Protein thiocarboxylate-dependent methionine biosynthesis in Wolinella succinogenes

Article abstract of DOI:10.1021/ja107424t

Thiocarboxylated proteins are important intermediates in a variety of biochemical sulfide transfer reactions. Here we identify a protein thiocarboxylate-dependent methionine biosynthetic pathway in Wolinella succinogenes. In this pathway, the carboxy terminal alanine of a novel sulfur transfer protein, HcyS-Ala, is removed in a reaction catalyzed by a metalloprotease, HcyD. HcyF, an ATP-utilizing enzyme, catalyzes the adenylation of HcyS. HcyS acyl-adenylate then undergoes nucleophilic substitution by bisulfide produced by Sir to give the HcyS thiocarboxylate. This adds to O-acetylhomoserine to give HcyS-homocysteine in a PLP-dependent reaction catalyzed by MetY. HcyD-mediated hydrolysis liberates homocysteine. A final methylation completes the biosynthesis. The biosynthetic gene cluster also encodes the enzymes involved in the conversion of sulfate to sulfide suggesting that sulfate is the sulfur source for protein thiocarboxylate formation in this system.

Full text of DOI:10.1021/ja107424t

Published on Web 12/16/2010
Protein Thiocarboxylate-Dependent Methionine Biosynthesis
in Wolinella succinogenes
Kalyanaraman Krishnamoorthy and Tadhg P. Begley*
Department of Chemistry, Texas A&M UniVersity, College Station, Texax 77840, United States
Received August 17, 2010; E-mail: begley@chem.tamu.edu
Abstract: Thiocarboxylated proteins are important intermediates in a variety of biochemical sulfide transfer
reactions. Here we identify a protein thiocarboxylate-dependent methionine biosynthetic pathway in Wolinella
succinogenes. In this pathway, the carboxy terminal alanine of a novel sulfur transfer protein, HcyS-Ala, is
removed in a reaction catalyzed by a metalloprotease, HcyD. HcyF, an ATP-utilizing enzyme, catalyzes
the adenylation of HcyS. HcyS acyl-adenylate then undergoes nucleophilic substitution by bisulfide produced
by Sir to give the HcyS thiocarboxylate. This adds to O-acetylhomoserine to give HcyS-homocysteine in a
PLP-dependent reaction catalyzed by MetY. HcyD-mediated hydrolysis liberates homocysteine. A final
methylation completes the biosynthesis. The biosynthetic gene cluster also encodes the enzymes involved
in the conversion of sulfate to sulfide suggesting that sulfate is the sulfur source for protein thiocarboxylate
formation in this system.
1. Introduction
Sulfur-containing natural products are widely distributed in
nature, for example, in amino acids, carbohydrates, proteins,
nucleic acids, antibiotics, cofactors, siderophores, alkaloids,
etc.1,2 Three major sulfur sources have been identified in
bacterial metabolism: free sulfide used for example in cysteine
biosynthesis, protein persulfides (R-S-SH) used for example in
iron sulfur cluster biosynthesis, and protein thiocarboxylates (R-
COSH). Protein thiocarboxylates are members of a growing
family of biosynthetic sulfide donors and are involved in a
variety of biosynthetic pathways, including vitamin B1 (ThiS-
COSH),3 molybdopterin (MoaD-COSH),4 cysteine (CysO-
COSH),5 thioquinolobactin (QbsE-COSH),6 2-thioribothymidine
(TtuB-COSH),7 and 5-methoxy-carbonyl-methyl-2-thiouridine
(Urm1p-COSH).8 A significant gap in our understanding of the
biosynthetic role of protein thiocarboxylates is the identification
of the sulfur source for thiocarboxylate formation. For ThiS-
COSH,9 MoaD-COSH,4,10 and Urm1p,8,11 a desulfurase per-
Figure 1. Protein thiocarboxylates function as sulfide donors in the
biosynthesis of a variety of natural products. In many cases, the sulfur source
for thiocarboxylate formation is unknown.
(1) Kessler, D. FEMS Microbiol. ReV. 2006, 30, 825.
(2) Mueller, E. G. Nat. Chem. Biol. 2006, 2, 185.
(3) Dorrestein, P. C.; Zhai, H.; McLafferty, F. W.; Begley, T. P. Chem.
Biol. 2004, 11, 1373.
sulfide is the sulfur donor; for the others shown in Figure 1,
the sulfur source is unknown.
(4) Leimkuhler, S.; Wuebbens, M. M.; Rajagopalan, K. V. J. Biol. Chem.
2001, 276, 34695.
(5) Burns, K. E.; Baumgart, S.; Dorrestein, P. C.; Zhai, H.; McLafferty,
F. W.; Begley, T. P. J. Am. Chem. Soc. 2005, 127, 11602.
(6) Godert, A. M.; Jin, M.; McLafferty, F. W.; Begley, T. P. J. Bacteriol.
2007, 189, 2941.
