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J Biol Chem. 2008 September 19; 283(38): 25829–25838. doi: 10.1074/jbc.M802674200. | PMCID: PMC2533785 |
Copyright © 2008, The American Society for Biochemistry and
Molecular Biology, Inc. Crenarchaeal Arginine Decarboxylase Evolved from an
S-Adenosylmethionine Decarboxylase
Enzyme *![[S with combining enclosing square]](/corehtml/pmc/pmcents/x0053x20DE.gif) Teresa N. Giles and David E. Graham1 Department of Chemistry and Biochemistry and the Institute for Cellular and Molecular Biology, University of Texas, Austin, Texas 78712 Received April 7, 2008; Revised June 19, 2008. The crenarchaeon Sulfolobus solfataricus uses arginine to produce
putrescine for polyamine biosynthesis. However, genome sequences from S.
solfataricus and most crenarchaea have no known homologs of the
previously characterized pyridoxal 5′-phosphate or pyruvoyl-dependent
arginine decarboxylases that catalyze the first step in this pathway. Instead
they have two paralogs of the S-adenosylmethionine decarboxylase
(AdoMetDC). The gene at locus SSO0585 produces an AdoMetDC enzyme, whereas the
gene at locus SSO0536 produces a novel arginine decarboxylase (ArgDC). Both
thermostable enzymes self-cleave at conserved serine residues to form
amino-terminal β-domains and carboxyl-terminal α-domains with
reactive pyruvoyl cofactors. The ArgDC enzyme specifically catalyzed arginine
decarboxylation more efficiently than previously studied pyruvoyl enzymes.
α-Difluoromethylarginine significantly reduced the ArgDC activity of
purified enzyme, and treating growing S. solfataricus cells with this
inhibitor reduced the cells' ratio of spermidine to norspermine by decreasing
the putrescine pool. The crenarchaeal ArgDC had no AdoMetDC activity, whereas
its AdoMetDC paralog had no ArgDC activity. A chimeric protein containing the
β-subunit of SSO0536 and the α-subunit of SSO0585 had ArgDC
activity, implicating residues responsible for substrate specificity in the
amino-terminal domain. This crenarchaeal ArgDC is the first example of
alternative substrate specificity in the AdoMetDC family. ArgDC activity has
evolved through convergent evolution at least five times, demonstrating the
utility of this enzyme and the plasticity of amino acid decarboxylases. The Crenarchaeota include the most hyperthermophilic cultivated
microorganisms. To help stabilize macromolecules in these extreme conditions,
some hyperthermophiles produce unusually long chain or branched polyamines
( 1). Chromatographic analyses
of extracts from the crenarchaeon Sulfolobus solfataricus identified
significant amounts of the linear polyamines 1,3-diaminopropane, putrescine,
sym-norspermidine (caldine), spermidine, and sym-norspermine
(thermine), with traces of spermine
( 2,
3). The biosynthesis of the
spermidine and spermine polyamines was predicted to follow a canonical
eukaryotic pathway, where the decarboxylation of l-ornithine
produces putrescine, and S-adenosyl- l-methionine
(AdoMet) 2 is
decarboxylated to form the propylamine donor
S-(5′-adenosyl)-3-methylthiopropylamine (dcAdoMet)
( 4). Subsequently, the AdoMet
decarboxylase (AdoMetDC) ( 5)
and propylamine transferase ( 6)
enzymes were purified from S. solfataricus. The latter enzyme
produces spermidine and spermine from putrescine and dcAdoMet, and it produces
norspermidine and norspermine from 1,3-diaminopropane and dcAdoMet
(). The complete genome sequences of 13 thermophilic crenarchaea encode
orthologs of propylamine transferase
( 7) and two homologs of the
archaeal type AdoMetDC (supplemental Table 1)
( 8). However, none of the
genomes encodes a recognizable ornithine decarboxylase enzyme to produce
putrescine. Instead they have homologs of the agmatine ureohydrolase enzyme
( 9), suggesting that putrescine
is derived from l-arginine as in the Euryarchaeota. Some
bacteria produce agmatine from arginine using a pyridoxal 5′-phosphate
(PLP)-dependent arginine decarboxylase (ArgDC)
( 10). Alternatively,
euryarchaea use a pyruvoyl-dependent ArgDC that is related to
Lactobacillus sp. histidine decarboxylase
( 11). No crenarchaea have
homologs of the PLP-dependent enzyme, and only three have homologs of the
euryarchaeal enzyme. Therefore, we predicted that members of the
Crenarchaeota have a new type of arginine decarboxylase that is
evolutionarily unrelated to the previously characterized enzymes. The two AdoMetDC proteins from S. solfataricus, SSO0536 and
SSO0585, share 47% amino acid sequence identity
(). mRNAs from the
respective genes were previously shown to have different half-lives (7 min for
SSO0536 and 17–20 min for SSO0585)
( 12). The same discrepancy was
observed in Sulfolobus acidocaldarius, and the two genes could have
different functions. Both AdoMetDC homologs from S. solfataricus were
