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J. Biol. Chem., Vol. 277, Issue 51, 49091-49100, December 20, 2002
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From the Laboratory of Biochemistry, NHLBI, National Institutes of Health, Bethesda, Maryland 20892-8012
I 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 (1). 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 (2). 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.
Anaerobic Metabolism Background
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Fig. 1.
H. A. Barker.
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 (3). The other was a rod-shaped organism that I later named Clostridium sticklandii (4).
Detailed studies on the morphology and biochemical properties of
M. vannielii (3) 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.
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Aerobic Metabolism of Cholesterol |
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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 aerobic
Nocardia 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 
-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.
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Amino Acid Fermentation Studies |
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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 with M. vannielii from San Francisco Bay mud (4). Initial studies on the anaerobic metabolism of lysine and ornithine (5) and the roles of vitamin B12 in these processes (6) provided examples of interesting new reactions and additional roles of B12 coenzyme. 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 of D-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 (7).
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 (8). Thus, in the
presence of orthophosphate and ADP, glycine was reduced to acetate and
ammonia and ATP was formed with the stoichiometry shown in Equation 1.
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(Eq. 1) |
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The Selenium Era |
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Much to my surprise the studies on glycine reductase from C. 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 (10), 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 of C. 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 (11). 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 (12) and birds (13), it was
known in biology mainly for its toxic properties.
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Identification of Selenocysteine in Glycine Reductase Protein A |
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I had determined previously (10) 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 (14). 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 (14). 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 (15).
To determine whether free added selenocysteine could be incorporated
into protein A, Gregory Dilworth, a postdoctoral fellow in my
laboratory, synthesized selenocysteine labeled either with 3H, 75Se, or 14C, and we grew
C. 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). 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 (17) and from liver (18), convert selenocysteine to an
atomic form of selenium and alanine.
|
(Eq. 2) |
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 (19), the
synthesis of this compound was achieved by Glass and his group, and
authentic selenophosphate was supplied to us as a reference compound
(20). 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.
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The UGA Codon Is Used for Specific Selenocysteine Incorporation |
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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 the
Escherichia coli formate dehydrogenase H gene directed selenocysteine incorporation into the protein (21) 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 (22). The amino acid
sequence of bovine glutathione peroxidase had been determined earlier
at Grunenthal GmbH in Aachen by Flohé and associates (23).
Eventually, I could verify that selenocysteine occurred in the formate
dehydrogenase protein in the position predicted by the TGA codon in the
gene (24). 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 (25-27). 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 (19, 27, 28). A unique elongation factor (the
selB 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 (29). 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 (30).
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E. coli Formate Dehydrogenase |
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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-kDa E. coli formate dehydrogenase H (31) 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 (32) and a comparison of the catalytic advantages afforded by selenium over sulfur revealed (33) 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 (34). 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 (35). Crystallization of the oxygen-labile enzyme
under strictly anaerobic conditions was achieved (36) and based on
analysis of the crystal structure (37), 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 (38). 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.
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Selenocysteine Incorporation in Clostridia |
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Despite the dearth of information concerning the genetic
makeup of anaerobic spore-forming members of the genus
Clostridium, Greg Garcia was able to isolate and clone the
glycine reductase selenoprotein A gene from two different clostridia
(39, 40) and establish that an in-frame TGA codon in each corresponded to selenocysteine at position 44 in the polypeptides (41). However, attempts to express the C. sticklandii cloned gene in
E. coli were only partially successful (40). 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 Clostridium
difficile should provide information on the SECIS stem-loop
structure involved and its location in the mRNA. In fact, both
glycine reductase and formate dehydrogenase were detected in extracts
of a strain of C. difficile that we used in our studies in
the 1950s, and the corresponding genes have been found in the published
genomic structure. It is clear that the statement commonly made in the
literature to the effect that in all prokaryotes the SECIS element is
identical to that in E. coli and is located in the same
orientation as in FdhF is incorrect. E. coli is not
representative of all prokaryotes.
