C. Chemical characterization of the selenoprotein component of clostridial glycine reductase: identification of selenocysteine as the organoselenium moiety..Proc. Natl. Acad. Sci. U. S. A. 1976; 73: 2659-2663Crossref PubMed Scopus (226) Google Scholar). The methods we developed for identification of selenocysteine in our bacterial protein were used later by other investigators to isolate and identify the selenium-containing moiety in mammalian glutathione peroxidase, another enzyme that had been reported in 1973 to contain selenium (15Forstrom J.W. Zakowski J.J. Tappel A.L. Identification of the catalytic site of rat liver glutathione peroxidase as selenocysteine..Biochemistry. 1978; 17: 2639-2644Crossref PubMed Scopus (356) Google Scholar). To determine whether free added selenocysteine could be incorporated into protein A, Gregory Dilworth, a postdoctoral fellow in my laboratory, synthesized selenocysteine labeled either with3H, 75Se, or 14C, and we grewC. sticklandii in the presence of these added labeled substrates. There was no detectable incorporation of the labeled carbon chain of the amino acid into protein A, but the [75Se]selenocysteine was used more efficiently as a selenium source than the normal supplement [75Se]selenite, which is reduced by thiols in the culture medium (16.Stadtman, T. C., Dilworth, G. L., and Chen, C. S. (1979) in Proceedings of the Third International Symposium on Organic Selenium and Tellurium Compounds (Cagniant, D., and Kirsch, G., eds) Selenium-dependent bacterial enzymes, pp. 115–130, Metz, France.Google Scholar). In retrospect, the facile utilization of selenium from selenocysteine for protein A biosynthesis observed in these experiments is indicative of the participation of a selenocysteine lyase. These lyases, first purified by Kenji Soda and his collaborators in Kyoto from bacteria (17Chocat P. Esaki N. Tanizawa K. Nakamura K. Tanaka H. Soda K. Purification and characterization of selenocysteine B-lyase from Citrobacter freundii..J. Bacteriol. 1985; 163: 669-676Crossref PubMed Google Scholar) and from liver (18Esaki N. Nakamura T. Tanaka H. Soda K. Selenocysteine lyase, a novel enzyme that specifically acts on selenocysteine. Mammalian distribution and purification and properties of pig liver enzyme..J. Biol. Chem. 1982; 257: 4386-4391Abstract Full Text PDF PubMed Google Scholar), convert selenocysteine to an atomic form of selenium and alanine.SeHCH2CHNH2COOH→Se+CH3CHNH2COOHEquation 2 For several years, we thought these proteins served solely as detoxification agents, but they now are considered to function as selenium transferases or selenium delivery proteins. Much of our current research is directed to elucidation of the chemical properties and biochemical roles of these selenium transferases. Dr. Richard Glass, a sulfur organic chemist, joined our group in 1987 while on sabbatical leave from the University of Arizona. We had met in 1984 in Lindau, Germany, at a Symposium on the Organic Chemistry of Sulfur. At this meeting the organizers had decided to enlarge the program to include biologically important selenium compounds, and my presentation on the small selenoprotein component of glycine reductase stimulated Dick Glass to become involved in selenium organic chemistry. During his year in Bethesda, we grew C. sticklandii in the presence of 77Se and isolated selenoprotein A labeled with the stable isotope. This was used to investigate conformational properties of the selenoprotein using 77Se NMR spectroscopy as a probe. Later, when we discovered that the biological donor for biosynthesis of selenuridine in tRNAs is selenophosphate (19Veres Z. Tsai L. Scholz T.D. Politino M. Balaban R.S. Stadtman T.C. Synthesis of 5-methylaminomethyl-2-selenouridine in tRNAs: 31P NMR studies show that the labile selenium donor synthesized by the selD gene product contains selenium bonded to phosphorus..Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 2975-2979Crossref PubMed Scopus (109) Google Scholar), the synthesis of this compound was achieved by Glass and his group, and authentic selenophosphate was supplied to us as a reference compound (20Glass R.S. Singh W.P. Jung W. Veres Z. Scholz T.D. Stadtman T.C. Monoselenophosphate: synthesis, characterization and identity with the prokaryotic biological selenium donor, compound SePX..Biochemistry. 