cloned in Escherichia coli, and the polyhistidine-tagged proenzymes
were heterologously expressed and purified. These proteins self-cleaved at a
conserved serine residue, forming aminoterminal β-subunits and active
pyruvoyl groups on their carboxyl-terminal α-subunits. The
His 10-SSO0585 protein catalyzed the decarboxylation of AdoMet in a
coupled assay with AdoMet synthetase enzyme, and it is probably the same
protein that was previously purified (but not sequenced) by Cacciapuoti et
al. ( 5). The
His 10-SSO0536 protein also self-cleaved, but it did not catalyze
AdoMet decarboxylation. Instead, it efficiently catalyzed the decarboxylation
of l-arginine. Therefore, this crenarchaeal protein represents a
new class of arginine decarboxylases that developed through convergent
evolution. A chimeric protein containing the β-subunit of SSO0536 and the
α-subunit of SSO0585 functioned specifically as an ArgDC, correlating
conserved changes in the amino-terminal part of this protein with the change
in substrate specificity. The SSO0536 protein is the first member of the
AdoMet decarboxylase family shown to catalyze another reaction. Phylogenetic
analysis suggests that an ancestral AdoMetDC gene duplicated once in the
crenarchaeal lineage. Subtle changes to the active site structure of one
paralog enabled the evolution of this new arginine decarboxylase activity and
displaced an ancestral euryarchaeal ArgDC. Synthesis of
dl-Difluoromethylarginine—2-Amino-5-carbamimidamido-2-(difluoromethyl)pentanoic
acid (α-difluoromethylarginine (DFMA)) was synthesized by the
guanylation of 2-(difluoromethyl)- dl-ornithine (DFMO) using a
modification of the procedure described by Bey et al.
( 13). DFMO hydrochloride (1.0
g, 4.2 mmol) was dissolved in 2.5 ml of 2 m sodium hydroxide.
S-Methylisothiourea hemisulfate (1.18 g, 8.5 mmol) was added with
stirring at room temperature. The pH was adjusted to 10.5 with sodium
hydroxide, and the solution was stirred for 5 days. The solution was adjusted
to pH 7 with HCl and evaporated to dryness under vacuum at 60 °C. The
residue was dissolved in 7 ml of water and applied to a Dowex 50
(H +) column (1 × 31 cm). The column was subsequently washed
with 4 bed volumes of water and 2 volumes of 1 m ammonium
hydroxide. DFMA was eluted with 2 m ammonium hydroxide.
Ninhydrin-positive fractions were combined and evaporated to dryness under
vacuum to produce 0.63 g of DFMA (2.8 mmol, 67%). Crystallization of the
hydrochloride salt afforded hygroscopic white crystals. The 1H NMR
and 39F NMR spectra were consistent with previous data
( 13). High resolution
electrospray ionization mass spectrometry (ESI-MS) confirmed the chemical
composition of the MH + ion: expected 225.11576
m/ z, observed 225.1160 m/z. DFMO was a gift from
Dr. Patrick Woster (Wayne State University) and was originally produced by Dow
Chemicals. All other chemicals were purchased from various distributors in
reagent grade and used without further purification. Strains and Growth Media— S. solfataricus P2 (DSM
1617) was obtained from the Deutsche Sammlung von Mikroorganismen und
Zellkulturen. Cultures were grown in complex DSMZ medium 182 containing yeast
extract (2 g), KH 2PO 4 (3.1 g),
(NH 4) 2SO 4 (2.5 g),
MgSO 4·7H 2O (0.2 g) and
CaCl 2·2H 2O (0.25 g) in 1 liter adjusted to pH 3.7
with sulfuric acid. Defined growth medium contained
(NH 4) 2SO 4 (1.3 g),
KH 2PO 4 (0.28 g), MgSO 4·7H 2O
(0.28 g), CaCl 2·2H 2O (0.25 g),
FeCl 3·6H 2O (0.02 g), and 100× mineral salt
solution (10 ml) ( 14) in 1
liter adjusted to pH 3.5 with sulfuric acid
( 15). Defined medium was
supplemented with either d-glucose (0.4%, w/v) or casein
hydrolysate (0.4%, w/v). Batch cultures were grown in 75 ml of medium in
150-ml medium bottles, incubated in a convection oven at 85 °C attached to
a linear motion shaker (80 rpm). Polyamine Analysis—For analysis by gas chromatographymass
spectrometry, cellular polyamines were converted to trifluoroacetamide
derivatives. Cells (0.15 g wet mass) were suspended in 0.75 ml of water and
lysed by sonication on ice. To the lysate was added 0.75 ml of trichloroacetic
acid (10% w/v), and the mixture was centrifuged for 10 min at 18,000 ×
g. The pellet was reextracted with 1 ml of trichloroacetic acid, and
the combined supernatant was extracted twice with 10 ml of diethyl ether. The
aqueous solution was evaporated to dryness under vacuum, and the solids were
suspended in 0.3 ml of trifluoroacetic anhydride for 30 min at 55 °C. The
trifluoroacetyl polyamine derivatives were analyzed by gas chromatographymass
spectrometry with chemical ionization in the positive ion mode. Mass spectra
were consistent with those reported previously
( 2,
16). For quantitative analysis by HPLC, cellular polyamines were converted to
dansyl sulfonamide derivatives
( 17). Extracts of S.
