Reaction mechanism and molecular basis for selenium/sulfur discrimination of selenocysteine lyase

Article abstract of DOI:10.1074/jbc.M109.084475

Selenocysteine lyase (SCL) catalyzes the pyridoxal 5′-phosphate- dependent removal of selenium from L-selenocysteine to yield L-alanine. The enzyme is proposed to function in the recycling of the micronutrient selenium from degraded selenoproteins containing selenocysteine residue as an essential component. The enzyme exhibits strict substrate specificity toward L-selenocysteine and no activity to its cognate L-cysteine. However, it remains unclear how the enzyme distinguishes between selenocysteine and cysteine. Here, we present mechanistic studies of selenocysteine lyase from rat. ESI-MS analysis of wild-type and C375A mutant SCL revealed that the catalytic reaction proceeds via the formation of an enzyme-bound selenopersulfide intermediate on the catalytically essential Cys-375 residue. UV-visible spectrum analysis and the crystal structure of SCL complexed with L-cysteine demonstrated that the enzyme reversibly forms a nonproductive adduct with L-cysteine. Cys-375 on the flexible loop directed L-selenocysteine, but not L-cysteine, to the correct position and orientation in the active site to initiate the catalytic reaction. These findings provide, for the first time, the basis for understanding how trace amounts of a selenium-containing substrate is distinguished from excessive amounts of its cognate sulfur-containing compound in a biological system.

Full text of DOI:10.1074/jbc.M109.084475

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 285, NO. 16, pp. 12133–12139, April 16, 2010
2010 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.
©
Reaction Mechanism and Molecular Basis for Selenium/Sulfur
□
S
*
Discrimination of Selenocysteine Lyase
Received for publication, November 12, 2009, and in revised form, February 13, 2010 Published, JBC Papers in Press, February 17, 2010, DOI 10.1074/jbc.M109.084475
‡1 §2
‡
1
¶
ʈ
ʈ
Rie Omi , Suguru Kurokawa , Hisaaki Mihara , Hideyuki Hayashi , Masaru Goto , Ikuko Miyahara ,
‡
ʈ3
‡4
Tatsuo Kurihara , Ken Hirotsu , and Nobuyoshi Esaki
‡
ʈ
From the Institute for Chemical Research, Kyoto University, Uji, Kyoto 611-0011, Japan, the Department of Chemistry, Graduate
School of Science, Osaka City University, Osaka 558-8585, Japan, the Department of Biotechnology, Institute of Science and
Engineering, College of Life Sciences, Ritsumeikan University, Kusatsu, Shiga 525-8577, Japan, and the Department of
§
¶
Biochemistry, Osaka Medical College, 2-7 Daigakumachi, Takatsuki 569-8686, Japan
Selenocysteine lyase (SCL) catalyzes the pyridoxal 5ꢀ-phos-
Selenium is an essential trace element required for the nor-
phate-dependent removal of selenium from L-selenocysteine to mal function of mammals (1). It is similar to sulfur in its chem-
yield L-alanine. The enzyme is proposed to function in the recy- ical properties and needs to be discriminated biologically from
cling of the micronutrient selenium from degraded selenopro- abundant sulfur during its metabolism. During the synthesis of
teins containing selenocysteine residue as an essential compo- selenoproteins, selenium is specifically incorporated into sel-
Sec
nent. The enzyme exhibits strict substrate specificity toward enocysteyl-tRNA and eventually into the specific position of
L-selenocysteine and no activity to its cognate L-cysteine. How- the nascent polypeptide chains as directed by the UGA codon
Sec
ever, it remains unclear how the enzyme distinguishes between (2). The specific synthesis of selenocysteyl-tRNA from seryl-
Sec
selenocysteine and cysteine. Here, we present mechanistic stud- tRNA and selenide involves coordinated sequential reactions
ies of selenocysteine lyase from rat. ESI-MS analysis of wild-type catalyzed by selenophosphate synthetase (3, 4), seryl-tRNA
and C375A mutant SCL revealed that the catalytic reaction pro- kinase (5), and selenocysteine synthase (6, 7). However, the
ceeds via the formation of an enzyme-bound selenopersulfide mechanism underlying selenium-specific substrate recognition
intermediate on the catalytically essential Cys-375 residue. UV- in these enzymes remains unknown.
visible spectrum analysis and the crystal structure of SCL com-
The most common selenium source in normal foods is sel-
plexed with L-cysteine demonstrated that the enzyme reversibly enocysteine and selenomethionine in proteins (8). Previous
forms a nonproductive adduct with L-cysteine. Cys-375 on the studies suggest that selenomethionine is transformed to seleno-
flexible loop directed L-selenocysteine, but not L-cysteine, to the cysteine through the trans-selenation pathway (9, 10). Specific
5
correct position and orientation in the active site to initiate cleavage of selenocysteine by selenocysteine lyase (SCL) is
the catalytic reaction. These findings provide, for the first time, thought to be the critical metabolic step for the recycling of
the basis for understanding how trace amounts of a selenium- selenocysteine, which is formed by proteolytic degradation of
containing substrate is distinguished from excessive amounts of selenoproteins.
its cognate sulfur-containing compound in a biological system.