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Seleno-tRNAs |
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Another type of biochemical process in which selenophosphate
is utilized as selenium donor is the synthesis of 2-selenouridine in
the "wobble position" of the anticodons of certain tRNAs. The 2-selenouridine residue in the form of
5-methylaminomethyl-2-selenouridine had been identified earlier in the
lysine and glutamate tRNAs of E. coli, Salmonella
typhimurium, C. sticklandii, and M. vannielii (42). When Dr. Zsuzsanna Veres, a young Hungarian
scientist from Budapest, came to my laboratory as a Visiting Fellow she decided to work on the biochemistry of 2-selenouridine. My good luck in
having Dr. Veres as a collaborator came about as a result of my visit
to Budapest in 1988 as a member of a USA National Academy of Sciences
committee commissioned to evaluate the mutual benefits of exchanges
between the United States and the Hungarian Academies of Sciences. When
my counterpart on the Hungarian Academy committee, Professor Geza
Denes, heard that I had an opening in my laboratory for a Foreign
Visiting Scientist, he suggested his best student as a possible
candidate. Dr. Veres was interested and after the usual formalities she
arrived in my laboratory in August 1989. Thus started a very fruitful
collaboration of almost 5 years and a deep friendship developed. During
Zsuzsa's stay in the laboratory, she demonstrated that the ATP
requirement for selenouridine synthesis in tRNAs was explained entirely
by its use in the generation of selenophosphate, the biological donor of selenium in the reaction (19, 20, 43). A subsequent step involved
the replacement of a sulfur in the 2-thiouridine precursor in tRNA with
selenium to form the 2-selenouridine residue, and the responsible
enzyme system was partially purified (44). Veres' detailed studies on
selenophosphate synthetase and the role of the enzyme in the generation
of the new high energy selenium donor compound were very important
contributions to the overall field of selenium biochemistry. When Dr.
Ick Young Kim, a young investigator from Korea, arrived, he joined
Veres in studies on selenophosphate synthetase, and by site-directed
mutagenesis he produced several useful mutant forms of the enzyme (45).
The Veres-Kim collaboration proved to be very productive in this investigation.
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Selenophosphate Synthetase Reaction Mechanism |
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Later studies in my laboratory on the selenophosphate
synthetase reaction mechanism by Dr. Heidi Walker demonstrated that a
group on the enzyme first is phosphorylated by reaction with ATP (46).
This phosphoryl group derived from the 
-phosphoryl group of ATP
subsequently is transferred to selenium to form selenophosphate. The
ADP moiety of ATP, which is tightly bound during the initial step, then
is cleaved in a hydrolytic step to form orthophosphate (derived from
the 
-phosphoryl group) and AMP (47). Earlier, we thought that a
pyrophosphoryl derivative of the enzyme might be formed in the initial
reaction with ATP leaving AMP as the other product. However, Song
Liu's experiments showed that retention of 32P from
-32P-labeled ATP was insignificant, whereas when
-32P-labeled ATP was used up to 0.6 eq of
32P was bound to the enzyme (48). Details of the mechanism
of the selenophosphate synthetase reaction are still under investigation.
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Selenium-dependent Enzymes That Do Not Contain Selenocysteine |
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A group of molybdopterin-dependent hydroxylases,
nicotinic acid hydroxylase (49, 50), xanthine dehydrogenase, and purine hydroxylase (51), that have been purified from anaerobic bacteria require selenium for activity. However, the selenium in these enzymes
is not present in selenocysteine residues in the polypeptides but
instead occurs in a labile cofactor. The selenium can be released from
the cofactor by treatment with cyanide and thus might be in the form of
a perselenide. The mechanism of incorporation of selenium in these
enzymes currently is being investigated by Dr. William Self, who
discovered purine hydroxylase, the most recent addition to the list of
selenium-dependent hydroxylases. It was shown earlier in
EPR studies that the selenium in nicotinic hydroxylase is coordinated
to the molybdenum of the molybdopterin cofactor (50). In contrast to
the hydroxylases from anaerobic bacteria, the corresponding enzymes
from eukaryotes have not been shown to be selenoenzymes.