1993; 32: 12555-12559Crossref PubMed Scopus (132) Google Scholar). I continue to rely on Dick Glass for advice and assistance concerning a wide variety of problems we encounter in the field of selenium chemistry. The presence of an unusual amino acid in two selenoenzymes, glycine reductase and glutathione peroxidase, that was not specified by the genetic code posed the problem of the method of specific incorporation of a selenoamino acid in the proteins. In fact, 13 years elapsed before it was recognized that one of the three stop codons, UGA, is used as the signal for selenocysteine insertion into a growing polypeptide chain. Simultaneously it was shown by August Böck and his collaborators in München that the TGA codon in theEscherichia coli formate dehydrogenase H gene directed selenocysteine incorporation into the protein (21Zinoni F. Birkmann A. Stadtman T.C. Böck A. Nucleotide sequence and expression of the selenocysteine-containing polypeptide of formate dehydrogenase (formate-hydrogen-lyase-linked) from Escherichia coli..Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 4650-4654Crossref PubMed Scopus (350) Google Scholar) and by P. R. Harrison and his collaborators in Glasgow that the TGA codon in the murine glutathione peroxidase gene corresponded to the position of selenocysteine in bovine glutathione peroxidase (22Chambers I. Frampton J. Goldfarb P. Affara N. McBain W. Harrison P.P. The structure of the mouse glutathione peroxidase gene: the selenocysteine in the active site is encoded by the “termination codon” TGA..EMBO J. 1986; 5: 1221-1227Crossref PubMed Scopus (529) Google Scholar). The amino acid sequence of bovine glutathione peroxidase had been determined earlier at Grunenthal GmbH in Aachen by Flohé and associates (23Gunzler W.A. Steffens G.J. Grossmann A. Kim S.-M.A. Otting F. Wendel A. Flohé L. The amino acid sequence of bovine glutathione peroxidase..Hoppe-Seyler's Z. Physiol. Chem. 1984; 365: 195-212Crossref PubMed Scopus (107) Google Scholar). Eventually, I could verify that selenocysteine occurred in the formate dehydrogenase protein in the position predicted by the TGA codon in the gene (24Stadtman T.C. Davis J.N. Ching W.-M. Zinoni F. Böck A. Amino acid sequence analysis of Escherichia coli formate dehydrogenase (FDHH) confirms that TGA in the gene encodes selenocysteine in the gene product..BioFactors. 1991; 3: 21-27PubMed Google Scholar). In a series of elegant experiments by August Böck and his associates, genes were isolated that complemented some of the mutant strains of E. coli defective in synthesis of formate dehydrogenase that had been isolated previously by Marie Andre Mandrand-Berthelot (25Haddock B.A. Mandrand-Berthelot M.-A. Escherichia coli formate-to-nitrate respiratory chain: genetic analysis..Biochem. Soc. Trans. 1982; 10: 478-480Crossref PubMed Scopus (16) Google Scholar, 26Leinfelder W. Zehelin E. Mandrand-Berthelot M.-A. Böck A. Gene for a novel tRNA species that accepts l-serine and cotranslationally inserts selenocysteine..Nature. 1988; 331: 723-725Crossref PubMed Scopus (317) Google Scholar, 27Leinfelder W. Forchhammer K. Zinoni F. Sawers G. Mandrand-Berthelot M.-A. Böck A. Escherichia coli genes whose products are involved in selenium metabolism..J. Bacteriol. 1988; 170: 540-546Crossref PubMed Google Scholar). Four genes that encoded four different products essential for the specific synthesis of selenocysteine and its insertion into protein were cloned and the expressed products characterized. In one step, a serine esterified to a special tRNA (selC product, anticodon UCA complementary to UGA) is converted to selenocysteinyl-tRNA by a pyridoxal phosphate-dependent selenocysteine synthase (selA gene product) using selenium from selenophosphate, produced by selenophosphate synthetase, the selD gene product (19Veres Z. Tsai L. Scholz T.D. Politino M. Balaban R.S. Stadtman T.C. Synthesis of 5-methylaminomethyl-2-selenouridine in tRNAs: 31P NMR studies show that the labile selenium donor synthesized by the selD gene product contains selenium bonded to phosphorus..Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 2975-2979Crossref PubMed Scopus (109) Google Scholar, 27Leinfelder W. Forchhammer K. Zinoni F. Sawers G. Mandrand-Berthelot M.-A. Böck A. Escherichia coli genes whose products are involved in selenium metabolism..J. Bacteriol. 1988; 170: 540-546Crossref PubMed Google Scholar, 28Böck A. Forchhammer K. Heider J. Leinfelder W. Sawers G. Veprek B. Zinoni F. Selenocysteine: the 21st amino acid..Mol. Microbiol. 1991; 5: 515-520Crossref PubMed Scopus (555) Google Scholar). A unique elongation factor (theselB gene product) that binds a secondary stem loop structure located 3′ to the UGA in the E. coli fdhF mRNA forms a complex with the selenocysteinyl-tRNA for delivery at the ribosome site and insertion of selenocysteine at UGA (29Forchhammer K. Leinfelder W. Böck A. Identification of a novel translation factor necessary for the incorporation of selenocysteine into protein..Nature. 1989; 342: 453-456Crossref PubMed Scopus (209) Google Scholar). Refinements of these groundbreaking discoveries still are being made by many investigators in the field, particularly with respect to the differing modes of recognition of UGA for selenocysteine incorporation in eukaryotes, archae, and E. coli. This process that prevents operation of the usual translation termination step and instead directs insertion of selenocysteine at a specific in-frame UGA codon is an important example of a growing list of exceptions to the established stop codon rules (30Hao B. Gong W. Ferguson T.K. James C.M. Krzycki J.A. Chan M.K. A new UAG-encoded residue in the structure of a methanogen methyltransferase..Science. 2002; 296: 1462-1466Crossref PubMed Scopus (323) Google Scholar). David Grahame and Milton Axley, working in the anaerobic laboratory at NIH, developed an elegant two-step chromatographic procedure for isolation of the markedly oxygen-sensitive 80-kDaE. coli formate dehydrogenase H (31Axley M.J. Grahame D.A. Stadtman T.C. Escherichia coli formate-hydrogen lyase. Purification and properties of the selenium-dependent formate dehydrogenase component..J. Biol. Chem. 1990; 265: 18213-18218Abstract Full Text PDF PubMed Google Scholar) in highly purified form. This enzyme contains molybdenum in a molybdopterin cofactor in addition to the selenocysteine in the polypeptide. Detailed kinetic analysis of the enzyme (32Axley M.J. Grahame D.A. Kinetics for formate dehydrogenase of Escherichia coli formate-hydrogen lyase..J. Biol. Chem. 1991; 266: 13731-13736Abstract Full Text PDF PubMed Google Scholar) and a comparison of the catalytic advantages afforded by selenium over sulfur revealed (33Axley M.J. Böck A. Stadtman T.C. Catalytic properties of an Escherichia coli formate dehydrogenase mutant in which sulfur replaces selenium..Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 8450-8454Crossref PubMed Scopus (181) Google Scholar) that the selenocysteine-containing native or wild-type enzyme was about 300 times more active than the selenocysteine/Cys mutant for oxidation of formate with benzyl viologen as the artificial electron acceptor. A few years later, it was shown in EPR studies that the selenium of the selenocysteine residue in formate dehydrogenase is coordinated directly to the molybdenum in the molybdopterin cofactor (34Gladyshev V.N. Khangulov S.V. Axley M.J. Stadtman T.C. Coordination of selenium to molybdenum in formate dehydrogenase H from Escherichia coli..Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 7708-7711Crossref PubMed Scopus (85) Google Scholar). The oxidation of formate by this enzyme does not involve a typical molybdenum-dependent hydroxylation mechanism. Instead, formate is converted directly to carbon dioxide without introduction of oxygen from solvent (35Khangulov S.V. Gladyshev V.N. Dismukes G.C. Stadtman T.C. Selenium-containing formate dehydrogenase H from Escherichia coli: a molybdopterin enzyme that catalyzes formate oxidation without oxygen transfer..Biochemistry. 