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A nifS-like Gene, csdB, Encodes anEscherichia coli Counterpart of Mammalian Selenocysteine Lyase

1999; Elsevier BV; Volume: 274; Issue: 21 Linguagem: Inglês

10.1074/jbc.274.21.14768

1083-351X

Hisaaki Mihara, Masaki Maeda, Tomomi Fujii, Tatsuo Kurihara, Yasuo Hata, Nobuyoshi Esaki,

Sulfur Compounds in Biology

Selenocysteine lyase is a pyridoxal 5′-phosphate (PLP)-dependent enzyme that catalyzes the exclusive decomposition of l-selenocysteine tol-alanine and elemental selenium. An open reading frame, named csdB, from Escherichia coli encodes a putative protein that is similar to selenocysteine lyase of pig liver and cysteine desulfurase (NifS) of Azotobacter vinelandii. In this study, the csdB gene was cloned and expressed inE. coli cells. The gene product was a homodimer with the subunit M r of 44,439, contained 1 mol of PLP as a cofactor per mol of subunit, and catalyzed the release of Se, SO2, and S from l-selenocysteine,l-cysteine sulfinic acid, and l-cysteine, respectively, to yield l-alanine; the reactivity of the substrates decreased in this order. Although the enzyme was not specific for l-selenocysteine, the high specific activity for l-selenocysteine (5.5 units/mg compared with 0.019 units/mg for l-cysteine) supports the view that the enzyme can be regarded as an E. coli counterpart of mammalian selenocysteine lyase. We crystallized CsdB, the csdB gene product, by the hanging drop vapor diffusion method. The crystals were of suitable quality for x-ray crystallography and belonged to the tetragonal space group P43212 with unit cell dimensions of a = b = 128.1 Å and c = 137.0 Å. Consideration of the Matthews parameter Vm (3.19 Å3/Da) accounts for the presence of a single dimer in the crystallographic asymmetric unit. A native diffraction dataset up to 2.8 Å resolution was collected. This is the first crystallographic analysis of a protein of NifS/selenocysteine lyase family. Selenocysteine lyase is a pyridoxal 5′-phosphate (PLP)-dependent enzyme that catalyzes the exclusive decomposition of l-selenocysteine tol-alanine and elemental selenium. An open reading frame, named csdB, from Escherichia coli encodes a putative protein that is similar to selenocysteine lyase of pig liver and cysteine desulfurase (NifS) of Azotobacter vinelandii. In this study, the csdB gene was cloned and expressed inE. coli cells. The gene product was a homodimer with the subunit M r of 44,439, contained 1 mol of PLP as a cofactor per mol of subunit, and catalyzed the release of Se, SO2, and S from l-selenocysteine,l-cysteine sulfinic acid, and l-cysteine, respectively, to yield l-alanine; the reactivity of the substrates decreased in this order. Although the enzyme was not specific for l-selenocysteine, the high specific activity for l-selenocysteine (5.5 units/mg compared with 0.019 units/mg for l-cysteine) supports the view that the enzyme can be regarded as an E. coli counterpart of mammalian selenocysteine lyase. We crystallized CsdB, the csdB gene product, by the hanging drop vapor diffusion method. The crystals were of suitable quality for x-ray crystallography and belonged to the tetragonal space group P43212 with unit cell dimensions of a = b = 128.1 Å and c = 137.0 Å. Consideration of the Matthews parameter Vm (3.19 Å3/Da) accounts for the presence of a single dimer in the crystallographic asymmetric unit. A native diffraction dataset up to 2.8 Å resolution was collected. This is the first crystallographic analysis of a protein of NifS/selenocysteine lyase family. Selenocysteine lyase (SCL) 1The abbreviations used are: SCL, selenocysteine lyase; PLP, pyridoxal 5′-phosphate; KPB, potassium phosphate buffer; PAGE, polyacrylamide gel electrophoresis; Tricine, N-tris(hydroxymethyl)methylglycine; NifS, cysteine desulfurase; CSD, cysteine sulfinate desulfinase; Mes, 2-(N-morpholino)ethanesulfonic acid. (EC 4.4.1.16) and cysteine desulfurase (commonly referred to as NifS) are pyridoxal 5′-phosphate (PLP)-dependent enzymes that catalyze the same type of reaction, i.e. the removal of a sulfur or selenium atom from l-cysteine or l-selenocysteine to produce l-alanine. NifS acts on both l-cysteine and l-selenocysteine (1Zheng L. White R.H. Cash V.L. Dean D.R. Biochemistry. 