SCL is a pyridoxal 5Ј-phosphate (PLP) enzyme that catalyzes
the removal of selenium from L-selenocysteine to yield L-ala-
nine. In previous studies, we identified and characterized SCL
from pig liver (11) and Citrobacter freundii (12) as the first
*
This work was supported in part by Grant-in-aid for Scientific Research on enzyme that discriminately acts on a selenium-containing com-
Priority Areas (B) 19370040 (to N. E.) from the Ministry of Education, Cul-
ture, Sports, Science, and Technology of Japan, by Grant-in-aid for Encour-
agement of Young Scientists 21780094 (to H. M.) from the Japan Society
pound but not on its cognate sulfur analog. The cDNA cloning
of mouse SCL (13) revealed that SCL belongs to subgroup V in
for the Promotion of Science, by the National Project on Protein Structural fold type I of aminotransferases (14). Among them, overall
and Functional Analyses, and by a grant-in-aid from the Ministry of Educa-
sequence similarity (ϳ30%) is found between mammalian SCL
tion, Culture, Sports, Science and Technology of Japan (21st Century COE
on Kyoto University Alliance for Chemistry) (to N. E.).
and cysteine desulfurases (13).
The atomic coordinates and structure factors (codes 3A9X, 3A9Y, and 3A9Z) have
been deposited in the Protein Data Bank, Research Collaboratory for Structural
Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).
The on-line version of this article (available at http://www.jbc.org) contains
supplemental Figs. S1–S4 and Experimental Procedures.
SCL and cysteine desulfurase catalyze the same type of reac-
tion, namely the removal of selenium or sulfur from selenocys-
teine or cysteine to yield alanine. Although bacterial cysteine
desulfurases exhibit activity to both cysteine and selenocysteine
□S
1
2
Both authors contributed equally to this work.
(
15), SCL shows significant specificity to selenocysteine. Mech-
To whom correspondence may be addressed: Dept. of Biotechnology, Insti-
tute of Science and Engineering, College of Life Sciences, Ritsumeikan Uni-
versity, Kusatsu, Shiga 525-8577, Japan. Tel.: 81-775-61-2732; Fax: 81-775-
anistic and crystal structure studies showed that the catalytic
reaction of cysteine desulfurase involves the formation of sul-
fane sulfur in the form of a cysteine persulfide in the active site
of the enzyme (16–18). Sulfur transfer from the persulfide to a
6
1-2659; E-mail: mihara@fc.ritsumei.ac.jp.
3
4
To whom correspondence may be addressed: RIKEN Spring-8 Center,
Harima Institute, 1-1-1 Kouto, Sayo, Hyogo 679-5148, Japan. Tel.: 81-791-
5
8-2891; Fax: 81-791-58-2892; E-mail: hirotsu@spring8.or.jp.
Recipient of 2009 Humboldt Research Award. To whom correspondence
may be addressed: Institute for Chemical Research, Kyoto University, Uji,
Kyoto 611-0011, Japan. Tel.: 81-774-38-3240; Fax: 81-774-38-3248; E-mail:
esakin@SCL.kyoto-u.ac.jp.
5
The abbreviations used are: SCL, selenocysteine lyase; PLP, pyridoxal
5Ј-phosphate; ESI-MS, electrospray ionization mass spectrometry; PDB,
Protein Data Bank.
APRIL 16, 2010•VOLUME 285•NUMBER 16
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Crystal Structure and Mechanism of Selenocysteine Lyase
cysteine residue of its partner proteins, such as IscU (19), ThiI mutant of SCL was performed in the same manner as described
(
20), TusA (21), and SufE (22, 23), is associated with direct for the wild-type enzyme.
protein-protein interaction, thereby ensuring the specific deliv-
Site-directed Mutagenesis—Site-directed mutagenesis was
ery of the persulfide sulfur to eventual target molecules, such as performed by using the QuikChange site-directed mutagenesis
an iron-sulfur cluster, thiamine, and thiolated nucleosides, kit (Stratagene, La Jolla, CA). The mutagenic oligonucleotides
without nonspecific spreading of reactive sulfur in the cell (24). used were 5Ј-ATCCGAGTGGGCTGAAGCTCC-3Ј and
Although SCL has a conserved cysteine residue at the position 5Ј-GGAGCTTCAGCCCACTCGGAT-3Ј.
corresponding to the persulfide-forming cysteine residue of
Enzyme Assay and Kinetic Analysis—SCL activity was as-
cysteine desulfurase, its role in the catalytic function of the sayed by the determination of selenide with lead acetate as
enzyme has not been established.
