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Selenocysteine Lyase

Thressa C. Stadtman

[Section Editor: Georges Cohen]

Posted July 27, 2004

Laboratory of Biochemistry, National Heart, Lung, and Blood Institute, National Institutes of Health, Building 50, Room 2120, Bethesda, MD 20892-8012

Phone: 301-496-3002, Fax: 301-496-0599, E-mail: This e-mail address is being protected from spambots. You need JavaScript enabled to view it

 

INTRODUCTION

Selenocysteine is a naturally occurring analog of cysteine in which the sulfur atom of the latter is replaced with selenium. This seleno-amino acid occurs as a specific component of various selenoproteins and selenium-dependent enzymes. Incorporation of selenocysteine into these proteins occurs cotranslationally as directed by the UGA codon. For this process, a special tRNA having an anticodon complimentary to UGA, tRNA^Sec, is utilized. In Escherichia coli and related bacteria, this tRNA first is amino acylated with serine, and the seryl-tRNA^Sec is converted to selenocysteyl-tRNA^Sec. The precursor tRNA derivative in mammals has not been identified, but selenophosphate serves as the specific selenium donor for generation of selenocysteyl-tRNA^Sec in systems known to date. Details of this overall process in E. coli are described in this volume by Bock and Thanbichler (1).

In contrast, nonspecific incorporation of selenocysteine in place of cysteine is common in many biological systems. When it occurs, the extent of this replacement depends on the relative concentrations of available sulfur and selenium compounds. Sulfur levels that are at least a thousandfold greater than selenium levels are common in nature. However, in areas such as the semiarid regions of the western United States where the selenium content of soils is unusually high, the S/Se ratio can be much lower. Cereal grains, including wheat and corn, grown in these soils have been shown to contain high to toxic levels of selenium in the form of selenocysteine and selenomethionine residues incorporated nonspecifically in their proteins. Free selenocysteine that is synthesized via sulfur pathways is esterified and charged to tRNA^Cys. Lack of effective discrimination between the cysteine and its selenium analog by cysteine tRNA synthetase allows random attachment of the two amino acids to tRNA^Cys. Both esterified amino acids then are incorporated into the growing polypeptide chains as directed by the cysteine mRNA codons, UGU or UGC.

As pointed out above, the specific incorporation of selenocysteine into proteins directed by the UGA codon depends on the synthesis of selenocysteyl-tRNA^Sec. This requires a unique selenium donor, selenophosphate, that is formed by E. coli selenophosphate synthetase from ATP and millimolar levels of selenide in vitro (4, 7, 15, 16). However, a less toxic elemental form of selenium supplied by a selenium transferase or selenium delivery enzyme is the preferred substrate for the enzyme. Included in the selenium delivery protein category are rhodaneses that mobilize selenium from inorganic sources and NIFS-like proteins that liberate elemental selenium from selenocysteine. The NIFS protein from Azotobacter vinelandii that converts cysteine to equimolar amounts of sulfur and alanine (6, 17, 18) was found to serve as an efficient catalyst in vitro for delivery of selenium from free selenocysteine to E. coli selenophosphate synthetase for selenophosphate formation (9). In similar experiments with NIFS-like proteins from E. coli, efficient selenium delivery from selenocysteine to selenophosphate sythetase was observed (10). Lacourciere showed that E. coli effectively utilizes added selenocysteine in vivo as a selenium donor (8) . In these experiments, incorporation of radiolabeled selenite into formate dehydrogenase was monitored as an index of selenophosphate biosynthesis. A concomitant decrease in incorporation of ⁷⁵Se in formate dehydrogenase observed upon addition of increasing concentrations of unlabeled selenocysteine to the cultures was consistent with mobilization of selenium from the added selenocysteine.