1994; 33: 4714-4720Crossref PubMed Scopus (358) Google Scholar, 2Lacourciere G.M. Stadtman T.C. J. Biol. Chem. 1998; 273: 30921-30926Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). Several enzymes participating in sulfur metabolism also act on the selenium analogs of the substrates (3Stadtman T.C. Adv. Enzymol. Relat. Areas Mol. Biol. 1979; 48: 1-28PubMed Google Scholar). In contrast, SCL exclusively decomposesl-selenocysteine. Selenium is specifically metabolized by such enzymes as selenophosphate synthetase (4Veres Z. Kim I.Y. Scholz T.D. Stadtman T.C. J. Biol. Chem. 1994; 269: 10597-10603Abstract Full Text PDF PubMed Google Scholar), selenocysteine synthase (5Forchhammer K. Böck A. J. Biol. Chem. 1991; 266: 6324-6328Abstract Full Text PDF PubMed Google Scholar), and selenocysteine methyl transferase (6Neuhierl B. Böck A. Eur. J. Biochem. 1996; 239: 235-238Crossref PubMed Scopus (152) Google Scholar). Discrimination of selenium from sulfur is important for establishing the role of selenium as an essential trace element in mammals and other organisms (7Stadtman T.C. Annu. Rev. Biochem. 1996; 65: 83-100Crossref PubMed Scopus (819) Google Scholar). SCL of pig liver (8Esaki N. Nakamura T. Tanaka H. Soda K. J. Biol. Chem. 1982; 257: 4386-4391Abstract Full Text PDF PubMed Google Scholar) was characterized as the first enzyme that specifically acts on a selenium-containing substrate. We have found that the peptide sequences obtained from the proteolysate of SCL are similar to those of NifS proteins. 2H. Mihara, T. Kurihara, T. Yoshimura, and N. Esaki, unpublished results. However, a gene encoding SCL has not been cloned yet, and the physiological role of the enzyme remains to be investigated. Selenoproteins such as formate dehydrogenase from Escherichia coli contain selenocysteine residues (9Zinoni F. Birkmann A. Stadtman T.C. Böck A. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 4650-4654Crossref PubMed Scopus (350) Google Scholar, 10Heider J. Böck A. Adv. Microb. Physiol. 1993; 35: 71-109Crossref PubMed Google Scholar). Selenocysteyl-tRNASec is required for the biosynthesis of these selenoproteins (5Forchhammer K. Böck A. J. Biol. Chem. 1991; 266: 6324-6328Abstract Full Text PDF PubMed Google Scholar, 11Leinfelder W. Stadtman T.C. Böck A. J. Biol. Chem. 1989; 264: 9720-9723Abstract Full Text PDF PubMed Google Scholar, 12Lee B.J. Worland P.J. Davis J.N. Stadtman T.C. Hatfield D.L. J. Biol. Chem. 1989; 264: 9724-9727Abstract Full Text PDF PubMed Google Scholar). Selenophosphate is a highly reactive selenium compound, and serves as a selenium donor for the selenocysteyl-tRNASec production (4Veres Z. Kim I.Y. Scholz T.D. Stadtman T.C. J. Biol. Chem. 1994; 269: 10597-10603Abstract Full Text PDF PubMed Google Scholar, 7Stadtman T.C. Annu. Rev. Biochem. 1996; 65: 83-100Crossref PubMed Scopus (819) Google Scholar, 12Lee B.J. Worland P.J. Davis J.N. Stadtman T.C. Hatfield D.L. J. Biol. Chem. 1989; 264: 9724-9727Abstract Full Text PDF PubMed Google Scholar). Selenophosphate is synthesized from selenide and ATP, which is catalyzed by selenophosphate synthetase (13Mullins L.S. Hong S.-B. Gibson G.E. Walker H. Stadtman T.C. Raushel F.M. J. Am. Chem. Soc. 1997; 119: 6684-6685Crossref Scopus (22) Google Scholar, 14Liu S.-Y. Stadtman T.C. Arch. Biochem. Biophys. 1997; 341: 353-359Crossref PubMed Scopus (9) Google Scholar). Recently, Lacourciere and Stadtman (2Lacourciere G.M. Stadtman T.C. J. Biol. Chem. 1998; 273: 30921-30926Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar) found that the replacement of selenide by NifS andl-selenocysteine in an in vitro selenophosphate synthetase assay resulted in an increased rate of formation of selenophosphate, indicating that selenium derived froml-selenocysteine by the action of NifS serves as a better substrate than selenide for selenophosphate synthetase. In Azotobacter vinelandii, NifS functions in nitrogen fixation by supplying sulfur to stabilize or repair the Fe-S cluster of the nitrogenase component protein (15Zheng L. White R.H. Cash V.L. Jack R.F. Dean D.R. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 2754-2758Crossref PubMed Scopus (503) Google Scholar). NifS homologs also occur in many nondiazotrophic procaryotes, including E. coli (16Mihara H. Kurihara T. Yoshimura T. Soda K. Esaki N. J. Biol. Chem. 1997; 272: 22417-22424Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar, 17Flint D.H. J. Biol. Chem. 1996; 271: 16068-16074Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar) and Bacillus subtilis (18Sun D. Setlow P. J. Bacteriol. 1993; 175: 1423-1432Crossref PubMed Google Scholar), and in eucaryotes, includingSaccharomyces cerevisiae (19Kolman C. Söll D. J. Bacteriol. 1993; 175: 1433-1442Crossref PubMed Google Scholar), Caenorhabditis elegans (20Wilson R. Ainscough R. Anderson K. Baynes C. Berks M. Bonfield J. Burton J. Connell M. Copsey T. Cooper J. Coulson A. Craxton M. Dear S. Du Z. Durbin R. Favello A. Fraser A. Fulton L. Gardner A. Green P. Hawkins T. Hillier L. Jier M. Johnston L. Jones M. Kershaw J. Kirsten J. Laister N. Latreille P. Lightning J. Lloyd C. Mortimore B. O'Callaghan M. Parsons J. Percy C. Rifken L. Roopra A. Saunders D. Shownkeen R. Sims M. Smaldon N. Smith A. Smith M. Sonnhammer E. Staden R. Sulston J. Thierry-Mieg J. Thomas K. Vaudin M. Vaughan K. Waterston R. Watson A. Weinstock L. Wilkinson-Sproat J. Wohldman P. Nature. 1994; 368: 32-38Crossref PubMed Scopus (1438) Google Scholar), mice (21Nakai Y. Yoshihara Y. Hayashi H. Kagamiyama H. FEBS Lett. 1998; 433: 143-148Crossref PubMed Scopus (50) Google Scholar), and humans.2 These NifS homologs are proposed to play a general role in the mobilization of sulfur for Fe-S cluster synthesis (22Zheng L. Cash V.L. Flint D.H. Dean D.R. J. Biol. Chem. 1998; 273: 13264-13272Abstract Full Text Full Text PDF PubMed Scopus (576) Google Scholar). However, the exact roles of thenifS-like genes in these non-nitrogen-fixing organisms have not been clarified, and it is possible that some of these NifS homologs act physiologically as selenocysteine-specific enzymes (e.g.SCL) to facilitate the selenophosphate synthesis, as proposed by Lacourciere and Stadtman (2Lacourciere G.M. Stadtman T.C. J. Biol. Chem. 1998; 273: 30921-30926Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). The E. coli genome contains three genes with sequence homology to nifS. Two enzymes, IscS (17Flint D.H. J. Biol. Chem. 1996; 271: 16068-16074Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar) and cysteine sulfinate desulfinase (CSD) (16Mihara H. Kurihara T. Yoshimura T. Soda K. Esaki N. J. Biol. Chem. 1997; 272: 22417-22424Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar), among the three NifS homologs have been isolated and characterized. E. coli IscS, which shows significant sequence identity (40%) to the A. vinelandiiNifS, can deliver the sulfur from l-cysteine for thein vitro synthesis of the Fe-S cluster of dihydroxyacid dehydratase from E. coli (17Flint D.H. J. Biol. Chem. 1996; 271: 16068-16074Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). CSD exhibits both selenocysteine lyase and cysteine desulfurase activities in addition to cysteine sulfinate desulfinase activity, and the enzyme is distinct from A. vinelandii NifS in its amino acid sequence, absorption spectrum, and lack of cysteine residues catalytically essential for the decomposition of l-selenocysteine (16Mihara H. Kurihara T. Yoshimura T. Soda K. Esaki N. J. Biol. Chem. 1997; 272: 22417-22424Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar). Neither enzyme shows strict specificity forl-selenocysteine, and both act on l-cysteine. Thus, we have explored the possibility that the last nifShomolog (csdB) 3The symbol, csd, was given to designate the postulatedcysteine-selenocysteine-decomposition function of a gene product, although the physiological relevance has not been proved. mapped at 37.9 min (23Rudd K.E. Microbiol. Mol. Biol. Rev. 1998; 62: 985-1019Crossref PubMed Google Scholar) in the chromosome encodes SCL, which plays a crucial role in selenophosphate synthesis. We have isolated the gene product (CsdB), studied its enzymatic properties, and carried out preliminary x-ray crystallographic studies. Restriction enzymes and other DNA modifying enzymes were purchased from New England Biolabs (Beverly, MA) and Takara Shuzo (Kyoto, Japan); molecular weight markers for SDS-PAGE and gel filtration were from Amersham Pharmacia Biotech (Uppsala, Sweden) and Oriental Yeast (Tokyo, Japan); oligonucleotides were from Biologica (Nagoya, Japan); Gigapite was from Seikagaku Corporation (Tokyo, Japan); DEAE-Toyopearl, Phenyl-Toyopearl and Butyl-Toyopearl were from Tosoh (Tokyo, Japan). l-Selenocystine was synthesized as described previously (24Tanaka H. Soda K. Methods Enzymol. 