A search for ThiS-COOH orthologs in the SEED database
(http://theseed.uchicago.edu/FIG/index.cgi), revealed in Wolinel-
la succinogenes the gene for a putative thiocarboxylate-forming
protein clustered with the sulfate assimilation proteins and
putative methionine biosynthetic genes (Figure 2). This protein
thiocarboxylate/sulfate assimilation clustering pattern, and varia-
tions of it, was seen in many other micro-organisms (e.g.,
Clostridium kluyVeri, Clostridium thermocellum, Desulfitobac-
terium hafniense, Carboxydothermus hydrogenoformans Z-2901,
Caldicellulosiruptor saccharolyticus DSM 8903, Alkaliphilus
metalliredigens QYMF, Geobacter metallireducens GS-15,
(7) Shigi, N.; Sakaguchi, Y.; Asai, S.; Suzuki, T.; Watanabe, K. Embo J.
2008, 27, 3267.
(8) Leidel, S.; Pedrioli, P. G.; Bucher, T.; Brost, R.; Costanzo, M.;
Schmidt, A.; Aebersold, R.; Boone, C.; Hofmann, K.; Peter, M. Nature
2009, 458, 228.
(9) Begley, T. P.; Xi, J.; Kinsland, C.; Taylor, S.; McLafferty, F. Curr.
Opin. Chem. Biol. 1999, 3, 623.
(10) Leimkuhler, S.; Rajagopalan, K. V. J. Biol. Chem. 2001, 276, 22024.
(11) Noma, A.; Sakaguchi, Y.; Suzuki, T. Nucleic Acids Res. 2009, 37,
1335.
9
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J. AM. CHEM. SOC. 2011, 133, 379–386 379

A R T I C L E S
Krishnamoorthy and Begley
Figure 2. Putative thiocarboxylate forming protein, WS1007 (shown in green), clustered with sulfate assimilation proteins, WS1004, WS1008, WS1009,
and WS1010, and homocysteine biosynthetic proteins, WS1012 and WS1015, in Wolinella succinogenes. Hypothetical functions for these genes are given
in Table 1.
Table 1. Hypothetical Functions of Proteins Adjacent to the Putative Thiocarboxylate-Forming Protein, HcyS-Ala (WS1007)
protein IDs
putative function of the proteins
WS1004, NP_907206 (sir)
WS1005, NP_907207 (hcyD)
Ferredoxin sulfite reductasea
Metalloprotease involved in the removal of C-terminal alanine of
HcyS-Ala prior to sulfur transfer and also in the release of
homocysteine from HcyS-Homocysteine adducta
ATP-utilizing enzyme involved in adenylating HcyS C-terminal prior to
sulfur transfera
WS1006, NP_907208 (hcyF)
WS1007, NP_907209 (hcyS-ala)
WS1008 and WS1009, NP_907210 and NP_907211 (cysD and cysN)
WS1010, NP_907212 (cysH)
Sulfur transfer protein involved in methionine biosynthesisa
Sulfate adenylyltransferase, subunits 1 and 2
Phosphoadenylyl-sulfate reductase/Adenylyl-sulfate reductase
Conserved hypothetical protein probably involved in assimilatory sulfate
reduction
WS1011, NP_907213
WS1012, NP_907214 (metY)
WS1013, NP_907215
O-acetylhomoserine sulfhydrylasea
Methyltransferase (EC 2.1.1.-)
WS1014, NP_907216
WS1015, NP_907217 (metZ)
Putative efflux protein
O-acetylhomoserine or O-succinylhomoserine sulfhydrylasea
a Experimentally characterized in this study.
boxylate-dependent methionine biosynthetic pathway in Wolinel-
la succinogenes and identify sulfate as the probable sulfur donor.
2. Materials and Methods
2.1. Materials. W. succinogenes genomic DNA (ATCC 29543D-
5) was purchased from ATCC (Manassas, VA). pTYB1, SapI, NdeI,
XhoI and chitin beads were bought from New England Biolabs
(Ipswich, MA). EMD biosciences (Gibbstown, NJ) supplied
Luria-Bertani, glycerol, chloroform, methanol, acetonitrile, potas-
sium phosphate and ammonium acetate. IPTG, amplicillin and
kanamycin were procured from Lab Scientific (Livingston, NJ).
L-Cysteine, arabinose, DL-methionine, D-glucose, M9 minimal salts,
formic acid, EDTA, urea, D2O, triton X-100, methyl viologen,
TCEP, O-acetyl-L-serine, O-succinyl-L-homoserine, DL-homocys-
teine, 10% Pt on activated carbon, hydroxycobalamin hydrochloride,
S-adenosylmethionine chloride, 3-mercaptopropionic acid and o-
phthalaldehyde were purchased from Sigma-Aldrich (St. Louis,
MO). Ferrous ammonium sulfate, nickel sulfate, chloramphenicol,
boric acid, Na2SO3 and Tris.HCl were acquired from Fisher
Scientific (Fairlawn, NJ). CaCl2, ZnSO4, MgCl2, MgSO4 and NaCl
were from Mallinckrodt. Microcon YM-10 (MWCO 10 kDa), YM-3
(MWCO 3 kDa) and amicon (MWCO 5 kDa) cellulose filters were
obtained from Millipore Corporation (Billerica, MA). Aminole-
vulinic acid, ATP and sodium sulfide were from Acros. O-Acetyl-
L-homoserine and 5-DL-methyltetrahydrofolic acid calcium salt
trihydrate was from TRC Canada (North York, Ontario). Typhoon
trio and chelating sepharose fast flow (used for Ni-NTA affinity
purification) were products of GE healthcare biosciences (Piscat-
away, NJ). All bacterial cultures were grown in a New Brunswick
Scientific (Edison, NJ) Excella E25 shaker incubator and lysed by
sonication using Misonix sonicator 3000 (Misonix Inc., Farm-
ingdale, NY). Absorbance data was obtained on a Cary 300 Bio
UV-visible spectrophotometer (Varian, Palo Alto, CA). The
glovebox was made by Coy Laboratory products (Grass lakes, MI).