solfataricus cells were deproteinized with perchloric acid, and then the
neutralized extracts were mixed with dansyl chloride. The derivatives were
extracted into cyclohexane, evaporated to dryness by heating under
N 2, and dissolved in 50% acetonitrile. These samples were applied
to a reversed phase HPLC column (Luna C18(2), 150 × 4.6 mm; Phenomenex)
and eluted using a gradient from 50 to 100% acetonitrile in 0.02%
trifluoroacetic acid. Dansyl sulfonamides were detected by their fluorescence
with an excitation wavelength of 340 nm and an emission wavelength of 515 nm.
These derivatives were identified by their co-elution with polyamine
standards, and they were quantified by integrating the peak areas of the
fluorescence chromatograms. 1,7-Diaminoheptane was used as an internal
standard to measure polyamine extraction and derivatization efficiency
(73–84% recovery). Arginine Decarboxylase Assay—The rate of arginine
decarboxylation was determined using a CO 2 capture assay to detect
the release of 14CO 2 from 14C-labeled
arginine ( 18). Standard
reactions (100 μl) contained 12 m m citric acid, 26 m m
sodium phosphate (pH 6.0), 10 m m l-arginine-HCl, 6.5–300 nCi
of l-[1- 14C]arginine (55 mCi mmol –1;
American Radiolabeled Chemicals), and enzyme. After a 5-min incubation at 80
°C, the reactions were terminated by the addition of 100 μl of 4
m HCl and heated at 70 °C for 15 min. One unit of activity
catalyzes the decarboxylation of 1 μmol of l-arginine/min. A
nonlinear regression program (KaleidaGraph version 3.6) estimated steady-state
kinetic parameters from the initial rate data fit to the hyperbolic
Michealis-Menten-Henri equation. Protein thermostability was tested by preincubating 2 μgof protein at
temperatures from 4 to 90 °C for 10 min in citrate-phosphate buffer (pH
6.0). Arginine decarboxylase activity was determined at room temperature as
described above. The temperature dependence of enzyme activity was determined
by preincubating enzyme in reaction buffer at temperatures from 4 to 70 °C
for 10 min. Reactions were initiated by the addition of arginine substrate and
incubated at the same temperature. The pH dependence of enzyme activity was analyzed at 70 °C in reactions
containing 2 μg of purified enzyme, 10 mm l-arginine, 200 nCi of
l-[U-14C]arginine (305 mCi mmol–1;GE
Healthcare), and buffer (citrate-phosphate buffer (pH 2.8–6.0), 25
mm sodium potassium phosphate (pH 7.0–8.0), or 6
mm sodium borate (pH 10)). Cloning and Molecular Biology—The MJ1208 gene was amplified
by PCR using oligonucleotide primers 5MJ1208BN and 3MJ1208B (supplemental
Table 2) and Methanocaldococcus jannaschii JAL-1 chromosomal DNA
( 19). The product was ligated
in the NdeI and BamHI sites of vector pET-19b to produce vector pDG395
(supplemental Table 3). Primers 5SSO0585BN and 3SSO0585B were similarly used
to clone the SSO0585 gene from S. solfataricus P2 chromosomal DNA
producing vector pDG401. Primers 5SSO0536BN and 3SSO0536B were used to clone
the S. solfataricus SSO0536 gene producing vector pDG398. Splicing
overlap extension PCR was used to construct two chimeric genes
( 20). For the
β536α585 chimera, the T7 promoter and SSO0536ChR primers were used
to amplify the 5′-fragment from pDG401, and the SSO0585ChF and T7
terminator primers were used to amplify the 3′-fragment from pDG398.