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Discovery of a New Mammalian Selenoenzyme |
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Our studies on thioredoxin reductase were initiated as the result of a serendipitous discovery made by Dr. Takashi Tamura, a young Japanese Visiting Fellow in our laboratory, during his one-year leave of absence from Okayama University. A cytochrome P-450 present in a human lung adenocarcinoma cell line had been predicted to contain selenocysteine based on the occurrence of a TGA codon in the open reading frame of the corresponding gene (52). The possibility that the putative selenocysteine residue, located at some distance from the conserved cysteine at the heme binding site, might have a novel role in the enzyme prompted Dr. Tamura to attempt isolation of the protein. The lung adenocarcinoma cells were cultured in the presence of [75Se]selenite, and the expected 57-kDa protein labeled with 75Se was isolated in near homogeneous form. Two different alkyl derivatives of the protein were prepared, and the corresponding alkyl [75Se]selenocysteines were isolated and identified. The chromophore bound to the protein, however, proved to be FAD instead of a heme group. The FAD could be reduced specifically by NADPH, and various disulfides, including thioredoxin, served as substrates for the enzyme. It was evident that this selenoprotein, a homodimer of 57-kDa subunits, must be thioredoxin reductase, an enzyme that had been purified from various mammalian tissues and studied by other investigators but never suspected to be a selenoenzyme. Although the reported sequence of a putative thioredoxin reductase gene from human placenta (53) contained a TGA codon near the C terminus, this had been interpreted as a termination signal. Subsequently, experiments carried out in my laboratory by Vadim Gladyshev (54) and by Song Liu (55) showed that the selenocysteine in thioredoxin reductase, previously identified by Tamura, occurs at the C terminus in the sequence -Cys-selenocysteine-Gly in a position corresponding to the TGA codon in the placental gene. Thioredoxin reductases purified from human Jurkat T cells (54), from HeLa cells, and from the human adenocarcinoma cells (55) were shown to have the same C-terminal triplet peptide sequences.
The importance of the potential C-terminal redox center for catalytic
activity was shown in experiments with the HeLa cell enzyme by Sergey
Gorlatov (56). Alkylation of the NADPH-reduced enzyme under conditions
that limited alkyl group incorporation to the one ionized selenol per
subunit was sufficient to inhibit catalytic activity 99% (56). When
HeLa cells were grown at higher than optimal oxygen levels, the
isolated enzyme consisted of significant amounts of species that
contained an average of 0.5 instead of 1 selenium atom per
subunit, and these forms exhibited correspondingly lowered catalytic
activities (57). Reduced forms of thioredoxin reductase were very
oxygen-labile in the absence of bound pyridine nucleotide, and
corresponding losses of selenium and catalytic activity were observed.
It now is generally agreed by a number of investigators that premature
termination of gene expression at the UGA codon gives rise to a
truncated inactive form of the enzyme, and mutant enzyme species in
which a cysteine residue replaces the selenocysteine exhibit very low
catalytic activity (58). It thus is evident that the additional
C-terminal redox center present in fully active mammalian thioredoxin
reductases is an important determinant of catalytic activity and is
essential in addition to the bound FAD and the redox active cysteine
pair near the NADPH binding site in the N-terminal region of the protein.
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Selenium Transport Proteins |
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One of the interesting recent developments in the selenium
field comes from the realization of the importance of selenium transport or delivery proteins in selenoprotein biosynthesis. Our
specific interest involves the participation of these proteins in
supplying selenium for selenophosphate biosynthesis. Even under in vitro conditions an atomic form of selenium provided by a
delivery protein is used more efficiently as substrate by
selenophosphate synthetase than millimolar levels of selenide normally
added to reaction mixtures (59). The atomic form of selenium can be
derived from free selenocysteine by selenocysteine lyases that are
structurally related to the NifS sulfur transferase family of proteins
(60) or from inorganic selenium compounds, i.e. selenite
after reaction with thiols to form RS-Se-SR adducts. Enzymes, such as
rhodanese, that can transfer the sulfane sulfur from thiosulfate to
cyanide generate a persulfide derivative of an active cysteine residue as the enzyme-bound intermediate (61). In an analogous process, a
perselenide derivative of a reactive cysteine residue could be
generated by reaction with a selenium substrate (62). An unusual
selenium-binding protein that I isolated recently from M. vannielii appears to be a candidate for such a role. The gene that
encodes this protein was isolated, cloned, and expressed in E. coli by Dr. William Self. Several properties of the isolated protein expressed in E. coli are identical to those of the
native M. vannielii protein, and preliminary studies by Dr.
Self suggest that the ability to bind selenium may be specific. If so,
this could imply a role in supplying the significant levels of selenium required for synthesis of multiple selenoenzymes involved in the energy
metabolism of M. vannielii (63). The ability to discriminate between selenium present at micromolar concentrations in most biological systems as compared with millimolar levels of sulfur compounds is essential for efficient biosynthesis of specific selenium-containing catalysts.