ESI-MS analysis was performed using an Esquire-LC_00146
instrument (Bruker, Billerica, MA) in the positive ion mode.
MALDI-MS data were recorded in positive mode on an Applied
Biosystems Voyager STR (matrix: sinapinic acid). LC-MS data
Figure 3. Three well-characterized pathways for L-methionine biosynthesis.
The transsulfuration pathway that makes homocysteine from cysteine
through cystathionine is absent in W. succinogenes (http://seed-viewer.
theseed.org/seedviewer.cgi).
Geobacter uraniireducens Rf4, AcidoVorax sp. JS42 and Pe-
lodictyon luteolum DSM 273), suggesting that the genes are
functionally related and that sulfate may be used as the sulfur
source for protein thiocarboxylate formation in these systems.
The well-characterized biosynthesis of methionine is outlined
in Figure 3. In this pathway homocysteine formation occurs
either by direct addition of sulfide to O-succinylhomoserine or
O-acetylhomoserine, or by breakdown of cystathionine.12 In this
paper, we describe the identification of a new protein thiocar-
(12) Belfaiza, J.; Martel, A.; Margarita, D.; Saint Girons, I. J. Bacteriol.
1998, 180, 250.
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A R T I C L E S
were obtained on an Agilent 1200 capillary HPLC system interfaced
to an API QSTAR Pulsar Hybrid QTOF mass spectrometer (Applied
Biosystems/MDS Sciex, Framingham, MA) equipped with an
electrospray ionization (ESI) source. Liquid chromatography (LC)
separation was achieved using a Phenomenex Jupiter C4 microbore
column (150 × 0.50 mm2, 300 Å) at a flow rate of 10 µL/min.
MALDI-MS and LC-MS data were provided by the Laboratory of
Biological Mass spectrometry at Texas A&M University, College
Station, Texas. All the protein concentrations were measured by
the Bradford assay.13 The protein stock samples are in 100 mM
Tris, 150 mM NaCl, 2 mM TCEP, 30% glycerol, pH 8.0 unless
otherwise mentioned. Methionine-auxotroph E. coli B834(DE3),
harboring the iron sulfur cluster biosynthetic genes in the vector
pDB1282, was a gift from Dr. Squire Booker.14
2.2. Methods. Cloning and Overexpression. The sir, hcyD,
hcyF, hcyS-ala, metY, metZ and metE genes, from Wolinella
succinogenes FDC 602W, were inserted between NdeI/XhoI
restriction sites of the THT vector (Table 1s, Supporting Informa-
tion). This vector is a pET-28 derived vector which allows
attachment of a modified 6xHisTag followed by a TEV protease
site onto the N-terminus of the expressed protein. Salmonella
typhimurium cysG (siroheme synthase) was cloned into the pA-
CYCDuet vector. All the genes, except sir, were overexpressed in
E. coli BL21(DE3). Luria-Bertani cultures containing 40 mg
kanamycin per liter were grown at 37 °C until an OD600 of 0.6,
cooled to 15 °C and then induced with a final concentration of 500
µM IPTG before continuing growth at 15 °C for another 12-16 h.
The sir gene was coexpressed with the S. typhimurium cysG and
the A. Vinelandii IscS cluster in E. coli B834(DE3). M9 minimal
medium (1.5 L) was supplemented with 30 mL 20% glucose, 3
mL of 1 M MgSO4, 150 µL of 1 M CaCl2, 120 mg DL-methionine,
150 mg ampicillin and 60 mg each of kanamycin and chloram-
phenicol, inoculated with a starter culture and grown at 37 °C to
an OD600 of 0.1. At this point, 3.75 g of L-arabinose, 88 mg of
ferrous ammonium sulfate and 90 mg of L-cysteine were added
and the culture was shaken at 100 rpm until the OD600 reached
0.6. The culture was then cooled for 4 h at 4 °C, 45 mg of
aminolevulinic acid and IPTG (final concentration ) 0.5 mM) were
added and the culture was incubated with shaking at 15 °C for
12-16 h.
yield HcyS-DL-homocysteine. Proteins were buffer exchanged into
100 mM Tris, 150 mM NaCl, 2 mM TCEP, 30% glycerol, pH 8.0
by dialysis using a Novagen D-tube dialyzer Maxi (MWCO 3.5
kDa) and stored as frozen aliquots at -80 °C.
Activity Assay for HcyD (Putative Metalloprotease). 200 µL
of 611 µM HcyS-Ala were treated with 6 µL of 2.4 mM HcyD
and 3 µL of 10 mM ZnSO4 at room-temperature for 2 h. The
samples were desalted into 200 µL of 50 mM NH4OAc. 200 µL of
acetonitrile and 2 µL of HCOOH were then added and the sample
was then analyzed for HcyS formation by positive-mode ESI-MS.