1987; 143: 240-243Crossref PubMed Scopus (38) Google Scholar). l-Selenocysteine was prepared from l-selenocystine according to the previous method (8Esaki N. Nakamura T. Tanaka H. Soda K. J. Biol. Chem. 1982; 257: 4386-4391Abstract Full Text PDF PubMed Google Scholar). The Kohara/Isono miniset clone No. 430 (25Kohara Y. Akiyama K. Isono K. Cell. 1987; 50: 495-508Abstract Full Text PDF PubMed Scopus (1110) Google Scholar) was a kind gift from Dr. Yuji Kohara, National Institute of Genetics, Japan. All other chemicals were of analytical grade. The DNA fragment containingcsdB was cloned from the chromosomal DNA of E. coli K-12 ICR130 by polymerase chain reaction in a manner identical to that used for cloning of csdA (16Mihara H. Kurihara T. Yoshimura T. Soda K. Esaki N. J. Biol. Chem. 1997; 272: 22417-22424Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar). Oligonucleotide primers used were 5′-GGAATTC AGGAGGTGCCATATGATTTTTTCCGTCGAC-3′ (upstream) and 5′-CCCAAGCTTATCCCAGCAAACGGTG-3′ (downstream); underlining indicates EcoRI andHindIII sites, respectively, and bold face indicates a putative ribosome binding sequence. The polymerase chain reaction product was ligated into pUC118, and then the resultant expression plasmid, pCSDB, was introduced into E. coli JM109 competent cells. The enzyme was assayed in 0.12 mTricine-NaOH buffer at pH 7.5. The enzymatic activities towardl-selenocysteine and l-cysteine were measured with lead acetate as described previously (16Mihara H. Kurihara T. Yoshimura T. Soda K. Esaki N. J. Biol. Chem. 1997; 272: 22417-22424Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar). The previously reported value (8Esaki N. Nakamura T. Tanaka H. Soda K. J. Biol. Chem. 1982; 257: 4386-4391Abstract Full Text PDF PubMed Google Scholar) for a molar turbidity coefficient of PbSe at 400 nm was corrected as 1.18 × 104m−1·cm−1, and this value was used in this study. Sulfite produced from l-cysteine sulfinic acid was determined with fuchsin (26Leinweber F.-J. Monty K.J. Methods Enzymol. 1987; 143: 15-17Crossref PubMed Scopus (24) Google Scholar). Production of alanine from substrates was determined with a Beckman 7300 high performance amino acid analyzer (Beckman Coulter, Fullerton, CA). Specific activity was expressed as units/mg of protein, with 1 unit of enzyme defined as the amount that catalyzed the formation of 1 μmol of the product in 1 min. Purification was carried out at 0–4 °C, and potassium phosphate buffer (KPB) (pH 7.4) was used as the buffer throughout the purification. E. coli JM109 cells harboring pCSDB were grown in 9 liters of LB medium containing 200 μg/ml ampicillin and 1 mmisopropyl-1-thio-β-d-galactopyranoside at 37 °C for 16 h. The cells were harvested by centrifugation, suspended in 10 mm KPB, and disrupted by sonication. The cell debris was removed by centrifugation, and the supernatant solution was fractionated by ammonium sulfate precipitation (25–50% saturation). The enzyme was dissolved in 10 mm KPB and dialyzed against the same buffer. The enzyme was applied to a DEAE-Toyopearl column (3 × 15 cm) equilibrated with the same buffer. After the column was washed with the same buffer, the enzyme was eluted with a 0.8-liter linear gradient of 0–0.25 m NaCl in the buffer. The active fractions were pooled (110 ml) and concentrated by ultrafiltration through an Advantec UP-20 membrane (Advantec, Naha, Japan). The enzyme was dialyzed against 10 mm buffer containing 0.65m ammonium sulfate and applied to a Phenyl-Toyopearl column (3 × 15 cm) equilibrated with the same buffer. The enzyme was eluted with a 0.7-liter linear gradient of 0.65–0.3 mammonium sulfate in the buffer, and the active fractions were pooled and concentrated as above. The enzyme was dialyzed against 10 mm buffer and applied to a Gigapite column (3 × 10 cm) equilibrated with the same buffer. The enzyme was eluted with a 1-liter linear gradient of 10–150 mm KPB, and the active fractions were collected and concentrated. The final preparation was further concentrated with Centriprep-10 (Millipore, Bedford, MA) to a volume of 2.7 ml. Purification of CSD fromE. coli JM109 transformed with a plasmid pCSD1 containing the csdA gene was performed as described previously (16Mihara H. Kurihara T. Yoshimura T. Soda K. Esaki N. J. Biol. Chem. 1997; 272: 22417-22424Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar). Expression and purification of recombinant IscS will be described elsewhere. 