described previously (25, 26). Protein was determined using the
Here, we investigate the mechanism of rat SCL. Site-directed CBB protein assay (Nacalai Tesque, Kyoto, Japan) with bovine
serum albumin as a standard.
mutagenesis and ESI-MS studies using wild-type and C375A
mutant of SCL show that Cys-375 is essential for the activity of
SCL as the site for cysteine selenopersulfide formation. UV-
visible spectrum analysis and crystal structure of SCL com-
plexed with L-cysteine in a non-productive adduct form shows
the structural basis for discrimination between selenocysteine
and cysteine by the enzyme. UV-visible spectrum analysis also
shows that loss of Cys-375 resulted in the formation of a non-
productive adduct with selenocysteine, suggesting that Cys-375
serves as a guide to direct the substrate in an appropriate ori-
entation for the catalytic reaction.
ESI-MS Sample Preparation and Measurements—Samples of
wild-type SCL and the C375A mutant for ESI-MS analysis were
prepared under a nitrogen atmosphere in a glove box (Miwa,
Osaka, Japan) at room temperature. SCL (230 ␮M) was incu-
bated with L-selenocysteine (70 ␮M) in a 50 mM Tris-HCl buffer
(
pH 7.0) and immediately separated with a MicroBioSpin 6 col-
umn (Bio-Rad). The desalted enzyme was introduced at a flow
rate of 18 ␮l/min into a PE Sciex API 2000 single quadrupole
mass spectrometer equipped with an electrospray ionization
source (PE-Sciex, Thornhill, ON, Canada). The mass spectrom-
eter scanned from m/z ϭ 800 to m/z ϭ 1800 with a 2.0 ms dwell
time and a 0.2 m/z step size.
EXPERIMENTAL PROCEDURES
Crystallization and Data Collection—The purified enzyme
was dialyzed against 10 mM HEPES-NaOH (pH 7.5). SCL crys-
tals were grown by the hanging-drop vapor-diffusion
Materials—L-Selenocystine and L-cysteine were purchased
from Sigma Aldrich. Selenopropionate was synthesized as
described in supplemental Experimental Procedures. Oligonu-
cleotides were synthesized by Hokkaido System Science (Sap-
poro, Japan). Restriction and DNA modification enzymes were
purchased from New England Biolabs (Beverly, MA), Takara
Shuzo (Kyoto, Japan) and Toyobo (Osaka, Japan). All other
chemicals were analytical grade reagents.
Ϫ1
method at 297 K. A 2 ␮l droplet of a 35 mgꢁml protein
solution mixed with the same amount of reservoir solu-
tion was equilibrated at 293 K against a 500 ␮l reservoir solution
(
100 mM citrate buffer pH 4.7 and 1.5 M ammonium dihydro-
gen phosphate). The native crystals appeared within 2 weeks of
incubation and grew to maximum dimensions of 0.3 ϫ 0.3 ϫ
Gene Expression and Purification of SCL—cDNA for the full-
0
.1 mm. The crystals (pH 4.7) were transferred to the reservoir
TM
length SCL protein of Rattus norvegicus (GenBank /EMBL
solution adjusted to pH 8.0 using 1.5 M di-ammonium hydrogen
phosphate instead of ammonium dihydrogen phosphate, to
produce the native crystals at pH 8.0. Crystals of SCLꢁL-cysteine
and SCLꢁselenopropionate were obtained at 293 K by soaking
the native crystals in the reservoir solution adjusted to pH 8.0.
These crystals are isomorphous with the orthorhombic space
group P2 2 2 , average unit cell parameters a ϭ 55.0, b ϭ 101.9,
accession number NM_001007755) was cloned by PCR using
the primers 5Ј-AGCGATCAGGCATATGGACGTGGCGCG-
GAATGGC-3Ј and 5Ј-CCGGAATTCCTAGACCGGGCTTC-
CAGCTGGTTC-3Ј. PCR products were cloned into the NdeI
and EcoRI sites of pET21a(ϩ) for heterologous expression in
Escherichia coli Rosetta(DE3). The E. coli cells harboring the
expression plasmid were grown in an LB medium containing 50
1
1 1
c ϭ 197.3 Å. Assuming two monomers in the asymmetric unit,
␮
g/ml ampicillin at 28 °C for 16 h and were harvested by cen-
the Matthews coefficient (V ) value was calculated to be 2.96
M
3
Ϫ1
trifugation. A Tris-HCl buffer (10 mM) (pH 7.4) containing 0.1
mM phenylmethylsulfonyl fluoride and 1 mM EDTA was used as
the standard buffer. The cells were disrupted by sonication. The
Å Da , indicating an estimated solvent content of 58.3% in
the unit cell (27).