Both gel-purified PCR products were mixed together, the annealed product was
extended with Vent DNA polymerase (New England Biolabs), and the chimeric
template was amplified with the T7 promoter and T7 terminator primers. This
chimeric product was ligated into the NdeI and BamHI sites of pET-19b to
create vector pDG453. To produce the β585α536 chimera, the T7
promoter and SSO0585ChR primers were used to amplify the 5′-fragment
from pDG398, and the SSO0536ChF and T7 terminator primers were used to amplify
the 3′-fragment from pDG401. The products were spliced as described
above and cloned into the NdeI and BamHI sites of pET-19b to create vector
pDG483. Plasmids were propagated in E. coli DH5α cells
(Invitrogen). Recombinant DNA was sequenced at the Institute for Cellular and
Molecular Biology Core Laboratories DNA Sequencing facility (University of
Texas, Austin, TX). Protein Expression and Purification—The polyhistidine-tagged
proteins His 10-MJ1208, His 10-SSO0536,
His 10-SSO0585, His 10-β536α585, and
His 10-β585α536 were heterologously expressed in E.
coli BL21 (DE3) strains transformed with the respective expression
vectors (supplemental Table 3). The cells were grown with continuous shaking
(250 rpm) at 37 °C in Luria-Bertani broth with ampicillin (100
μgml –1). Protein expression and cell lysis were performed
by standard methods ( 18).
Extracts were heated at 75 °C for 20 min and then centrifuged at 18,000
× g for 10 min. Ni 2+ affinity chromatography
separated the polyhistidine-tagged proteins. The concentrated, desalted
proteins were further purified by strong anion exchange chromatography using a
Mono Q column (5 × 50 mm, 10 μm; GE Healthcare). This column was
equilibrated with buffer A containing 25 m m Tris-HCl (pH 7.6), and
protein was eluted using a gradient to 100% buffer B (buffer A with 1
m NaCl). Purified protein was desalted using a HiTrap Sephadex G-25
column (5 ml; GE Healthcare) in 50 m m ammonium bicarbonate (pH 7.5)
and was concentrated by ultrafiltration (3000 molecular weight cut-off). Protein Size Measurements—Apparent masses of purified,
denatured proteins were measured by SDS-PAGE using the Schägger and von
Jagow Tris-Tricine system with 12% total and 3.3% cross-linked acrylamide
( 21). Protein bands were
identified in the gels by silver staining (Pierce) or staining with Coomassie
Brilliant Blue R dye. Measurements of native protein mass and Stokes radius
were made by analytical size exclusion chromatography
( 18). Precipitation with
trichloroacetic acid was used to concentrate proteins from collected
fractions; these were analyzed by SDS-PAGE to determine the subunit
composition of oligomers. For mass spectrometry, affinity-purified proteins
were desalted into 50 m m ammonium bicarbonate (pH 7.5) using a
Sephadex G-25 desalting column (5 ml; GE Healthcare). Protein ESI-MS was
performed by the CRED Analytical Instrumentation Facility at the University of
Texas at Austin. S-Adenosylmethionine Decarboxylase Assay—AdoMet
decarboxylase activity was measured in a coupled reaction using AdoMet
synthetase to produce the
S-adenosyl- l-[ carboxyl- 14C]methionine
substrate. Reactions contained His 10-MJ1208 protein (6 μg), 25
m m Hepes [4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid]KOH
(pH 8), 50 m m KCl, 10 m m MgCl 2, 2 m m
l-methionine, 100 nCi of l-[1- 14C]methionine (55
mCi mmol –1; American Radiolabeled Chemicals), and 9.5
m m ATP in a volume of 100 μl
( 19). After a 30-min
incubation at 70 °C, purified decarboxylase enzyme (1 μg) was added,
and reactions were incubated for 5 min. The reactions were terminated by the
addition of 100 ml of 4 m HCl, and released
14CO 2 was determined using a CO 2 capture
assay ( 8). Identification of Alternative Substrates and
Inhibitors—Substrate analogs and carbonyl-reactive compounds were
preincubated with 1 μgofHis 10-SSO0536 protein at 80 °C for
10 min before 0.2 m m arginine was added. After 5 min, the reactions
were stopped, and the released 14CO 2 was measured. The
potential inhibitors tested included the arginine analogs
l-arginine O-methyl ester,
Nα-acetyl l-arginine,
l-argininamide, l-canavanine, l-citrulline,
d-arginine, l-homoarginine,
NG-nitro- l-arginine methyl ester,
NG-methyl l-arginine, l-ornithine,
l-histidine, l-lysine, and l-methionine.