4H. Mihara, T. Kurihara, T. Yoshimura, and N. Esaki, manuscript in preparation. Briefly, theiscS gene was amplified by polymerase chain reaction with the Kohara miniset clone No. 430 (25Kohara Y. Akiyama K. Isono K. Cell. 1987; 50: 495-508Abstract Full Text PDF PubMed Scopus (1110) Google Scholar) as a template and inserted into the NdeI and HindIII sites in pET21a (Novagen, Madison, WI) to yield pEF1. IscS was isolated from BL21 (DE3) pLysS cells harboring pEF1 by sonication, ammonium sulfate fractionation, and chromatography with Phenyl-Toyopearl, DEAE-Toyopearl, Butyl-Toyopearl, Gigapite, and Superose 12 (Amersham Pharmacia Biotech, Uppsala, Sweden) columns. Protein was quantified by the Bradford method (27Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (217544) Google Scholar) using Protein Assay CBB solution (Nacalai Tesque, Kyoto, Japan) with bovine serum albumin as a standard. The concentration of the purified enzyme was determined with the value εM = 4.8 × 104m−1·cm−1 at 280 nm, which was calculated from the content of tyrosine, tryptophan, and cysteine (28Perkins S.J. Eur. J. Biochem. 1986; 157: 169-180Crossref PubMed Scopus (546) Google Scholar). The subunit and the native M r of CsdB were determined by SDS-PAGE (29Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207537) Google Scholar) and gel filtration with Superdex 200 (Amersham Pharmacia Biotech, Uppsala, Sweden), respectively. The PLP content of the enzyme was determined fluorometrically with KCN according to the method of Adams (30Adams E. Methods Enzymol. 1979; 62: 407-410Crossref PubMed Scopus (60) Google Scholar). Crystals of CsdB were grown by the hanging drop vapor diffusion method. Each droplet was prepared by mixing 5 μl of 20 mg/ml enzyme in 10 mm KPB (pH 7.4) with an equal volume of each reservoir solution of the Crystal Screen™ (Hampton Research, CA) initially and of a modified reservoir solution subsequently. The yellow crystals of CsdB were mounted in glass capillaries with the crystallographic c* axis along the rotation axis of the spindle and subjected to x-ray experiments. Native data for structure determination were collected at 20 °C with a Rigaku R-AXIS IIC imaging plate detector using double focusing mirror-monochromated CuK α radiation that was generated with a 0.3-mm focal cup of an x-ray generator RU300 (Rigaku, Tokyo, Japan) operated at 40 kV and 100 mA. The crystal-to-detector distance was set to 130.0 mm. Data reduction was carried out using the R-AXIS IIC software package. For the production of a large amount of CsdB, expression plasmids were constructed as described under "Experimental Procedures" with chromosomal DNA isolated from E. coliK-12. The nucleotide sequence of csdB in the expression vector (pCSDB) was confirmed to be identical with that registered in GenBank™ accession number D90811 (open reading frame o320#17). The clone provided overexpression of the cloned gene: about 10% of the total protein in the extract of E. coli JM109 recombinant cells. In the representative purification (TableI), about 8 mg of homogeneous preparation of CsdB was obtained per liter of culture.Table IPurification of CsdBStepTotal proteinTotal activityaDetermined with l-selenocysteine as a substrate.Specific activityPurificationYieldmgunitsunits/mg-fold%Crude extract310016000.521100Ammonium sulfate10009200.921.858DEAE-Toyopearl3708902.44.656Phenyl-Toyopearl854305.19.827Gigapite703905.61124a Determined with l-selenocysteine as a substrate. Open table in a new tab CsdB provided a single band corresponding to the M r of 43,000 on SDS-PAGE (Fig. 1). The N-terminal