Data collection was performed at 100 K using a wavelength of
cell debris was removed by centrifugation, and the supernatant 1.00 Å from the synchrotron-radiation source and an ADSC
solution was applied to a Q-Sepharose column (5 ϫ 5 cm). After Quantum CCD Camera. X-ray diffraction data sets for the unli-
the column was washed, the enzyme was eluted with a 1.0-liter ganded SCL (pH 8.0) were collected to 2.0 Å resolution on the
linear gradient of 0–0.5 M NaCl in the buffer. The fractions BL5A station at the Photon Factory, KEK (Tsukuba, Japan).
containing SCL were pooled, mixed with an equal volume of X-ray diffraction data sets for the SCLꢁL-cysteine crystal and the
standard buffer containing 2.0 M ammonium sulfate, and SCLꢁselenopropionate crystal were collected to 1.85 and 1.55 Å
applied to a Butyl-Toyopearl column (3 ϫ 15 cm). After the resolution on the BL41XU and the BL38B2 station, respec-
column was washed, the enzyme was eluted with a 1.0-liter tively, at SPring8 (Hyogo, Japan). Data were processed using
linear gradient of 1.0–0.6 M ammonium sulfate in the buffer. DENZO/SCALEPACK (28). Data collection statistics are pre-
The enzyme fractions were concentrated with Centriprep sented in Table 1.
YM-30 (Millipore, Bedford, MA) and stored at Ϫ80 °C until use
Structure Determination and Refinement—The structure of
without significant inactivation. Purification of the C375A the unliganded SCL (pH 8.0) was solved by the molecular
1
2134 JOURNAL OF BIOLOGICAL CHEMISTRY
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Crystal Structure and Mechanism of Selenocysteine Lyase
replacement method with the program AMoRe (29) using The water molecules whose thermal factors were above the
the IscS structure (PDB ID: 1P3W) as the search model. The maximum thermal factor of the main chain after refinement
modeling of the polypeptide chain was performed using were removed from the list. Model building and refinement
program O (30), and the structure was refined with program cycles resulted in an R
of 0.197 and an R
of 0.236% at
factor
free
CNS (31). Water molecules were picked up on the basis of 2.0 Å resolution, calculated for 69817 reflections. The same
the peak heights (1.5 ␴) and distance criteria (4.0 Å from refinement procedure was applied to SCLꢁL-cysteine and
protein and solvent) from the sigma-weighted 2F Ϫ F map. SCLꢁselenopropionate but using the coordinates of the native
o
c
enzyme as the initial model. Refinement cycles resulted in an
R
of 0.187 and 0.211 and R of 0.187 and 0.209 for SCLꢁL-
TABLE 1
factor
free
cysteine and SCLꢁselenopropionate, respectively. Structure dia-
grams were drawn using MOLSCRIPT (32), POVSCRIPTϩ
Data collection and refinement statistics
SCL
SCLꢁL-cysteine SCLꢁselenopropionate
(33), and PyMol (34).
Diffraction data
Wavelength (Å)
1.0
2.00
1.0
1.85
1.0
1.55
Resolution (Å)
RESULTS
Unique no. of reflections
69817
86205
159678
98.7 (94.4)
4.4 (29.9)
36.4
b
b
b
b
b
b
Completeness (%)
91.8 (85.0)
4.3 (29.9)
28.3
90.3 (85.2)
5.8 (25.6)
38.2
Purification and Properties of Rat SCL—Because our attempt
to obtain crystals of SCL cloned from mouse liver (13) was
unsuccessful, we constructed an expression system for rat SCL.
The recombinant SCL was purified to homogeneity. Steady-
a
R
merge (% )
I/␴(I)
Refinement
Resolution limits (Å)
20.0–2.00
20.0–1.85
20–1.55
b
b
b
R
R
(%)
19.7 (25.7)
23.6 (27.4)b
18.7 (24.1)
21.1 (27.5)b
18.7 (23.4)
factor
free (%)
20.9 (26.4)b
state kinetic analysis revealed that V
and K values for
max
m
Deviations
Ϫ1
Ϫ1
L-selenocysteine were 26 ␮molꢁmin ꢁmg
and 5.5 mM,
Bond length (Å)
Bond angles (deg)
Mean B factor
0.022
2.1
0.007
1.3
0.029
2.4
respectively (supplemental Fig. S1). The enzyme showed no
detectable activity toward L-cysteine (examined at 5–50 mM).
An inhibition study showed that L-cysteine is a competitive
2
2
Main chain atoms (Å )
32.1
33.4
22.0
21.1
Side-chain atoms (Å )
23.6
22.7
2
2
Water atoms (Å )
36.3
31.1
31.3
Hetero atoms (Å )
PLP 31.1
PLP 16.7
PO4 37.4
GOL 45.3
CYS 21.8
PLP 17.0
PO4 34.6
GOL 34.4
SLP 10.2
inhibitor of the enzyme with K ϭ 9.6 mM (supplemental
i
PO 61.0
4
Fig. S1). Thus, the fundamental properties SCL are similar to
those reported for the enzymes from pig (11) and mouse (13).