Histidine, homoarginine, and canavanine were also tested as potential
substrates in reactions without arginine. Primary amines in the reaction
product were derivatized with naphthalene 2,3-dicarboxaldehyde and cyanide to
produce fluorescent cyanobenz[ f]isoindole derivatives, which were
analyzed by HPLC as described previously
( 22). The nucleophiles
O-methyl hydroxylamine hydrochloride and
O-nitrobenzylhydroxylamine hydrochloride were also tested as
inhibitors. Phylogenetic Analysis of AdoMetDC Homologs—Amino acid
sequences from 13 ArgDC and 19 AdoMetDC homologs were separately aligned using
the T-Coffee program (version 4.96)
( 23). The alignments were
manually combined using the CIN-EMA5 alignment editor program
( 24). From the full alignment,
118 positions that were confidently aligned were chosen for phylogenetic
analysis. The proml program from the Phylip package (version 3.66; J.
Felsenstein, University of Washington) was used to infer a maximum likelihood
phylogeny from this alignment, with the Jones-Taylor-Thornton model of amino
acid changes and a γ-distribution of rates (α = 2.4) approximated
by three states. Bootstrap analysis was performed with 100 replicates. A
similar phylogeny was inferred using the protdist and neighbor programs from
the same software package. Full organism names and sequence accession numbers
are listed in the supplemental materials. Polyamines of S. solfataricus P2—Previous studies identified
spermidine and norspermine as the predominant polyamines in Caldariella
acidophila (now S. solfataricus DSM 5833)
( 2) and S.
solfataricus P1 ( 3), with
traces of norspermidine. However, cells in those experiment were grown on a
complex medium containing yeast extract and casamino acids, which could have
introduced exogenous polyamines. To identify the polyamines specifically
produced by S. solfataricus P2, we grew the cells on a defined
glucose minimal medium and extracted polyamines for gas chromatography-mass
spectrometry analysis as the trifluoroacetyl derivatives
( 2). The polyamine pool
comprised spermidine (76%), norspermine (10%), putrescine (8%), and
norspermidine (5%). For comparison, cells grown with casamino acids instead of
glucose contained higher total levels of polyamines, including spermidine
(72%), norspermine (12%), norspermidine (11%), and putrescine (5%). Both
cultures also contained high levels of the osmolyte trehalose
( 25). Therefore amino acids in
the growth medium slightly changed the distribution and abundance of
polyamines in S. solfataricus, although the types of polyamines
detected were consistent with those reported previously. Arginine Decarboxylase Activity in P2 Cells—A metabolic
reconstruction of polyamine biosynthesis in S. solfataricus P2 from
its genome sequence led us to predict that these cells decarboxylate arginine
to produce agmatine, and hydrolyze the guanidinium group to produce putrescine
(). To establish that
arginine decarboxylase is expressed in this strain, we measured ArgDC activity
at 80 °C in cell-free extracts of S. solfataricus grown to
stationary phase in defined medium with glucose or casamino acids. Extract
from glucose-grown cells catalyzed arginine decarboxylation with a specific
activity of 160 pmol min–1 mg–1, compared
with a specific activity of 400 pmol min–1
mg–1 for extract from cells grown on casamino acids. These
cells catalyzed l-arginine decarboxylation at pH 6, consistent with
the pathway proposed for polyamine biosynthesis in
. The physiological significance of arginine decarboxylase was tested by
adding DFMA to S. solfataricus cultures growing in glucose minimal
medium. DFMA irreversibly inhibits arginine decarboxylases, and DFMO
irreversibly inhibits ornithine decarboxylases
( 26,
27). Cells inoculated into
medium containing 2 m m DFMA had a prolonged lag phase of growth
compared with cells inoculated into medium with 2 m m DFMO or no
inhibitor. An actively growing culture that was split into three samples
(DFMA, DFMO, or control) showed only a small decrease in the growth rate with
DFMA. Cells growing in DFMA medium had a specific growth rate of 0.040
± 0.0013 h –1, compared with cells in DFMO medium
(0.045 ± 0.0034 h –1) or control medium (0.044 ±
0.0018 h –1). Cells grown with DFMA contained lower
concentrations of putrescine and spermidine than cells grown in control or
DFMO media ( Table 1). However,
cells grown in all three conditions had similar total polyamine contents.
S. solfataricus cells adapted to DFMA by substantially increasing
their relative levels of norspermine, which does not contain a putrescine
core. | TABLE 1Polyamine contents of S. solfataricus cells |
Expression, Purification, and Cleavage of AdoMetDC
Homologs—Both AdoMetDC homologs from S. solfataricus were
heterologously expressed in E. coli as soluble, heat-stable proteins.