For alanine detection, HcyS-Ala and HcyD were buffer ex-
changed twice into 50 mM potassium phosphate, pH 8.0 using
Biorad biospin 6 columns. Eighty-five microliters of 28 mM HcyS-
Ala was treated with 100 µL of 1.62 mM HcyD at room-temperature
for 2 h. The sample was freeze-dried and redissolved in D2O. The
proteins were removed using YM-3 microcon (washed extensively
with D2O to remove glycerol before loading the protein) and the
filtrate was analyzed by 1H NMR on a Varian 500 MHz spectrometer.
Activity Assay for HcyF (Putative HcyS Adenylating
Enzyme). Thirty microliters of 3.98 mM HcyS-Ala, 23 µL of 1.76
mM HcyF, 30 µL of 0.88 mM HcyD, 3 µL of 10 mM ATP and 6
µL of 10 mM MgCl2 were mixed and incubated at room-
temperature for 15 min. The reaction was quenched with an equal
volume of 12 M urea, the proteins were removed using a YM-10
microcon and the samples analyzed for AMP formation by HPLC
(Agilent 1200, Supelco supelcosil 15 cm × 4.6 mm, 3 µm LC-
18-T column) using the following gradient at a flow rate of 1 mL/
min: solvent A is water, solvent B is 100 mM potassium phosphate,
pH 6.6, solvent C is methanol. 0 min: 100% B; 7 min: 10% A,
90% B; 12 min: 25% A, 60% B, 15% C; 17 min: 25% A, 10% B,
65% C; 19 min: 100% B, 25 min: 100% B. Controls lacking HcyF,
HcyD and HcyS were similarly run and analyzed.
Preparation of Reduced Methyl Viologen. Methyl viologen
(149.5 mg) was dissolved in 12.5 mL of 50 mM potassium
phosphate, pH 8.0 in a 15 mL centrifuge tube. 10% Pt on activated
carbon (15 mg) was added. Argon was bubbled through the solution
for 5 min followed by hydrogen for 30 min. The sample was quickly
sealed with parafilm and centrifuged for 5 min to remove the
catalyst. The supernatant containing the reduced methyl viologen
was then transferred to a new 15 mL centrifuge tube in an oxygen
free glovebox. The concentration of the reduced methyl viologen
was measured at 600 nm (extinction coefficient 1.3 × 104
M-1cm-1).16
Sir-Mediated HcyS-COSH Formation Detected using
Lissamine Rhodamine Sulfonyl Azide and LC-MS. Thirty
microliters of 3.98 mM HcyS-Ala, 30 µL of 1.33 mM HcyF, 10
µL of 1.61 mM HcyD and 9 µL of 379 µM Sir were mixed. The
sample was then transferred to a glovebox and allowed to stand
for 2 h to allow for the sample to become anerobic. Thirty
microliters of 10 mM ATP, 4 µL of 1 M MgCl2, 2.5 µL of 100
mM Na2SO3 and 100 µL of 3.5 mM reduced methyl viologen were
then added. After 15 min, the sample was quenched by exposing
to air with shaking.
All the cultures were harvested by centrifugation and the cell-
pellets were lysed by sonication on ice. The proteins were purified
by Ni-NTA affinity chromatography at 4 °C. All buffers contained
1 mM TCEP. After purification, all proteins were buffer exchanged
into 100 mM Tris, 150 mM NaCl, 2 mM TCEP, 30% glycerol, pH
8.0 and stored in frozen aliquots at -80 °C.
To make HcyS-COSH and HcyS-DL-homocysteine, hcyS (with
the C-terminal alanine removed, see below) was inserted between
the NdeI/SapI restriction sites in pTYB1, an intein encoding
plasmid.15 The HcyS-Intein fusion was overexpressed in E. coli
BL21(DE3) as follows: Luria-Bertani cultures containing 100 mg
of ampicillin per liter were grown at 37 °C until an OD600 of
0.6-0.8 when the temperature was reduced to 15 °C and the
cultures were induced with IPTG (final concentration ) 0.5 mM).
Further growth was carried out at 15 °C for 12-16 h with constant
shaking. The cells were harvested by centrifugation and lysed by
sonication on ice in 20 mM Tris, 500 mM NaCl, 1 mM EDTA,
0.1% Triton X-100, pH 7.8. The samples were then loaded onto a
chitin column (20 mL) at a flow rate of 0.5 mL/min and washed
with 300 mL of 20 mM Tris, 500 mM NaCl, 1 mM EDTA, pH 7.8
at a flow rate of 2 mL/min. Cleavage of the HcyS-Intein fusion
was carried out at 4 °C for 12-16 h with 30 mL of 50 mM Na2S
to give HcyS-COSH or with 30 mL of 50 mM DL-homocysteine to
For gel analysis, the sample was buffer exchanged into 100 µL
of 50 mM NH4OAc, 6 M urea, pH 6.0 using a Biorad biospin 6
column and then treated with 7.4 µL of 15 mM lissamine rhodamine
sulfonyl azide for 15 min in the dark at 26 °C.17 The sample was
then desalted by chloroform/methanol precipitation and analyzed
by SDS-PAGE (15% tris-glycine gel). The fluorescence image of
the labeled protein in the gel was obtained on a Typhoon Trio
imager (excitation: 532 nm green laser; emission: 580-nm band-
pass filter (580 BP 30)). A similar sample, lacking HcyD, was
prepared as a control.
For MS analysis, the sample was heated at 100 °C for 5 min
(13) Bradford, M. M. Anal. Biochem. 1976, 72, 248.