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The Famous NIH Anaerobic Laboratory |
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An anaerobic laboratory facility (Nitrogen Laboratory) that was constructed in the 1960s in Building 3 at NIH at a cost of $250,000 was used for numerous studies on oxygen-sensitive organic catalysts and oxygen-labile enzymes until March 2001 when the occupants of Building 3 were moved to Building 50. The walls of the anaerobic facility are made of 3/8-inch carbon steel plates that were welded into place and supported by a framework of I-beams. Floor plates were riveted to a concrete floor. All joints were welded and sealed. A nitrogen atmosphere containing hydrogen, introduced to remove the last traces of oxygen by passage through a palladium catalyst bed, was maintained at less than 10 ppm of oxygen.
This laboratory is still the only one of its kind in the world. For one
of the first tests of the new anaerobic facility, I inoculated Petri
dishes containing a formate-mineral salts agar medium with a culture of
M. vannielii and placed them in an ordinary 37 °C
incubator. When I inspected the plates the next day, I was so delighted
to find colonies of this extremely oxygen-sensitive organism on the
surface of the agar that I laughed, and this caused enough nitrogen to
leak into my mask to set off the alarm system. I then made a rule not
to laugh in the anaerobic laboratory. Seriously, over the years, we and
investigators from other NIH laboratories and several universities have
carried out large scale isolations of oxygen-labile enzymes and
characterized oxygen-sensitive flavoproteins, B12
coenzyme-dependent enzymes, and selenoproteins in this
anaerobic facility (64, 65). To conduct many of these procedures in an
anaerobic glove box is either cumbersome or impossible as we are
learning to our sorrow. Sadly, this important anaerobic laboratory facility will soon be demolished to convert Building 3 into
administrative office space.
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FOOTNOTES |
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Published, JBC Papers in Press, October 25, 2002, DOI 10.1074/jbc.X200005200
Address correspondence to: tcstadtman{at}nih.gov.
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REFERENCES |
|---|
| 1. | Stadtman, T. C., and Barker, H. A. (1949) Studies on the methane fermentation. VII. Tracer experiments on the mechanism of methane formation. Arch. Biochem. 21, 256-264 |
| 2. |
Stadtman, T. C.,
and Barker, H. A.
(1951)
Studies on the methane fermentation. IX. The origin of methane in the acetate and methanol fermentations by Methanosarcina.
J. Bacteriol.
61,
81-86 |
| 3. |
Stadtman, T. C.,
and Barker, H. A.
(1951)
Studies on the methane fermentation X. A new formate-decomposing bacterium, Methanococcus vannielii.
J. Bacteriol.
62,
269-280 |
| 4. |
Stadtman, T. C.
(1954)
On the metabolism of an amino acid fermenting Clostridium.
J. Bacteriol.
67,
314-320 |
| 5. | Stadtman, T. C. (1962) Lysine fermentation to fatty acids and ammonia: A cobamide coenzyme-dependent process. J. Biol. Chem. 237, 2409-2411[Medline] [Order article via Infotrieve] |
| 6. | Stadtman, T. C. (1972) B12 coenzyme-dependent amino group migrations. The Enzymes 6, 539-563 |
| 7. | Stadtman, T. C. (1983) Some vitamin B12- and selenium-dependent enzymes. Ann. N. Y. Acad. Sci. 41, 233-236[CrossRef] |
| 8. |
Stadtman, T. C.,
Elliott, P.,
and Tiemann, L.
(1958)
Studies on the enzymic reduction of amino acids III. Phosphate esterification coupled with glycine reduction.
J. Biol. Chem.
231,
961-973 |
| 9. | Arkowitz, R. A., and Abeles, R. H. (1989) Identification of acetyl phosphate as the product of clostridial glycine reductase: evidence for an acyl enzyme intermediate. Biochemistry 28, 4639-4644 |
| 10. | Stadtman, T. C. (1966) 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. 113, 9-19[CrossRef][Medline] [Order article via Infotrieve] |
| 11. | Turner, D. C., and Stadtman, T. C. (1973) Purification of protein components of clostridial glycine reductase system and characterization of protein A as a selenoprotein. Arch. Biochem. Biophys 154, 366-381[CrossRef][Medline] [Order article via Infotrieve] |
| 12. | Schwarz, K., and Foltz, C. M. (1957) Selenium as an integral part of Factor 3 against dietary necrotic liver degeneration. J. Am. Chem. Soc. 79, 3292-3293 |
| 13. | Patterson, E. L., Milstrey, R., and Stokstad, E. L. R. (1957) Effect of selenium in preventing exudative diathesis in chicks. Proc. Soc. Exp. Biol. Med 95, 617-620[Medline] [Order article via Infotrieve] |
| 14. |
Cone, J. E.,
Martin del Rio, R.,
Davis, J. N.,
and Stadtman, T. C.