sequence of the purified enzyme, MIFSVDKVRA, agreed with that deduced from the nucleotide sequence of csdB. The M rof the native enzyme was determined to be 88,000 by gel filtration. Consequently, the enzyme is a dimer composed of two identical subunits. The spectrophotometric properties of the enzyme were very similar to those of CSD with an absorption maximum at 420 nm (Fig. 2) at pH 7.4. No significant changes in the absorption spectrum were observed in the range of pH 6–8. This absorption peak is characteristic of PLP enzymes, which contain the cofactor bound to the ε-amino group of a lysine residue at the active site. However, CsdB is distinct from either of two A. vinelandii proteins, NifS and IscS, and also from IscS of E. coli, all of which have an absorption maximum around 390 nm (15Zheng L. White R.H. Cash V.L. Jack R.F. Dean D.R. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 2754-2758Crossref PubMed Scopus (503) Google Scholar,17Flint D.H. J. Biol. Chem. 1996; 271: 16068-16074Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar, 22Zheng L. Cash V.L. Flint D.H. Dean D.R. J. Biol. Chem. 1998; 273: 13264-13272Abstract Full Text Full Text PDF PubMed Scopus (576) Google Scholar). Reduction with sodium borohydride resulted in a decrease in the absorption peak at 420 nm with a concomitant increase in the absorbance at 335 nm (Fig. 2). This result is consistent with that this is a PLP enzyme. The PLP content of CsdB was determined to be 1.0 mol per mol of subunit by the fluorometric method (30Adams E. Methods Enzymol. 1979; 62: 407-410Crossref PubMed Scopus (60) Google Scholar).Figure 2Absorption spectra of CsdB. The conditions were as follows: 10 mm KPB, pH 7.4, 25 °C, 0.64 mg/ml of the enzyme. Curve 1, native enzyme;curve 2, 1 min after addition of sodium borohydride (1 mm) to the enzyme solution.View Large Image Figure ViewerDownload Hi-res image Download (PPT) CsdB catalyzed the removal of a substituent at the β-carbon ofl-selenocysteine, l-cysteine, andl-cysteine sulfinic acid to yield l-alanine. The production of elemental selenium and elemental sulfur froml-selenocysteine and l-cysteine, respectively, in the reaction was confirmed in the same manner as reported previously (31Esaki N. Karai N. Nakamura T. Tanaka H. Soda K. Arch. Biochem. Biophys. 1985; 238: 418-423Crossref PubMed Scopus (27) Google Scholar). The optimal pH value for the removal of selenium froml-selenocysteine was between 6.5 and 7.5 in Tricine-NaOH or Mes buffer. The substrate specificity of the enzyme is summarized in Table II; l-selenocysteine was the best substrate followed by l-cysteine sulfinic acid and l-cysteine, in that order. The specific activity of CsdB on l-selenocysteine (5.5 units/mg) was comparable with that of CSD and IscS (Table III) but was about 7 times lower than that of SCL (37 units/mg) (8Esaki N. Nakamura T. Tanaka H. Soda K. J. Biol. Chem. 1982; 257: 4386-4391Abstract Full Text PDF PubMed Google Scholar). The cysteine desulfurase activity of CsdB was about 2 and 5% of that of CSD and IscS, respectively, at a substrate concentration of 12 mm(Table III). The specific activity of CsdB for l-cysteine was about 1/290 of the activity with l-selenocysteine (Table III). This value is much lower than those of CSD and IscS (TableIII). In contrast with CsdB, A. vinelandii NifS favorsl-cysteine as a substrate over its selenium analog (2Lacourciere G.M. Stadtman T.C. J. Biol. Chem. 1998; 273: 30921-30926Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). CsdB acted on l-cystine, l-selenocystine, andl-aspartic acid, although at extremely low rates (<0.08% of the rate for l-selenocysteine) (Table II).Table IISubstrate specificity of CsdBSubstrateSpecific activityaActivity was measured in the reaction buffer containing one of the following substrates:l-selenocysteine, 12 mm; l-cysteine sulfinic acid, 12 mm; l-cysteine, 12 mm; l-selenocystine, 20 mm;l-cystine, 20 mm; l-aspartic acid, 20 mm.Relative activityunits/mg%l-Selenocysteine5.5100l-Cysteine sulfinic acid0.8215l-Cysteine0.0190.35l-Selenocystine0.00440.080l-Cystine0.00310.056l-Aspartic acid0.00440.080The following amino acids were inert as the substrates when production of alanine was examined in the