(14) Chatterjee, A.; Li, Y.; Zhang, Y.; Grove, T. L.; Lee, M.; Krebs, C.;
Booker, S. J.; Begley, T. P.; Ealick, S. E. Nat. Chem. Biol. 2008, 4,
758.
(15) Kinsland, C.; Taylor, S. V.; Kelleher, N. L.; McLafferty, F. W.; Begley,
T. P. Protein Sci. 1998, 7, 1839.
and left to cool to room-temperature for an hour. The precipitated
(16) Thorneley, R. N. Biochim. Biophys. Acta 1974, 333, 487.
(17) Krishnamoorthy, K.; Begley, T. P. J. Am. Chem. Soc. 2010, 132,
11608.
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A R T I C L E S
Krishnamoorthy and Begley
Figure 5. ESI-MS analysis of the HcyD-catalyzed removal of alanine from
HcyS-Ala (+7 charge state). (a) HcyS-Ala (observed deconvoluted mass,
10304.98 Da; expected mass, 10304 Da; error, 0.01%) (b) HcyS formed
after treatment of HcyS-Ala with HcyD. (observed deconvoluted mass,
10235.61 Da; expected mass, 10232.92 Da; error, 0.03%).
Figure 4. Absorbance spectrum of Sir with its maxima at 388 and 590
nm characteristic of a ferredoxin-sulfite reductase.
protein was removed by filtration. The filtrate, containing small
proteins, was then concentrated and buffer exchanged into 50 mM
NH4OAc, pH 6.0 using a Biorad biospin 6 column and analyzed
by LC-MS.
HcyS-COSH is the Sulfur Donor for Homocysteine Biosy-
nthesis. Ninety-five microliters of 159 µM HcyS-COSH and 11
µL of 1.4 mM MetY were incubated with 1.3 µL of 10 mM
O-acetyl-L-serine or O-acetyl-L-homoserine. The samples were
incubated at room-temperature for 1 h. They were then buffer
exchanged into 50 mM NH4OAc, pH 6.0 and analyzed by MALDI-
MS. The time-period of incubation was later reduced to 2 min to
avoid formation of the 7912 Da adduct (Supporting Information,
Figure 3s).
Figure 6. Adenylating activity of HcyF. HcyF adenylates HcyS and the
resulting acyl-adenylate undergoes hydrolysis in the absence of bisulfide
to give AMP.
sample was treated with methanolic o-phthalaldehyde18-20 (1 mM
final concentration) and analyzed immediately by HPLC (Agilent
1200 using Supelco supelcosil LC-18-T (15 cm ×4.6 mm, 3 µm))
and the following gradient at a flow rate of 1 mL/min: solvent A
is water, solvent B is 100 mM potassium phosphate, pH 6.6, solvent
C is methanol; 0 min, 100% B; 7 min, 10% A, 90% B; 12 min,
25% A, 60% B, 15% C; 17 min, 25% A, 10% B, 65% C; 19 min,
100% B; 25 min, 100% B.
HcyD Catalyzed Hydrolysis of HcyS-COSH. Ninety-five
microliters of 42 µM HcyS-COSH were mixed with 3.6 µL of 88
µM HcyD and incubated at room-temperature for 5 min. The sample
was then treated with 99.5 µL of 9 M urea, buffer exchanged into
50 mM NH4OAc, pH 6.0 using a Biorad biospin 6 chromatography
column and analyzed by MALDI-MS. An identical reaction mixture
lacking HcyD was prepared as a control.
WS0269 (MetE)-Catalyzed Conversion of Homocysteine to
Methionine. One-hundred microliters of 169 µM of WS0269
(annotated as MetE) were incubated with 20 µL of 50 mM
potassium phosphate, pH 8.0, 1.7 µL of saturated 100 mM 5-DL-
methyltetrahydrofolate, 1.7 µL of 10 mM MgSO4 and 1.7 µL of
100 mM DL-homocysteine.21 The sample was incubated at room-
temperature for 1 h. The protein was then removed by passing the
samples through a Millipore ultrafree centrifugal filter device
(NMWL ) 5000 Da). 150 µL of the flow-through was treated with
50 µL of o-phthalaldehyde reagent (1 mL of 37 mM o-phthalal-
dehyde in MeOH, 4 mL of 0.1 M boric acid, pH 9.3, 162 µL of
3-mercaptopropionic acid were mixed together and the pH was
adjusted to 9.3 with NaOH). After 5 min at room-temperature, 20
µL of 1 M potassium phosphate, pH 6.0 were added and the reaction
Sulfide at High Concentrations is also a Sulfur Source for
Homocysteine Biosynthesis. Eighty microliters of 1.4 mM MetY
(in 50 mM potassium phosphate, pH 8.0) were mixed with 320 µL
of 50 mM potassium phosphate, 80% D2O, pH 8.0 along with 50
µL each of 100 mM sodium sulfide and 100 mM O-acetyl-L-
homoserine. The sample was incubated for 1 h at 26 °C. An
identical sample lacking MetY was prepared as a control. Samples
1
were freeze-dried, redissolved in 100% D2O and analyzed by H
NMR on Varian 300 MHz instrument.