(1976)
Chemical characterization of the selenoprotein component of clostridial glycine reductase: identification of selenocysteine as the organoselenium moiety.
Proc. Natl. Acad. Sci. U. S. A.
73,
2659-2663 |
| 15. | Forstrom, J. W., Zakowski, J. J., and Tappel, A. L. (1978) Identification of the catalytic site of rat liver glutathione peroxidase as selenocysteine. Biochemistry 17, 2639-2644[CrossRef][Medline] [Order article via Infotrieve] |
| 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 |
| 17. |
Chocat, P.,
Esaki, N.,
Tanizawa, K.,
Nakamura, K.,
Tanaka, H.,
and Soda, K.
(1985)
Purification and characterization of selenocysteine B-lyase from Citrobacter freundii.
J. Bacteriol.
163,
669-676 |
| 18. |
Esaki, N.,
Nakamura, T.,
Tanaka, H.,
and Soda, K.
(1982)
Selenocysteine lyase, a novel enzyme that specifically acts on selenocysteine. Mammalian distribution and purification and properties of pig liver enzyme.
J. Biol. Chem.
257,
4386-4391 |
| 19. |
Veres, Z.,
Tsai, L.,
Scholz, T. D.,
Politino, M.,
Balaban, R. S.,
and Stadtman, T. C.
(1992)
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.
89,
2975-2979 |
| 20. | Glass, R. S., Singh, W. P., Jung, W., Veres, Z., Scholz, T. D., and Stadtman, T. C. (1993) Monoselenophosphate: synthesis, characterization and identity with the prokaryotic biological selenium donor, compound SePX. Biochemistry 32, 12555-12559[CrossRef][Medline] [Order article via Infotrieve] |
| 21. |
Zinoni, F.,
Birkmann, A.,
Stadtman, T. C.,
and Böck, A.
(1986)
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.
83,
4650-4654 |
| 22. | Chambers, I., Frampton, J., Goldfarb, P., Affara, N., McBain, W., and Harrison, P. P. (1986) The structure of the mouse glutathione peroxidase gene: the selenocysteine in the active site is encoded by the "termination codon" TGA. EMBO J. 5, 1221-1227[Medline] [Order article via Infotrieve] |
| 23. | Gunzler, W. A., Steffens, G. J., Grossmann, A., Kim, S.-M. A., Otting, F., Wendel, A., and Flohé, L. (1984) The amino acid sequence of bovine glutathione peroxidase. Hoppe-Seyler's Z. Physiol. Chem 365, 195-212[Medline] [Order article via Infotrieve] |
| 24. | Stadtman, T. C., Davis, J. N., Ching, W.-M., Zinoni, F., and Böck, A. (1991) Amino acid sequence analysis of Escherichia coli formate dehydrogenase (FDHH) confirms that TGA in the gene encodes selenocysteine in the gene product. BioFactors 3, 21-27[Medline] [Order article via Infotrieve] |
| 25. | Haddock, B. A., and Mandrand-Berthelot, M.-A. (1982) Escherichia coli formate-to-nitrate respiratory chain: genetic analysis. Biochem. Soc. Trans 10, 478-480[Medline] [Order article via Infotrieve] |
| 26. | Leinfelder, W., Zehelin, E., Mandrand-Berthelot, M.-A., and Böck, A. (1988) Gene for a novel tRNA species that accepts L-serine and cotranslationally inserts selenocysteine. Nature 331, 723-725[CrossRef][Medline] [Order article via Infotrieve] |
| 27. |
Leinfelder, W.,
Forchhammer, K.,
Zinoni, F.,
Sawers, G.,
Mandrand-Berthelot, M.-A.,
and Böck, A.
(1988)
Escherichia coli genes whose products are involved in selenium metabolism.