Role of Cys-375—SCL has a conserved cysteine residue that
corresponds to the persulfide-forming cysteine residue in cys-
teine desulfurases (supplemental Fig. S2) (16, 35). To investi-
gate the role of the conserved cysteine residue in SCL, we pre-
pared a mutant SCL protein (C375A) in which Cys-375 was
replaced by an alanine residue. The purified C375A mutant
exhibited no activity to L-selenocysteine, suggesting that Cys-
Ramachandran plot
Favored
92.7
7.3
91.5
8.5
92.3
7.7
Additional allowed
Generously allowed
Disallowed
0.0
0.0
0.0
0.0
0.0
0.0
a
b
R
i i
merge ϭ ⌺hkl ⌺ ͉Ihkl,i Ϫ ϽIhklϾ͉ /⌺hkl ⌺ Ihkl,i, where I ϭ observed intensity and
ϽIϾ ϭ average intensity for multiple measurements.
The values in the parentheses are for the highest resolution shells (2.01–2.00,
1
.85–1.86, 1.55–1.56 Å) in SCL, SCLꢁL-cysteine, and SCLꢁselenopropionate,
respectively.
3
75 is a catalytically essential resi-
due of the enzyme similar to the
conserved catalytic cysteine residue
of cysteine desulfurases (35).
ESI-MS Analysis of Enzyme-
bound Selenopersulfide Intermedi-
ate—To further elucidate the cata-
lytic role of Cys-375, the formation
of an SCL-bound selenopersulfide
intermediate was assessed by ESI-
MS analysis. The observed masses
of the wild-type (47,273 Da) and the
mutant (47,243 Da) SCL were con-
sistent with their masses calculated
from the amino acid sequences
(47,261 and 47,229 Da, respectively)
within acceptable error of ESI-MS
measurement (Ͻ0.1%) (Fig. 1, A and
D). Incubation of the wild-type SCL
with L-selenocysteine resulted in the
formation of a new species with a
mass of [M ϩ 80] (Fig. 1C). In con-
trast, incubation with L-cysteine
resulted in no formation of such
species (Fig. 1B). The data suggest
FIGURE 1. Deconvoluted ESI-mass spectra of SCL. Wild-type SCL (A–C) or the C375A mutant enzyme (D–F)
was incubated with L-cysteine (B, E), L-selenocysteine (C, F), or none (A, D) and analyzed by ESI-mass spectrom-
eterasdescribedunder“ExperimentalProcedures.”Thepeakatthemassof47,353forthe[Mϩ80]speciesthat
Ϫ
corresponds to the selenopersulfide form of SCL (SCL-S-Se ) is indicated by an asterisk.
APRIL 16, 2010•VOLUME 285•NUMBER 16
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Crystal Structure and Mechanism of Selenocysteine Lyase
Leu-30 and from Thr-306 to the C
terminus) and large (from Glu-31 to
Glu-305) domains. The small do-
main has an ␣/␤ structure which
consists of a two-stranded parallel
␤
␤
-sheet, three-stranded antiparallel
-sheet, and six helices (supple-
mental Fig. S3B). The large domain
exhibits a typical ␣/␤ structure in
which seven parallel ␤-strands (S2–
S8), except for S8 form an open
twisted ␤-sheet as a core sur-
rounded by six ␣-helices from the
solvent side and two ␣-helices from
the protein side. The seven-
stranded ␤-sheet forms the base of
the active site that binds the coen-
zyme PLP, and is characteristic of
PLP-dependent fold type I enzyme
(
14, 36). The molecule has two
active site cavities around a molecu-
lar 2-fold axis. Each cavity is located
at the domain interface of one sub-
unit and at the subunit interface.
The program DALI (37) was used
to detect the enzymes possessing
three-dimensional structures most
FIGURE 2. UV-visible spectroscopic analysis of SCL. A, absorption spectra of wild-type SCL incubated with
various concentrations (0, 15, and 30 ␮M, indicated by lines 1, 2, and 3, respectively) of L-selenocysteine.
B, absorption spectra of wild-type SCL incubated with various concentrations (0, 0.027, 0.1, 0.26, 0.6, 1.25, 2.5,
5
, 10, 20, and 40 mM, indicated by lines 1–11, respectively) of L-cysteine. C, absorption spectra of the C375A
mutant incubated with various concentration (0, 1.5, 15, and 30 ␮M, indicated by lines 1–4) of L-selenocysteine.
D, absorption spectra of the C375A mutant incubated with various concentrations (0, 0.0024, 0.0073, 0.027, 0.1,
0
.26, 0.6, 1.25, 2.5, 5, 10, 20, and 40 mM, indicated by lines 1–13) of L-cysteine. Absorption spectra of SCL were similar to that of the unliganded
recorded with a Shimadzu UV-visible spectrophotometer (UV-2450, Shimadzu, Kyoto, Japan) at pH 7.0.