Reaction of HcyS-COSH with MetZ. Two and nine-tenths
microliters of 1.4 mM MetZ and 95 µL of 42 µM HcyS-COSH
were incubated with the appropriate substrate -0.4 µL of 10 mM
O-acetyl-L-serine, 0.4 µL of 10 mM O-acetyl-L-homoserine and
0.4 µL of 10 mM O-succinyl-L-homoserine for 3 min at room-
temperature. The samples were buffer exchanged into 50 mM
NH4OAc, pH 6.0 and analyzed by MALDI-MS.
HcyD-Catalyzed Homocysteine Release from HcyS-Homo-
cysteine. Ninety-five microliters of 337 µM HcyS-(DL)-homocys-
teine were mixed with 3.6 µL of 880 µM HcyD and incubated at
room-temperature for 5 min. The sample was then treated with 98.6
µL of 9 M urea, buffer exchanged into 50 mM NH4OAc, pH 6.0
using a Biorad biospin 6 chromatography column and analyzed by
MALDI-MS. An identical reaction mixture lacking HcyD was
prepared as a control.
For small molecule analysis, 190 µL of 42 µM HcyS-COSH and
8 µL of 1 mM MetY were mixed, buffer-exchanged into 50 mM
potassium phosphate, pH 8.0 and concentrated to 100 µL using a
Millipore ultrafree centrifugal filter device (NMWL ) 5000 Da).
Four-tenths microliters of 250 mM TCEP and 0.8 µL of 10 mM
O-acetyl-L-homoserine were then added. After incubation at room-
temperature for 5 min, 10 µL of 76 µM HcyD in 50 mM potassium
phosphate, pH 8.0 was added. After an additional 5 min at room-
temperature, protein was removed using a YM-3 microcon and the
(18) Benson, J. R.; Hare, P. E. Proc. Natl. Acad. Sci. U.S.A. 1975, 72,
619.
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Nat. Prod. 2009, 72, 172.
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(21) Gonzalez, J. C.; Peariso, K.; Penner-Hahn, J. E.; Matthews, R. G.
Biochemistry 1996, 35, 12228.
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Thiocarboxylate-Dependent Methionine Biosynthesis
A R T I C L E S
Figure 7. Reconstitution of HcyS-COSH biosynthesis using methyl viologen as the electron donor and sulfite as the sulfur source.
Figure 8. Reconstitution of HcyS-COSH biosynthesis from HcyS-Ala monitored by LC-MS analysis. (a) Reconstitution reaction mixture from which HcyD
was omitted showing that HcyS-Ala remains unmodified. (b) Full reaction mixture showing conversion of HcyS-Ala (observed mass, 10303.8 Da; expected
mass, 10304 Da; error, 0.002%) to HcyS-COSH (observed mass, 10248.8 Da; expected mass, 10248.92 Da; error, 0.001%) via the intermediacy of HcyS
(observed mass, 10232.8; expected mass, 10232.92 Da; error, 0.001%).
mixture was analyzed by HPLC (Agilent 1200 using Phenomenex
Gemini 5 µ C18 110A (15 cm ×4.6 mm, 5 µm)) using the following
gradient at a flow rate of 1 mL/min: solvent A is water, solvent B
is 100 mM potassium phosphate, pH 6.6, solvent C is methanol; 0
min, 100% B; 7 min, 10% A, 90%B; 12 min, 25% A, 60% B, 15%
C; 17 min, 25% A, 10% B, 65% C; 19 min, 100% B; 25 min,
100% B.
HcyD is a Metalloprotease Involved in C-Terminal Pro-
cessing of HcyS-Ala. HcyD removes the C-terminal alanine from
HcyS-Ala in the presence of exogenous Zn2+. ESI-MS analysis
of the reaction mixture demonstrates the conversion of HcyS-
Ala to HcyS (observed mass change: 69.37 Da, expected mass
change upon removal of alanine: 71.04 Da, Figure 5). 1H NMR
analysis of the small-molecule product confirmed the release
of alanine from HcyS-Ala (Supporting Information, Figure 2s).
HcyF Activates HcyS-COOH by Adenylation. HcyF was
shown to adenylate HcyS-COOH, generated by HcyD catalyzed
hydrolysis of HcyS-Ala. The adenylating reaction was monitored
by the release of AMP from the unstable HcyS-COAMP. AMP
formation was dependent on HcyF, HcyS-Ala and HcyD (Figure
6).
3. Results
Growth and Overexpression. The sir, hcyD, hcyF, hcyS-ala,
metY, metZ and WS0269 (metE) genes (Table 1) were cloned
into the THT vector. All the proteins, except Sir, were
overexpressed in E. coli BL21 (DE3) in LB medium. The sir
gene was overexpressed in the methionine-auxotroph E. coli
B834 (DE3). In this strain, Sir, annotated as ferredoxin-sulfite
reductase, was coexpressed with siroheme synthase (cloned into
pACYDuet) and the IscS-cluster assembly proteins (cloned into
pDB1282) in M9 medium. Coexpression with these proteins is
essential for good reconstitution of active Sir, which utilizes
siroheme and [4Fe-4S] cofactors. All proteins were purified on
a Ni-NTA affinity column. Sir was purified aerobically (this
[4Fe-4S] containing protein did not require anaerobic purifica-
tion) at 4 °C. It had the characteristic absorbance of a siroheme-
[4Fe-4S] cluster protein22 (Figure 4). MetY and MetZ were
yellow suggestive of bound PLP. SDS-PAGE analysis of all
the homocysteine biosynthetic proteins is given in the Support-
ing Information, Figure 1s.