J. Bacteriol
170,
540-546 |
| 28. | Böck, A., Forchhammer, K., Heider, J., Leinfelder, W., Sawers, G., Veprek, B., and Zinoni, F. (1991) Selenocysteine: the 21st amino acid. Mol. Microbiol. 5, 515-520[CrossRef][Medline] [Order article via Infotrieve] |
| 29. | Forchhammer, K., Leinfelder, W., and Böck, A. (1989) Identification of a novel translation factor necessary for the incorporation of selenocysteine into protein. Nature 342, 453-456[CrossRef][Medline] [Order article via Infotrieve] |
| 30. |
Hao, B.,
Gong, W.,
Ferguson, T. K.,
James, C. M.,
Krzycki, J. A.,
and Chan, M. K.
(2002)
A new UAG-encoded residue in the structure of a methanogen methyltransferase.
Science
296,
1462-1466 |
| 31. |
Axley, M. J.,
Grahame, D. A.,
and Stadtman, T. C.
(1990)
Escherichia coli formate-hydrogen lyase. Purification and properties of the selenium-dependent formate dehydrogenase component.
J. Biol. Chem.
265,
18213-18218 |
| 32. |
Axley, M. J.,
and Grahame, D. A.
(1991)
Kinetics for formate dehydrogenase of Escherichia coli formate-hydrogen lyase.
J. Biol. Chem.
266,
13731-13736 |
| 33. |
Axley, M. J.,
Böck, A.,
and Stadtman, T. C.
(1991)
Catalytic properties of an Escherichia coli formate dehydrogenase mutant in which sulfur replaces selenium.
Proc. Natl. Acad. Sci. U. S. A.
88,
8450-8454 |
| 34. |
Gladyshev, V. N.,
Khangulov, S. V.,
Axley, M. J.,
and Stadtman, T. C.
(1994)
Coordination of selenium to molybdenum in formate dehydrogenase H from Escherichia coli.
Proc. Natl. Acad. Sci. U. S. A.
91,
7708-7711 |
| 35. | Khangulov, S. V., Gladyshev, V. N., Dismukes, G. C., and Stadtman, T. C. (1998) Selenium-containing formate dehydrogenase H from Escherichia coli: a molybdopterin enzyme that catalyzes formate oxidation without oxygen transfer. Biochemistry 37, 3518-3528[CrossRef][Medline] [Order article via Infotrieve] |
| 36. |
Gladyshev, V. N.,
Boyington, J. C.,
Khangulov, S. V.,
Grahame, D. A.,
Stadtman, T. C.,
and Sun, P. D.
(1996)
Characterization of crystalline formate dehydrogenase H from Escherichia coli.
J. Biol. Chem.
271,
8095-8100 |
| 37. |
Boyington, J. C.,
Gladyshev, V. N.,
Khangulov, S. V.,
Stadtman, T. C.,
and Sun, P. D.
(1997)
Crystal structure of formate dehydrogenase H: catalysis involving Mo, molybdopterin, selenocysteine, and an Fe4S4 cluster.
Science
275,
1305-1308 |
| 38. | George, G. N., Colangelo, C. M., Dong, J., Scott, R. A., Khangulov, S. V., Gladyshev, V. N., and Stadtman, T. C. (1998) X-ray absorption spectroscopy of the molybdenum site of Escherichia coli formate dehydrogenase. J. Am. Chem. Soc. 120, 1267-1273 |
| 39. |
Garcia, G. E.,
and Stadtman, T. C.
(1991)
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.
173,
2093-2098 |
| 40. |
Garcia, G. E.,
and Stadtman, T. C.
(1992)
Clostridium sticklandii glycine reductase selenoprotein A gene: cloning, sequencing and expression in Escherichia coli.
J. Bacteriol.
174,
7080-7089 |
| 41. |
Sliwkowski, M. X.,
and Stadtman, T. C.
(1988)
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.
85,
368-371 |
| 42. | Wittwer, A. J., Tsai, L., Ching, W.-M., and Stadtman, T. C. (1984) Identification and synthesis of a naturally occurring selenonucleoside in bacterial tRNAs: 5-[(methylamino)methyl]-2-selenouridine. Biochemistry 23, 4650-4655[CrossRef][Medline] [Order article via Infotrieve] |
| 43. |
Veres, Z.,
Kim, I. Y.,
Scholz, T. D.,
and Stadtman, T. C.
(1994)
Selenophosphate synthetase. Enzyme properties and catalytic reaction.
J. Biol. Chem.
269,
10597-10603 |
| 44. |
Veres, Z.,
and Stadtman, T. C.
(1994)
A purified selenophosphate-dependent enzyme from Salmonella typhimurium catalyzes the replacement of sulfur in 2-thiouridine in tRNAs with selenium.