SDS-PAGE analysis of the affinity-purified His10-SSO0585 protein
preparation showed three prominent bands with apparent molecular masses of 21,
15, and 5 kDa (). These
bands corresponded to the proenzyme, β-subunit, and α-subunit,
respectively, and densitometry indicated that 77% of the proenzyme had cleaved
in the preparation shown. ESI-MS identified an ion series corresponding to the
proenzyme (16,602 Da observed, 16,680 Da expected), the methionine
aminopeptidase-cleaved proenzyme (16,546 Da observed, 16,549 Da expected) and
the α-subunit (6,258 Da observed, 6,260 Da expected). Peaks
corresponding to unidentified proteins with apparent masses of 11,758, 13,649,
and 13,780 Da were also observed, at lower ion abundances. Size exclusion
chromatography indicated that the His10-SSO0585 protein formed a
dimeric complex with an apparent mass of 33 kDa and a Stokes radius of 26
Å. The purified His10-SSO0536 protein preparation also formed three
predominant bands on an SDS-polyacrylamide gel with apparent molecular masses
of 21, 15, and 6 kDa. Up to 54% of the proenzyme was cleaved in preparations
purified from heat-treated cell lysate; however, only 21% of the protein was
cleaved in preparations from unheated lysates. ESI-MS analysis identified ion
series corresponding to the proenzyme (17,937 Da observed, 18,014 Da expected)
and the methionine aminopeptidase-cleaved proenzyme (17,880 Da observed,
17,883 Da expected), as well as the α-subunit (6,123 Da observed, 6,125
Da expected) and the β-subunit (11,759 Da observed, 11,760 Da expected
for aminopeptidase-cleaved subunit). The His10-SSO0536 protein
eluted from a size exclusion column with an apparent mass of 80 kDa and a
Stokes radius of 35 Å, suggesting that it forms a tetramer. SDS-PAGE
analysis of fractions eluting from the column showed that the oligomeric
protein contained ~50% uncleaved proenzyme. Although heating the
unpurified proenzyme in E. coli lysate promoted cleavage, incubating
the purified protein at 80 °C did not increase the portion of protein
cleaved; nor did incubation with methoxyamine or
O-nitrobenzylhydroxylamine at 80 °C for 20 min. S-Adenosylmethionine Decarboxylase Activity of SSO0585—In a
coupled reaction with AdoMet synthetase, the His 10-SSO0585 protein
catalyzed AdoMet decarboxylation with a net specific activity of 0.11 μmol
min –1 mg –1 at 70 °C, corresponding to a
rate of 2.3 min –1 (). This activity compares favorably with the AdoMet decarboxylase
activity of the native enzyme purified previously from S.
solfataricus (0.012 μmol min –1 mg –1)
( 5). In contrast, AdoMet
decarboxylase activities were no higher than the background level in reactions
containing His 10-SSO0536. AdoMet decomposes through a number of
spontaneous reactions ( 28); we
have not characterized the volatile products of background activity detected
in these assays. Arginine Decarboxylase Activity of SSO0536—The purified
His10-SSO0536 protein catalyzed l-arginine
decarboxylation with a specific activity of 0.5 units/mg at 70 °C.
Agmatine was the only amine produced in reaction mixtures containing purified
protein and l-arginine (data not shown). In standard assays, using
either l-[U-14C]arginine or
l-[1-14C]arginine as substrates for the enzyme,
14CO2 was trapped, confirming the decarboxylation
reaction. This enzyme had maximal activity at pH 6
(), and it was not
stimulated by potassium chloride as observed for other decarboxylases. The
SSO0536 decarboxylase was highly thermostable: after a 10-min incubation at 90
°C, the SSO0536 protein retained 80% of its original ArgDC activity. It
had maximal decarboxylase activity at 80 °C, but remained active above the
practical limits of the assay (). This reaction is first order with respect to arginine, so the
Michaelis-Menten-Henri equation fit a plot of initial rates of decarboxylase
activity with parameters Km of 150 ± 28
μ m and Vmax of 1.1 ± 0.042 units
min –1 mg –1
( Table 2). SDS-PAGE
densitometry indicated that 46% of this protein was cleaved to form an active
pyruvoyl group. Therefore, the enzyme had a turnover number
( kcat) of 2.6 s –1, similar to the
euryarchaeal enzyme. No arginine decarboxylase activity was detected in
purified preparations of His 10-SSO0585 (<0.001 units/mg
activity). | TABLE 2Decarboxylase activities in five classes of ArgDC |
Preincubation of the SSO0536 protein with 1 mm O-methyl
hydroxylamine or O-(4-nitrobenzyl) hydroxylamine reduced activity by
50%, consistent with the pyruvoyl group modifications observed previously. In
contrast, 1 mm phenylhydrazine did not inactivate the enzyme.