reaction mixture containing 10 mm substrate at 37 °C for 12 h:d-selenocysteine, d-cysteine,d-selenocystine, d-cystine,dl-kynurenine, l-djenkolic acid,dl-selenocysteamine,S-benzyl-l-cysteine.a Activity was measured in the reaction buffer containing one of the following substrates:l-selenocysteine, 12 mm; l-cysteine sulfinic acid, 12 mm; l-cysteine, 12 mm; l-selenocystine, 20 mm;l-cystine, 20 mm; l-aspartic acid, 20 mm. Open table in a new tab Table IIIDiscrimination of l-selenocysteine froml-cysteine in the reaction catalyzed by CsdB, CSD, and IscSEnzymeaThe amino acid sequence of the proteins can be accessed through NCBI Protein Database under NCBI Accession numbers 1742766 (CsdB), 1789175 (CSD), and 1788879 (IscS).Map positionbThe map positions were from Ref. 23.Specific activityDiscrimination factorcDiscrimination factor was calculated from the specific activity of the enzymes for l-selenocysteine divided by that for l-cysteine. Activity was measured in the reaction mixture containing 120 mm Tricine-NaOH (pH 7.5), 50 mm dithiothreitol, 0.2 mm PLP, and 12 mm substrate.l-Selenocysteinel-Cysteineminunits/mgunits/mgratio (l-selenocysteine/ l-cysteine)CsdB37.95.50.019290CSD63.46.20.906.9IscS57.33.10.388.2a The amino acid sequence of the proteins can be accessed through NCBI Protein Database under NCBI Accession numbers 1742766 (CsdB), 1789175 (CSD), and 1788879 (IscS).b The map positions were from Ref. 23Rudd K.E. Microbiol. Mol. Biol. Rev. 1998; 62: 985-1019Crossref PubMed Google Scholar.c Discrimination factor was calculated from the specific activity of the enzymes for l-selenocysteine divided by that for l-cysteine. Activity was measured in the reaction mixture containing 120 mm Tricine-NaOH (pH 7.5), 50 mm dithiothreitol, 0.2 mm PLP, and 12 mm substrate. Open table in a new tab The following amino acids were inert as the substrates when production of alanine was examined in the reaction mixture containing 10 mm substrate at 37 °C for 12 h:d-selenocysteine, d-cysteine,d-selenocystine, d-cystine,dl-kynurenine, l-djenkolic acid,dl-selenocysteamine,S-benzyl-l-cysteine. CsdB was crystallized at 25 °C within 2 days by hanging drop vapor diffusion against a 100 mm cacodylate solution (pH 6.8) containing 1.4 m sodium acetate, which corresponds to the solution No.7 in the Crystal Screen™. The crystals were also obtained in 100 mm KPB (pH 6.8) containing 1.4m sodium acetate and 10 μm PLP, and these conditions were further used for the crystallization of the enzyme. The yellow crystals (0.5 × 0.5 × 0.4 mm3) had tetragonal-bipyramidal shapes (Fig. 3). They were grown in amorphous debris, which was removed from the crystals before they were sealed in thin-walled glass capillaries. The space group of the CsdB wasP43212 with the cell dimensions ofa = b = 128.1 Å, and c= 137.0 Å. The assumption that a single dimer (89 kDa) exists in the asymmetric unit of the crystal gives a Vm value of 3.19 Å3/Da, which is equivalent to a solvent content of 62%. These values lie within the range of values commonly found for proteins (32Matthews B.W. J. Mol. Biol. 1968; 33: 491-497Crossref PubMed Scopus (7927) Google Scholar). A set of native data was collected to 2.8 Å resolution on a Rigaku R-AXIS IIC using 1.5° oscillation over a range of 45° (94.2% complete with 23,770 independent reflections). TheR merge value for the intensity data was 7.22%. The data collection statistics obtained for the native CsdB crystals are given in Table IV. The x-ray crystal structure determination of the enzyme is now under way by the multiple isomorphous replacement method.Table IVData collection statisticsUnit cell dimensions (Å)a = b = 128.1; c = 137.0Space groupP43212Resolution limit (Å)2.8Number of reflections73,138Number of unique reflections23,770Percent completeness94.2RmergeaRmerge = ∑ ‖Ii− 〈I〉‖∑Ii. (%)7.22a Rmerge = ∑ ‖Ii− 〈I〉‖∑Ii. Open table in a new tab We also obtained crystals of CSD at 25 °C by hanging drop vapor diffusion against a 100 mm sodium acetate solution (pH 4.6) containing 200 mm ammonium acetate and 30% (w/v) polyethylene glycol 4000. However, these crystals were small and not suitable for x-ray analysis. Further optimization of crystallization conditions by changing pH, polyethylene