SCL from among PLP-dependent
fold type I enzymes in the Protein
that the [M ϩ 80] species corresponds to a selenium-bound Data Bank data base. The highest Z scores were calculated to be
form of SCL. The C375A mutant incubated with L-selenocys- 62.4 for human SCL (an as yet unpublished structure, PDB
teine or L-cysteine did not produce the [M ϩ 80] molecule (Fig. 2HDY), 49.5 for E. coli IscS (38), 48.2 for Thermotoga maritima
1
, E and F), indicating that selenium eliminated from L-seleno- NifS-like protein (39), 40.6 for Synechocystis sp. PCC 6803 SufS
cysteine is bound to Cys-375 of the enzyme in the form of a (40), 38.9 for E. coli CsdB (17, 18), 35.2 for Synechocystis cystine
Ϫ
cysteine selenopersulfide intermediate (SCL-S-Se ).
C-S lyase (41), and 30.5 for Pseudomonas fluorescens kynureni-
UV-visible Spectrum Analysis—SCL shows an absorption nase (42). This result shows that the structure of SCL is quite
maximum at 416 nm, which is characteristic of the internal similar to that of human SCL, and more similar to those of
aldimine form of PLP enzymes. Incubation of SCL with L-sel- group I cysteine desulfurases (IscS and NifS-like protein) than
enocysteine resulted in a broad increase in the absorbance at those of group II cysteine desulfurases (SufS and CsdB) (35, 43)
ϳ420 nm (Fig. 2A). In contrast, the addition of L-cysteine to the (Fig. 3B).
enzyme caused a significant decrease in the absorption peak at
SCL changes its overall conformation from the open form to
16 nm, concomitantly with the increase in that at 350 nm (Fig. the closed form by a 3.8° rotation of the small domain as a rigid
B). The incubation with D-cysteine, D-selenocysteine, or 2-mer- body upon binding of cysteine to the active site crevice to form
4
2
captoethanolalsoresultedinanabsorptionpeakshiftfrom416nm the cofactor-cysteine complex (Fig. 3A). In addition to the rigid
to 330–340 nm in the same manner as observed in the enzyme body rotation of the small domain, the cysteine binding induces
incubated with L-cysteine (data not shown). These results indicate the local conformational change of the extended lobe (the small
that L-cysteine, D-cysteine, D-selenocysteine, and 2-mercaptoeth- domain residues from Ser-374 to Ile-392). The extended lobe of
anol can enter the active site of the enzyme to affect the absorption SCL in the unliganded open form was disordered and was not
properties of the PLP moiety even though they do not serve as a located on the electron density map. In contrast, the lobe in the
substrate of the enzyme. Interestingly, the C375A mutant enzyme liganded closed form was ordered to form the helix 11 (3
1
0
incubated with L-selenocysteine or L-cysteine also showed a peak helix)–loop–␣-helix 12-loop structure which covers the active
shift from 416 nm to 350 nm (Fig. 2, C and D). These results indi- site as a lid. In contrast to SCL, the open-closed conformational
cate that Cys-375 is essential for the proper capture of the sub- change on ligand binding was not observed in cysteine desul-
strate L-selenocysteine.
furases (17, 18, 38, 39, 44) (see Open-Closed Conformational
Overall Structure—The overall structure of SCL is shown in Change in “Discussion”).
Fig. 3A. The polypeptide chain is folded into a dimeric form
Active Site Structure of SCLꢁL-Cysteine Complex—PLP is
with each subunit consisting of the small (from N terminus to located at the bottom of the active site pocket formed at the
1
2136 JOURNAL OF BIOLOGICAL CHEMISTRY
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Crystal Structure and Mechanism of Selenocysteine Lyase
FIGURE 3. Overall structure of SCL. A, a dimeric form viewed down the crystallographic 2-fold axis. The upper half of the molecules is the superimposition of
one subunit in the liganded closed form onto that in the unliganded open form by least-squares fitting of C␣ atoms in the large domain. The open form is pink,
andtheclosedformis bluewitharedextendedlobe. Thelowerhalfofthemoleculerepresentstheothersubunitintheclosedform. Thesmalldomain, thelarge
domain, and the N-terminal loop are drawn in gray, green, and orange, respectively. The coenzyme PLP and L-cysteine located in the domain interface are
represented by CPK models. The extended lobe in the open form of SCL is disordered and, therefore, not shown in the model. B, superpositioning of C␣ atoms
of SCL onto those of cysteine desulfurases. SCL complexed with L-cysteine, human SCL, T. maritima NifS-like protein complexed with L-cysteine (PDB ID: 1ECX),
and E. coli CsdB complexed with L-propargylglycine (1I29) are presented in red, orange, blue, and green, respectively. The extended lobes are shown in thick
lines. The PLP moieties, bound ligands, and active site lysine and cysteine residues are represented by stick models. A part of the extended lobe of T. maritima
NifS-like protein is disordered and not shown in the model.