Incubation with 1 mm DFMO reduced activity by 20%, but incubation
with 1 mm DFMA at 80 °C reduced activity by 64% over 15
min. The most potent competitive inhibitor tested was l-argininamide,
which almost completely abolished arginine decarboxylase activity at a 1
mm concentration. Incubation with 1 mm l-arginine methyl
ester reduced activity by more than 70%, whereas 1 mm l-canavanine
reduced activity by 46%. The same concentration of l-histidine,
l-homoarginine, and
Nα-acetyl-l-arginine reduced activity by
20–30%. No inhibition was observed with d-arginine,
l-citrulline, l-lysine,
NG-methyl-l-arginine, l-methionine,
NG-nitro-l-arginine methyl ester, or
l-ornithine. The SSO0536 protein also catalyzed the decarboxylation
of l-canavanine with 40% relative activity compared with
l-arginine. No other substrates were identified for this
enzyme. Modeling and Activity of a Chimeric Protein—The crystal
structure of the (αβ) 2 dimer of Thermotoga
maritima AdoMetDC (TmAdoMetDC) showed that this protein is homologous to
human AdoMetDC, despite their low sequence similarity
( 29). The TmAdoMetDC
Ser 63 site of protein cleavage and pyruvoyl group formation is
highly conserved in this protein family, as are nearby active site residues
Ser 55, His 68, and Cys 83
(). From a model of human
AdoMetDC bound to the methyl ester of AdoMet, Phe 49,
Cys 83, and Glu 72 (TmAdoMetDC positions) were predicted
to be important for substrate binding and decarboxylation
( 30). These residues are
conserved in both crenarchaeal AdoMetDC and ArgDC paralogs; therefore, other
factors must determine the proteins' substrate specificities. From an
alignment of crenarchaeal paralogs, we identified two regions where amino acid
substitutions correlated with phylogenetically predicted protein function.
Cys 15 in most crenarchaeal and bacterial AdoMetDC proteins is
replaced by a hydrophobic residue in ArgDC paralogs, and a series of charged
residues is inserted in the loop between β-strands 2 and 3 of the ArgDC
proteins. In the TmAdoMetDC model, Cys 15 is at the opposite end of the
β-sheet from the pyruvoyl group active site of the monomer (22 Å
apart), but in the dimeric model, this position is 16 Å from the
pyruvoyl group of the adjacent protomer, which lies in a pocket at the dimer
interface. This Cys 15 residue could affect the position of
β-strands 4 and 5, including Glu 72, which forms hydrogen bonds
to the AdoMet ribose hydroxyl groups
( 30). Similarly, the loop
region between β-strands 2 and 3 is ~13 Å from the pyruvoyl
group of the adjacent TmAdoMetDC protomer, so residues inserted into this loop
could directly contact the substrate. Because both characteristic regions lie in the proteins' β-subunits,
we constructed chimeric proteins by swapping the subunits. The
His 10-β536α585 protein self-cleaved to form subunits
with apparent molecular masses of 21, 18, 15, and 7 kDa
(), although it had a
reduced cleavage efficiency (32%) compared with His 10-SSO0536
protein. Purified His 10-β536α585 catalyzed arginine
decarboxylation with a Km identical to the wild-type
protein ( Table 2). The
chimera's turnover was more than 100-fold lower, even after accounting for the
reduced cleavage efficiency. The opposite chimera,
His 10-β585α536, self-cleaved with only 5% efficiency to
produce subunits with apparent molecular masses of 18, 16, and 14 kDa. The
purified protein had no detectable arginine decarboxylase activity. No
AdoMetDC activity was detected in either chimeric protein preparation
(), although that assay
has a higher limit of detection than the ArgDC assay. We would not detect
activity if the turnover was 100-fold lower, as observed for the
His 10-β536α585 chimera. Therefore, residues in the
amino-terminal β-subunit of SSO0536 are sufficient to confer arginine
decarboxylase activity on this AdoMetDC scaffold, although additional factors
in the α-subunit are required for efficient cleavage and turnover. Evolution of Arginine Decarboxylase Activity in
Crenarchaea—The phylogeny of the crenarchaeal homologs suggests
that the ArgDC gene evolved from a single duplication of an ancestral AdoMetDC
gene early in the Crenarchaeota
(). Bootstrap analysis
offers low support for most branches, due to the limited information content
of the short protein sequence alignment. Three crenarchaeal genomes lack
orthologs of the SSO0536 gene. Instead, the marine mesophiles Cenarchaeum
symbiosum and Candidatus Nitrosopumilus maritimus as well as the
thermophile Thermophilum pendens have homologs of the euryarchaeal
pyruvoyl-dependent arginine decarboxylase (supplemental Table 1). All three
organisms branch early in the Crenarchaeota, so the common archaeal
ancestor could have had a homolog of the euryarchaeal ArgDC that was lost in
most crenarchaea. Nevertheless, a phylogeny of euryarchaeal type arginine