Figure 9. SDS-PAGE analysis of HcyS-COSH formation after labeling
with lissamine rhodamine sulfonyl azide (a) Coomassie image (b) Fluo-
rescent image. (Lane 1) Sample containing all componentssHcyD, HcyF,
HcyS-Ala, Sir, SO32-, reduced methyl viologen (electron donor), ATP,
Mg2+. (Lane 2) Same as Lane 1 except HcyD was omitted from the reaction
mixture. PMT voltage: 400 V.
(22) Koguchi, O.; Tamura, G. Agric. Biol. Chem. 1988, 52, 373.
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J. AM. CHEM. SOC. VOL. 133, NO. 2, 2011 383

A R T I C L E S
Krishnamoorthy and Begley
Figure 10. MALDI-MS analysis of the MetY-catalyzed homocysteine biosynthesis (a) HcyS-COSH in the presence of MetY and O-acetyl-L-homoserine,
forms HcyS-homocysteine (observed mass, 7809.83 Da; expected mass, 7805.96 Da; error, 0.05%; net mass change of protein, 100 Da). (b) HcyS-COSH
(observed mass, 7709.83 Da; expected mass, 7704.82 Da; error, 0.07%) in the presence of MetY and O-acetyl-L-serine, 1 h incubation time, showing that
HcyS-COSH cannot transfer sulfide to O-acetyl-L-serine. HcyS-COSH and HcyS-homocysteine (see below) do not have the His6-tag and hence, their masses
are different from the His-tagged HcyS used in other experiments in this work.
Figure 11. MALDI-MS analysis of HcyD treated HcyS-DL-Homocysteine: (a) HcyS-DL-Homocysteine (observed mass, 7812.11 Da; expected mass, 7805.96
Da; error, 0.08%) in the presence of HcyD is converted to HcyS-homocysteine to HcyS-COOH (observed mass, 7695.42 Da; expected mass, 7688.82 Da;
error, 0.09%; net mass change, 116.69 Da). (b) Reference sample of HcyS-DL-homocysteine (observed mass, 7812.49 Da; expected mass, 7805.96 Da; error,
0.08%).
Sir Catalyzes the Formation of HcyS-COSH from Sulfite. The
formation of HcyS-COSH from HcyS-Ala requires HcyD
catalyzed removal of alanine, HcyF-catalyzed adenylation and
AMP displacement by sulfide generated by the Sir-catalyzed
reduction of SO3 (Figure 7). LC-MS analysis of the recon-
stitution reaction mixture demonstrated that this conversion was
successfully achieved (Figure 8).
HcyS-COSH formation was also detected using the thiocar-
boxylate specific fluorescent reagent lissamine rhodamine sul-
fonyl azide.17 The resulting fluorescent protein was analyzed
by SDS-PAGE and scanned for fluorescence using a Typhoon
trio (Figure 9).
HcyS-COSH is the Sulfur Source for MetY-Catalyzed
Homocysteine Biosynthesis. WS1012, annotated as a putative
O-acetyl-L-homoserine sulfhydrylase (MetY), catalyzed the
reaction of HcyS-COSH with O-acetyl-L-homoserine to form
HcyS-Homocysteine (Figure 10a). MetY did not accept O-
acetyl-L-serine as a substrate even after extended incubation time
(Figure 10b). Longer incubation time or higher concentrations
of O-acetyl-L-homoserine resulted in the formation of a higher
molecular weight adduct (7911 Da), consistent with ho-
molanthionine formation (Supporting Information, Figure 3s).23
MetY can also use sulfide to convert O-acetyl-L-homoserine to
homocysteine (Supporting Information, Figure 4s).
WS1015, annotated as a putative O-acetyl/succinyl-L-ho-
moserine sulfhydrylase (MetZ), did not catalyze the reaction
of HcyS-COSH with O-acetyl-L-homoserine or O-succinyl-L-
homoserine to form HcyS-Homocysteine (Supporting Informa-
tion, Figure 5s).
2-
HcyD Catalyzes the Release of Homocysteine from HcyS-
Homocysteine. HcyS-homocysteine was prepared by cleaving
the HcyS-intein construct with (DL)-homocysteine. HcyD cata-
lyzes the release of homocysteine from HcyS-homocysteine as
Figure 13. WS0269 is a methyltetrahydrofolate dependent methionine
synthase. HPLC analysis of the reaction mixture after derivitization with
o-phthalaldehyde. The peak at 19.5 min comigrated with a reference
methionine/o-phthalaldehyde adduct.
Figure 12. Characterization of the amino acid released from HcyS-
homocysteine by o-phthalaldehyde derivatization. The peak at 19.1 min
comigrated with a reference homocysteine/o-phthalaldehyde adduct.