subunit interface and the domain interface (Fig. 4A). The side
Structure of SCLꢁSelenopropionate Complex—We solved the
chain S␥ atom of the bound cysteine, but not the ␣-amino crystal structure of an SCLꢁselenopropionate complex. Seleno-
group, makes a covalent bond with the C4Ј atom of PLP. The propionate is the substrate analog in which the ␣-amino group
residual electron density of the S␥ atom of the bound cysteine of the substrate L-selenocysteine is replaced by a hydrogen
was so close to C4Ј of PLP that the S␥ atom made a covalent atom. There is no significant difference in the overall and active
bond with C4Ј (supplemental Fig. S3C). When the C4Ј-N bond site fold between selenopropionate and the L-cysteine complex.
between PLP and Lys-247 was refined as a double bond (Schiff The residual electron density showed that the bound seleno-
base), C4Ј-N linkage deviated from the corresponding electron propionate is disordered over two sites such that the structure
density compared with the Schiff base in the unliganded form. is comprised of two conformers, A and B (Fig. 4B). Conformer
The C4Ј-N and C4Ј-S␥ were refined as single bonds for a good A is just like the bound L-cysteine in SCLꢁL-cysteine in that the
fit with the electron density, with distances of 1.51 and 1.86 Å, Se␥ atom of selenopropionate forms a covalent bond with
respectively. The bond angles around the C4Ј atom are 106, the C4Ј atom of the cofactor, although the distance of 2.28 Å
1
11, and 112°, confirming the tetrahedral geometry of the C4Ј is slightly longer than the normal Se-C bond length (1.98–
atom. The peak shift in the absorption spectrum from 416 to 2.00 Å) (45). The geometry around the C4Ј atom is tetrahe-
3
50 nm observed in the SCLꢁL-cysteine complex crystal (sup- dral with bond angles of 109, 110, and 113°. On the other
plemental Fig. S3A) and that in solution (Fig. 2) are thus due to hand, conformer B directs its Se␥ atom toward the extended
the formation of the tetrahedral adduct of cysteine-PLP-Lys247 lobe and forms a hydrogen bond with the thiol group of the
(
supplemental Fig. S3D).
lobe residue Cys-375 (Se␥—S␥ ϭ 3.29 Å) because the selenol
Upon binding of L-cysteine, the small domain moves toward group (pK of ϳ5.2) is deprotonated resulting in the seleno-
a
the active site and the extended lobe (small domain residues lato-thiol interaction (46). The Se␥ atom further interacts
Ser-374 to Ile-392), which is disordered in the unliganded form, with His-133 NE2 with an interatomic distance of 3.5 Å. The
shows its ordered structure, encapsulating L-cysteine within the spectrum of SCL measured in solution in the presence of
active site cavity (Figs. 3 and 4A). The carboxylate of the bound selenopropionate showed peaks at 350 and 416 nm, suggest-
cysteine makes a salt bridge with the guanidino group of Arg- ing that selenopropionate is bound to the active site in the
4
02 and is further fixed by hydrogen bond interactions with two conformers as observed in the SCLꢁselenopropionate
Ser-374 (extended lobe) and Asn-186. The protonated ␣-amino crystal (supplemental Fig. S4).
group of the bound cysteine, which is supposed to form a Schiff
DISCUSSION
base (external aldimine) with PLP in place of Lys-247 in cys-
teine desulfurases (15, 16), is hydrogen-bonded to the side
Catalytic Mechanism—The C375A mutant of SCL does
chain OH group of Ser-374, the main chain CϭO group of not catalyze the removal of selenium from L-selenocysteine.
Ala-26, and water molecule W1.
C375AꢁL-selenocysteine shows an absorption spectrum that is
APRIL 16, 2010•VOLUME 285•NUMBER 16
JOURNAL OF BIOLOGICAL CHEMISTRY 12137

Crystal Structure and Mechanism of Selenocysteine Lyase
ketimine intermediate. The sub-
strate-ketimine then transfers sele-
nium to Cys-375 to form cysteine
selenopersulfide (Cys375-S–Se(H))
and the enamine. Cys-375, which
approaches and interacts with the
substrate Se atom, plays a critical role
in the formation of the selenopersul-
fide as well as the productive form.
It remains unclear whether sele-
nium is directly released from Cys375-
S–Se(H) or trapped by a selenium-
transferringproteinandsubsequently
released by a reductant in the reac-
tion system. If selenopersulfide sele-
nium, which is more sensitive to
oxygen than persulfide sulfur, is
released directly from an intermedi-
ate and diluted by the bulk solvent,
then delivery to a specific acceptor
molecule would be inefficient. Plau-
sibly, selenium is shipped to the tar-
get protein through interaction with
the selenium-transferring protein in
a manner similar to the reaction of
cysteine desulfurases (16, 35).