Proc. Natl. Acad. Sci. U. S. A.
91,
8092-8096 |
| 45. |
Kim, I. Y.,
Veres, Z.,
and Stadtman, T. C.
(1993)
Biochemical analysis of Escherichia coli selenophosphate synthetase mutants.
J. Biol. Chem.
268,
27020-27025 |
| 46. | Mullins, L. S., Hong, S.-B., Gibson, G. E., Walker, H., Stadtman, T. C., and Raushel, F. M. (1997) Identification of a phosphorylated enzyme intermediate in the catalytic mechanism for selenophosphate synthetase. J. Am. Chem. Soc. 119, 6684-6685 |
| 47. |
Walker, H.,
Ferretti, J. A.,
and Stadtman, T. C.
(1998)
Isotope exchange studies on the Escherichia coli selenophosphate synthetase mechanism.
Proc. Natl. Acad. Sci. U. S. A.
95,
2180-2185 |
| 48. |
Liu, S.-Y.,
and Stadtman, T. C.
(1997)
Selenophosphate synthetase: enzyme labeling studies with [-32P]ATP, [-32P]ATP, [8-14C]ATP and [75Se]selenide.
Arch. Biochem. Biophys.
341,
353-359[CrossRef][Medline]
[Order article via Infotrieve]
|
| 49. | Dilworth, G. L. (1982) Properties of the selenium-containing nicotinic acid hydroxylase from Clostridium barkeri. Arch. Biochem. Biophys. 219, 30-38[CrossRef][Medline] [Order article via Infotrieve] |
| 50. |
Gladyshev, V. N.,
Khangulov, S. V.,
and Stadtman, T. C.
(1994)
Nicotinic acid hydroxylase from Clostridium barkeri: electron paramagnetic resonance studies show that selenium is coordinated with molybdenum in the catalytically active selenium-dependent enzyme.
Proc. Natl. Acad. Sci. U. S. A.
91,
232-236 |
| 51. |
Self, W. T.,
and Stadtman, T. C.
(2000)
Selenium-dependent metabolism of purines: a selenium-dependent purine hydroxylase and xanthine dehydrogenase were purified from Clostridium purinolyticum and characterized.
Proc. Natl. Acad. Sci. U. S. A.
97,
7208-7213 |
| 52. |
Tamura, T.,
and Stadtman, T. C.
(1996)
A new selenoprotein from human lung adenocarcinoma cells: purification, properties and thioredoxin reductase activity.
Proc. Natl. Acad. Sci. U. S. A.
93,
1006-1011 |
| 53. | Gasdaska, P. Y., Gasdaska, J. R., Cochran, S., and Powis, G. (1995) Cloning and sequencing of a human thioredoxin reductase. FEBS Lett. 373, 5-9[CrossRef][Medline] [Order article via Infotrieve] |
| 54. |
Gladyshev, V. N.,
Jeang, K.-T.,
and Stadtman, T. C.
(1996)
Selenocysteine, identified as the penultimate C-terminal residue in human T-cell thioredoxin reductase, corresponds to TGA in the human placental gene.
Proc. Natl. Acad. Sci. U. S. A.
93,
6146-6151 |
| 55. |
Liu, S.-Y.,
and Stadtman, T. C.
(1997)
Heparin-binding properties of selenium-containing thioredoxin reductase from HeLa cells and human lung adenocarcinoma cells.
Proc. Natl. Acad. Sci. U. S. A.
94,
6138-6141 |
| 56. |
Gorlatov, S. N.,
and Stadtman, T. C.
(1998)
Human thioredoxin reductase from HeLa cells: selective alkylation of selenocysteine residue in the protein inhibits enzyme activity and reduction with NADPH influences affinity to heparin.
Proc. Natl. Acad. Sci. U. S. A.
95,
8520-8525 |
| 57. | Gorlatov, S. N., and Stadtman, T. C. (1999) Human selenium-dependent thioredoxin reductase from HeLa cells: properties of forms with differing heparin affinities. Arch. Biochem. Biophys. 369, 133-142[CrossRef][Medline] [Order article via Infotrieve] |
| 58. | Lee, S.-R., Bar-Noy, S., Kwon, J., Levine, R. L., Stadtman, T. C., and Rhee, S. G. (2000) Mammalian thioredoxin reductase: oxidation of the C-termin |
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