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384 J. AM. CHEM. SOC. VOL. 133, NO. 2, 2011

Thiocarboxylate-Dependent Methionine Biosynthesis
A R T I C L E S
4. Discussion
Thiocarboxylated proteins are important intermediates in a
variety of biochemical sulfide transfer reactions. For ThiS-
COSH, MoaD-COSH, and Urm1p, the thiocarboxylate sulfur
is derived from cysteine in a cysteine desulfurase catalyzed
reaction.4,9,10 The sulfur donor for QbsE, PdtH and CysO is
unknown. The identification of a putative protein thiocarboxylate
dependent methionine biosynthetic gene cluster in Wolinella
succinogenes, also encoding the enzymes involved in the
reduction of sulfate to sulfide, suggested that sulfite reductase
might function as the sulfide source for protein thiocarboxylate
formation. The successful reconstitution of the new methionine
biosynthetic pathway described in Figure 14 supports this
conclusion. In this pathway, the carboxy terminal alanine is
removed from HcyS-Ala in a reaction catalyzed by HcyD. HcyF
then catalyzes the adenylation of HcyS. HcyS acyl-adenylate
undergoes nucleophilic substitution by bisulfide produced by
Sir to form HcyS thiocarboxylate, which adds to O-acetylho-
moserine to give HcyS-homocysteine in a reaction catalyzed
by MetY. This reaction is likely to proceed via a PLP-mediated
substitution followed by an S/N acyl shift analogous to cysteine
biosynthesis in M.tuberculosis.5 In that pathway, CysO-thio-
carboxylate is converted to CysO-cysteine upon treatment with
the PLP-dependent O-phospho-L-serine sulfhydrylase and
O-phospho-L-serine.5,24 HcyD mediated hydrolysis liberates
homocysteine. A final methylation, catalyzed by MetE, com-
pletes the methionine biosynthesis.
Figure 14. New protein-thiocarboxylate-mediated methionine biosynthesis
in Wolinella succinogenes.
shown by MALDI-MS analysis of the reaction mixture (Figure
11a). This reaction proceeded to greater than 50% indicating
that in addition to alanine (see above) HcyD can remove both
isomers of homocysteine. HcyD also catalyzes the hydrolysis
of HcyS-COSH (Supporting Information, Figure 6s).
Several features of this pathway merit further comment. All
known thiocarboxylate forming proteins have a Gly-Gly C-
terminus in their active form (Figure 15). HcyS-Ala ends in a
Gly-Gly-Ala sequence that requires processing by the HcyD
metalloprotease. The physiological significance of this process-
ing event is unknown. Pathway regulation is one possibility.
We have previously observed analogous carboxy terminal
processing: QbsD removes Cys-Phe from the carboxy terminal
of QbsE during thioquinolobactin biosynthesis in P. fluorescens6
and Mec+ removes cysteine from CysO-Cys during cysteine
biosynthesis in M. tuberculosis.5 HcyD shows broad substrate
tolerance and also catalyzes the removal of both isomers of
homocysteine from HcyS-homocysteine as well as sulfide from
HcyS-COSH.
To further characterize the released amino acid, HcyS-L-
homocysteine was prepared by reacting HcyS-COSH with
O-acetyl-L-homoserine in the presence of MetY and then treated
with HcyD. The resulting reaction mixture was then treated with
o-phthalaldehyde to yield a fluorescent homocysteine derivative
(Supporting Information, Figure 7s) which was analyzed by
HPLC18-20 (Figure 12). This detected product was identified
as the homocysteine derivative by coelution with a reference
homocysteine/o-phthalaldehyde adduct.
WS0269 is a Methionine Synthase. WS0269 (MetE) catalyzes
the methyl transfer from methyltetrahydrofolate to homocysteine
to form methionine (Figure 13). Methionine production was
assayed by HPLC after derivatization with o-phthalaldehyde/
mercaptopropionic acid.
Figure 15. Sequence alignment of biochemically characterized thiocarboxylate-forming proteins (done using Escript 2.2). All have a diglycyl C-terminus
in their active form. Both HcyS-Ala and QbsE are activated by carboxy terminal processing. Sequence alignment of putative HcyS-like proteins from various
organisms is shown in Supporting Information, Figure 8s.
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J. AM. CHEM. SOC. VOL. 133, NO. 2, 2011 385

A R T I C L E S
Krishnamoorthy and Begley
Acknowledgment. We thank Dr. Cynthia Kinsland at the
Protein facility, Cornell University for overexpression of all genes
used in this study and Dr. Squire Booker for the methionine-
auxotroph E. coli B834(DE3) harboring the iscS-cluster assembly
genes. MALDI-MS and LC-MS were obtained by the Laboratory
of Biological Mass spectrometry at Texas A&M University. This
research was supported by the NIH (DK44083) and by the Robert
A. Welch Foundation (A-0034).
We assayed for formation of HcyS thiocarboxylate using an
MS based assay as well as a recently developed reagent for the
detection of protein thiocarboxylates in bacterial proteomes
involving a click reaction with lissamine rhodamine B sulfonyl
azide.17 The fact that Sir is clustered with the other methionine
biosynthetic genes suggests that it is functioning as the sulfur
source for methionine biosynthesis in ViVo in Wolinella succi-
nogenes. However, we do not have evidence at this time for
sulfide channeling between Sir and the HcyF/HcyS complex.
Kinetics studies to address this are in progress.
Supporting Information Available: Additional sequence
analysis, SDS-PAGE of purified proteins, reaction schemes,
reaction mixture, and product spectra and the primers used for
cloning. This material is available free of charge via the Internet
at http://pubs.acs.org.
(23) Kromer, J. O.; Heinzle, E.; Schroder, H.; Wittmann, C. J. Bacteriol.
2006, 188, 609.
(24) O’Leary, S. E.; Jurgenson, C. T.; Ealick, S. E.; Begley, T. P.
Biochemistry 2008, 47, 11606.
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