Open-Closed Conformational
Change—Cysteine desulfurases thus
far determined by x-ray methods
exhibit no marked change in their
FIGURE 4. Active site structures. A, SCL complexed with L-cysteine and B, SCL complexed with selenopropi- overall or local conformation be-
onate. The PLP moiety, active site residues, and bound ligands (A, L-cysteine; B, selenopropionate) are shown as
ball-and-stick models. Water molecules (W1-W5) are presented as red circles. Backbone structures of the large
domain, thesmalldomain, andtheextendedlobeinonesubunitaredrawningreen, gray, andred, respectively.
cause the liganded forms have the
same structures as those of the unli-
C␣ structures and residues (indicated by asterisks) in the other subunit of the dimer are shown in blue. The 2Fo ganded forms (17, 18, 38–40). In
Ϫ F electron density map contoured at 1.0 ␴ is calculated from the final model with the bound ligand omitted.
c
contrast, upon binding of L-cysteine
Both conformers of selenopropionate are drawn in B.
or selenocysteine, the disordered
extended lobe of SCL in the unligan-
quite similar to that of the SCLꢁL-cysteine complex (Fig. 2), ded form shows its ordered structure composed of the helix 11
which suggests that L-selenocysteine forms the tetrahedral (3 helix)–loop- ␣-helix 12-loop to close the active site in con-
1
0
adduct with a mutant enzyme, such as L-cysteine bound to the cert with a 3.8° rotation of the small domain as a rigid body
wild-type enzyme. With consideration of the foregoing obser- (Figs. 3 and 4). The accessible surface area of the bound L-cys-
2
vations and this fact, we propose that the interaction of the thiol teine was calculated to be 0 Å , indicating that the open-closed
group of Cys-375 with the selenolate of the substrate selenocys- conformational change completely shields bound ligands from
teine brings the substrate L-selenocysteine into a favorable the solvent region. The disorder-order transition of the
arrangement to form a Schiff base with PLP, as was observed in extended lobe is deeply involved in the catalytic event in that
the conformer B of SCLꢁselenopropionate. SCL is designed to the lobe residue Cys-375 is oriented favorably for interaction
form the productive Michaelis complex by the thiol-selenolate with the selenol group of the substrate and the oxygen-sensitive
interaction as the guide, although the nonproductive form selenopersulfide is synthesized in the cavity shielded from the
might be in equilibrium with the productive form.
solvent region.
The productive L-selenocysteine is encapsulated in the
In contrast to SCL, the extended lobe of E. coli IscS (38) or the
active site by a small domain rotation and the ordering of the T. maritimaNifS-likeprotein(39)(groupIcysteinedesulfurase)in
extended lobe with its Se␥ atom interacting with the thiol the unliganded form is only one residue shorter than that of SCL,
group of Cys-375. The deprotonated amino group of the and is disordered in the region bearing the catalytic cysteine resi-
substrate makes a nucleophilic attack on the C4Ј of PLP to due (Fig. 3B). Upon binding of cysteine to the T. maritima NifS-
produce the external aldimine and release the neutral side chain like protein, the lobe remains disordered and does not change its
of Lys-247. The ␣-proton of the substrate is eliminated by Lys- conformation. The accessible surface area of the bound cysteine is
2
2
47 to yield a quinonoid intermediate. Lys-247 adds a proton to 49.3Å .E. coliCsdB(groupII)hasashorterextendedlobethanthe
the C4Ј of the quinonoid intermediate, producing the substrate- group I enzymes. The lobe is ordered and maintains the same
1
2138 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 285•NUMBER 16•APRIL 16, 2010

Crystal Structure and Mechanism of Selenocysteine Lyase
conformation regardless of whether it has the unliganded or ligan-
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Acknowledgments—We thank Dr. Wolfgang B u¨ ckel (Max Planck
Institute for Terrestrial Microbiology, Marburg), Dr. Rudolf K.
Thauer (Max Planck Institute for Terrestrial Microbiology, Mar-
burg), Dr. Roland Lill (Philipps University Marburg, Marburg), and
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kind suggestions concerning this study.
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APRIL 16, 2010•VOLUME 285•NUMBER 16
JOURNAL OF BIOLOGICAL CHEMISTRY 12139

Enzymology:
Reaction Mechanism and Molecular Basis
for Selenium/Sulfur Discrimination of
Selenocysteine Lyase
Rie Omi, Suguru Kurokawa, Hisaaki Mihara,
Hideyuki Hayashi, Masaru Goto, Ikuko
Miyahara, Tatsuo Kurihara, Ken Hirotsu and
Nobuyoshi Esaki
J. Biol. Chem. 2010, 285:12133-12139.
doi: 10.1074/jbc.M109.084475 originally published online February 17, 2010
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