glycol concentration, and salt has resulted in little improvement. Grishin et al. (33Grishin N.V. Phillips M.A. Goldsmith E.J. Protein Sci. 1995; 4: 1291-1304Crossref PubMed Scopus (343) Google Scholar) classified PLP enzymes into seven distinct fold types on the basis of primary structure, secondary structure prediction, and biochemical function. NifS proteins have been classified as "aminotransferases class V" in the fold type I together with serine-pyruvate aminotransferase (EC 2.6.1.51), phosphoserine aminotransferase (EC 2.6.1.52), isopenicillin N epimerase, and the small subunit of the soluble hydrogenase. Recently, three-dimensional structures of phosphoserine aminotransferases fromBacillus circulans sbsp.Alkalophilus 5G. Hester, T. N. Luong, M. Moser, and J. N. Jansonius, unpublished results. andE. coli 6G. Hester, W. Stark, M. Moser, J. Kallen, Z. Markovic-Housley, and J. N. Jansonius, unpublished results. were solved and deposited in the Protein Data Bank, Brookhaven National Laboratory, with the codes 1BT4 and 1BJN, respectively. Comparison of the structures of phosphoserine aminotransferases with that of CsdB will contribute to the understanding of how the related proteins confer separate reaction specificities on the same coenzyme. The reaction of CsdB shares some common features with that of other PLP-dependent enzymes such as aspartate β-decarboxylase (EC 4.1.1.12) (34Tate S.S. Meister A. Adv. Enzymol. Relat. Areas Mol. Biol. 1971; 35: 503-543PubMed Google Scholar), kynureninase (EC 3.7.1.3) (35Soda K. Tanizawa K. Adv. Enzymol. Relat. Areas Mol. Biol. 1979; 49: 1-40PubMed Google Scholar), and SCL. These enzymes catalyze removal of β-substituent from the substrate to form alanine. None of their structures have been solved. The solution of the three-dimensional structure of CsdB would contribute to the understanding of the mechanisms of these PLP-dependent enzymes. Genome sequencing projects have revealed that homologs of A. vinelandii nifS are widespread throughout nature and that some organisms contain more than one copy of a nifS homolog (16Mihara H. Kurihara T. Yoshimura T. Soda K. Esaki N. J. Biol. Chem. 1997; 272: 22417-22424Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar, 22Zheng L. Cash V.L. Flint D.H. Dean D.R. J. Biol. Chem. 1998; 273: 13264-13272Abstract Full Text Full Text PDF PubMed Scopus (576) Google Scholar). Some of the "NifS-like proteins" characterized so far preferl-cysteine to l-selenocysteine, and some of them show the opposite preference. Further experiments will need to be done to determine whether putative NifS-like proteins can play a role in Fe-S cluster assembly. Lacourciere and Stadtman (2Lacourciere G.M. Stadtman T.C. J. Biol. Chem. 1998; 273: 30921-30926Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar) have pointed out that in vivoconcentrations of sulfur-containing compounds are on the order of a thousand times greater than those of their selenium analogs (36Sliwkowski M.X. Stadtman T.C. J. Biol. Chem. 1985; 260: 3140-3144Abstract Full Text PDF PubMed Google Scholar). Thus, enzymes showing higher activity toward l-cysteine, such asA. vinelandii NifS, will preferentially utilizel-cysteine over l-selenocysteine in vivo (2Lacourciere G.M. Stadtman T.C. J. Biol. Chem. 1998; 273: 30921-30926Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). Therefore, it may be reasonable to assume that enzymes which are specific toward l-selenocysteine probably function as a physiological selenide delivery system in E. coli. Although CsdB is not strictly specific to selenocysteine, its discrimination factor (290 times over the activity on cysteine) is much higher than those of other NifS homologs of E. coli. Accordingly, the enzyme can be regarded as an E. colicounterpart of mammalian selenocysteine lyase. It would be particularly intriguing to determine whether CsdB is more effective than CSD and IscS as a selenide delivery protein in the formation of selenophosphate catalyzed by E. coli selenophosphate synthetase. Thein vivo function of CsdB is now being studied by disrupting its gene. We thank Dr. Yuji Kohara (National Institute of Genetics, Mishima, Japan) for providing us with the ordered λ phage clone No. 430.