1998; 37: 3518-3528Crossref PubMed Scopus (114) Google Scholar). Crystallization of the oxygen-labile enzyme under strictly anaerobic conditions was achieved (36Gladyshev V.N. Boyington J.C. Khangulov S.V. Grahame D.A. Stadtman T.C. Sun P.D. Characterization of crystalline formate dehydrogenase H from Escherichia coli..J. Biol. Chem. 1996; 271: 8095-8100Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar) and based on analysis of the crystal structure (37Boyington J.C. Gladyshev V.N. Khangulov S.V. Stadtman T.C. Sun P.D. Crystal structure of formate dehydrogenase H: catalysis involving Mo, molybdopterin, selenocysteine, and an Fe4S4 cluster..Science. 1997; 275: 1305-1308Crossref PubMed Scopus (497) Google Scholar), it was deduced that the selenium serves as the immediate proton acceptor in the reaction. This would suggest an effect of neighboring protein groups because usually at neutral pH a selenol is almost fully ionized. In contrast, from x-ray absorption spectroscopy (EXAFS) studies of oxidized and reduced forms of the enzyme, a novel selenosulfide ligation to the molybdenum was proposed as the proton acceptor (38George G.N. Colangelo C.M. Dong J. Scott R.A. Khangulov S.V. Gladyshev V.N. Stadtman T.C. X-ray absorption spectroscopy of the molybdenum site of Escherichia coli formate dehydrogenase..J. Am. Chem. Soc. 1998; 120: 1267-1273Crossref Scopus (81) Google Scholar). A possible alternative mechanism involving hydride or hydrogen atom transfer from formate to the selenosulfide instead of proton transfer also was suggested. Based on these somewhat differing types of evidence, the exact mechanism of action of the E. coli formate dehydrogenase and the precise role of selenium in the enzyme remain to be established. In vivo, the reducing equivalents from formate oxidation are transferred via an iron sulfur cluster eventually to a hydrogenase, and hydrogen gas is evolved. Despite the dearth of information concerning the genetic makeup of anaerobic spore-forming members of the genusClostridium, Greg Garcia was able to isolate and clone the glycine reductase selenoprotein A gene from two different clostridia (39Garcia G.E. Stadtman T.C. Selenoprotein A component of the glycine reductase complex from Clostridium purinolyticum: nucleotide sequence of the gene shows that selenocysteine is encoded by UGA..J. Bacteriol. 1991; 173: 2093-2098Crossref PubMed Google Scholar, 40Garcia G.E. Stadtman T.C. Clostridium sticklandii glycine reductase selenoprotein A gene: cloning, sequencing and expression in Escherichia coli..J. Bacteriol. 1992; 174: 7080-7089Crossref PubMed Google Scholar) and establish that an in-frame TGA codon in each corresponded to selenocysteine at position 44 in the polypeptides (41Sliwkowski M.X. Stadtman T.C. Selenoprotein A of the clostridial glycine reductase complex: purification and amino acid sequence of the selenocysteine-containing peptide..Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 368-371Crossref PubMed Scopus (14) Google Scholar). However, attempts to express the C. sticklandii cloned gene inE. coli were only partially successful (40Garcia G.E. Stadtman T.C. Clostridium sticklandii glycine reductase selenoprotein A gene: cloning, sequencing and expression in Escherichia coli..J. Bacteriol. 1992; 174: 7080-7089Crossref PubMed Google Scholar). The full-length, 18-kDa immunologically reactive protein was produced in good yield, but the catalytic activity as a component of glycine reductase was only about 10% that of native selenoprotein. A full-length protein produced in the absence of selenium or in a SelD mutant unable to synthesize selenophosphate was inactive. Detailed analysis showed that read-through and suppression of the UGA codon involved a cysteine-tRNA, and either cysteine or occasionally selenocysteine esterified to the tRNA was inserted. It was concluded that the mRNA secondary stem-loop structure required by E. coli for UGA-directed specific selenocysteine insertion was not present in the clostridial mRNA structure. Although details of rules concerning clostridial selenoprotein gene expression are still lacking, the now available genomic sequence of Clostr
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