 

EARLY IDENTIFICATION OF SELENOCYSTEINE LYASE

Selenocysteine β-lyase was originally described in 1982 by Soda and his collaborators at Kyoto University (5). The enzyme was purified from pig liver and characterized. The liver enzyme, molecular weight of about 85,000, appeared to be a dimer of 48,000 subunits and contained bound pyridoxal phosphate as cofactor. The enzyme exhibited high specificity for selenocysteine as substrate (K_m = 0.83 mM), and L-cysteine was a competitive inhibitor (K_i = 1.0 mM). Selenocysteine was decomposed to equimolar amounts of H₂Se and alanine in the presence of an added reducing agent, DDT. The widespread distribution of selenocysteine lyase in numerous bacterial species was reported (2) and the bacterial enzymes, like the pig liver enzyme, required pyridoxal phosphate as cofactor. A more detailed study of the enzyme isolated from Citrobacter freundii (3) showed that, in the absence of an added reducing agent, selenocysteine was converted to equimolar amounts of elemental selenium and alanine. High K_m and K_i values for L-selenocysteine as substrate and for L-cysteine as inhibitor also were exhibited by this bacterial enzyme. β-Chloro-L-alanine was decomposed to Cl^–, ammonia, and pyruvate by a process that irreversibly inactivated the enzyme. Although the bacterial and pig liver enzymes differed in subunit structure and certain physicochemical properties, their enzymological properties were comparable. Because of the high substrate concentrations required for full catalytic activity, these earlier investigators concluded that these L-selenocysteine lyases probably served primarily as detoxifying agents.

 

CYSTEINE LYASES AND NIFS PROTEINS

In the 1990s, interest in the selenocysteine lyase enzymes was renewed when information became available on a comparable group of enzymes, known as NIFS proteins. The NIFS proteins that decomposed L-cysteine to elemental sulfur and alanine were characterized from Azotobacter species by Zheng et al. (17, 18) and Flint (6) and termed L-cysteine lyases. They were shown to furnish sulfur for biosynthesis of iron-sulfur clusters in enzymes of nitrogen-fixing bacteria. Moreover, selenocysteine could serve as an alternate substrate for these NIFS proteins. A NIFS enzyme from A. vinelandii was shown to function as a selenium delivery protein for the in vitro synthesis of selenophosphate (9). In this case, selenocysteine in the presence of the NIFS protein was used as source of selenium for selenophosphate synthetase. In the absence of the NIFS protein, selenocysteine was inactive as a substrate. The transfer of selenium from free selenocysteine by the NIFS protein to selenophosphate synthetase occurred in aqueous solution without equilibration with a solvent.

 

SELENOCYSTEINE LYASE EXPRESSION IN E. COLI

Esaki and coworkers (11) isolated a cDNA clone for selenocysteine lyase from mouse liver that corresponded in sequence to the enzyme previously isolated from pig liver (5), and the protein was expressed in E. coli. Pyridoxal phosphate was required for catalytic activity. The gene product, a homodimer of 47-kDa subunits, was related in sequence to the NIFS family, but cysteine was a relatively poor substrate. The K _cat/K_m value determined for the mouse liver enzyme with L-selenocysteine as substrate was about 4,200 times greater than that with L-cysteine. Such selectivity for trace levels of selenium in the presence of millimolar concentrations of sulfur compounds is essential for specific selenoprotein biosynthesis in cells.

 

SELENOCYSTEINE LYASES FROM E. COLI

Three NIFS-like genes were isolated from E. coli by Esaki and coworkers and the expressed gene products were isolated and characterized (13, 14). One of these NIFS-like proteins also exhibited a high preference for selenocysteine over cysteine. Detailed studies on the release of elemental sulfur and selenium from the corresponding amino acid substrates by the lyase enzymes indicate that different reaction mechanisms are involved (12).

 

SELENOCYSTEINE LYASE FROM METHANOCOCCUS VANNIELII

M. vannielii, an anaerobic methane-producing organism that grows in a mineral medium containing formate as sole organic carbon source, synthesizes several specific selenoenzymes required for growth and energy production under these conditions. The apparent need for an especially active selenium metabolism in this organism suggested that selenium transferase enzymes should be present in significant amounts. In fact, two similar lyase proteins could be separated from cell extracts after several anionic and hydrophobic interaction chromatographic steps (T. C. Stadtman, unpublished data). L-Selenocysteine was the most active substrate for one of these lyase proteins, and L-cysteine was the preferred substrate for the other. Determination of the precise substrate specificities of these M. vannielii enzymes awaits the availability of additional amounts of highly purified preparations of the two proteins. Presumably the lyase protein that exhibited much higher activity on selenocysteine than on cysteine as substrate should be virtually specific for selenocysteine in homogeneous preparations unless an additional factor is required for the effective discrimination of selenium over sulfur.