decarboxylase proteins groups the crenarchaeal homologs with two different
branches of euryarchaea (data not shown). Therefore, we cannot rule out the
acquisition of a euryarchaeal ArgDC gene by horizontal gene transfer to these
microbes. Most organisms have an AdoMetDC that is required to produce the propylamine
donor for long-chain polyamines. The sequences of AdoMetDC homologs have
diverged significantly in the Bacteria, Archaea, and Eukarya. Yet the homologs
adopt similar structures ( 29),
and they share a common origin. Previously, all members of this
pyruvoyl-dependent protein family were presumed to catalyze the
decarboxylation of AdoMet. We have identified the first alternative reaction
catalyzed by this protein scaffold. An AdoMetDC was previously purified from S. solfataricus MT-4, and
it was shown to be a thermostable, pyruvoyl-dependent enzyme with a relatively
low specific activity (0.012 units/mg)
( 5). Although no sequence data
were reported for this enzyme, its native molecular mass (32 kDa) and activity
are similar to those of His 10-SSO0585. The MT-4 protein was not
resolved into its component subunits, and the heterologously expressed protein
migrated anomalously by SDS-PAGE. Therefore, we cannot determine whether the
native protein was fully cleaved. The SSO0585 sequence identifies it as member
of the bacterial and archaeal type AdoMetDC family
()
( 8). Human AdoMetDC co-crystal structures with AdoMet analogs show a limited
number of interactions between the substrate's adenosine moiety and the
protein active site residues
( 30). The Glu 247
γ-carboxylate forms hydrogen bonds with the ribosyl 2′- and
3′-hydroxyl groups of AdoMet. Human Glu 67, at the
COOH-terminal end of the β-subunit, forms hydrogen bonds with the adenine
base. Both residues are conserved in the two S. solfataricus
homologs. Therefore, the specificity determinants in the S.
solfataricus enzymes are ambiguous. ArgDC activity in the
β536α585 chimera suggests that residues in the β-subunit are
sufficient for arginine recognition, although more specific mutagenesis
studies and structural models will be required to identify the new mode of
substrate binding in the SSO0536 protein. DFMA was shown to modify a cysteine thiol in the Chlorella virus
PLP-dependent arginine decarboxylase
( 26). Although the mode of
DFMA inhibition of the SSO0536 protein cannot be determined without additional
experiments, the only cysteine in that protein, Cys 102, is a likely
target for adduct formation near the active site. Similar to E. coli
polyamine-deficient mutants
( 31), DFMA-treated S.
solfataricus had a reduced growth rate in minimal medium and a reduced
putrescine content. 1,3-Diaminopropane can replace putrescine as the core
polyamine for S. solfataricus, producing norspermidine and
norspermine, yet the source of diaminopropane has not been identified. No
diaminopropane was identified in polyamines extracted from these cells. Polyamine-producing organisms have convergently evolved different ways to
produce putrescine. Most eukaryotes and bacteria decarboxylate ornithine to
directly form putrescine, whereas archaea and some bacteria decarboxylate
arginine and hydrolyze agmatine to form putrescine. Although all known
arginine decarboxylases use either a PLP or a pyruvoyl cofactor, ArgDC
activity has evolved at least five times
( Table 2). Three classes of
PLP-dependent ArgDC enzymes share little sequence similarity, and the two
classes of pyruvoyl-dependent ArgDC enzymes are also nonhomologous. The
PLP-dependent ArgDCs have higher specificity constants than their
pyruvoyl-dependent analogs. However, the differences in rate constants among
the ArgDCs are small compared with the difference between catalyzed and
uncatalyzed reaction rates. Amino acid decarboxylases have some of the highest
catalytic rate enhancements observed in single substrate enzymes
( Table 2)
( 32,
33). Class 1 and 2 ArgDC enzymes belong to the
ornithine/arginine/diaminopimelate decarboxylase family of PLP-dependent
enzymes, which were called group IV amino acid decarboxylases by Sandmeier
et al. ( 34). Class 1
ArgDC enzyme are exemplified by the E. coli biosynthetic
PLP-dependent arginine decarboxylase (SpeA)
( 10). The Class 2 enzyme from
Paramecium bursaria chlorella virus evolved from a eukaryotic
ornithine decarboxylase ( 26).
Class 3 ArgDC enzymes belong to the ornithine/lysine/arginine decarboxylase
family that includes the E. coli inducible ArgDC (AdiA), which
functions in an arginine-dependent acid resistance system
( 35). This class is part of
the group III PLP-dependent amino acid decarboxylase family
( 34). Both Class 4 and 5 ArgDCs self-cleave to form pyruvoyl cofactors.
Euryarchaea use Class 4 enzymes for polyamine biosynthesis
( 11), but homologs have been
recruited by Chlamydia spp. and other bacteria to function in a
system analogous to the bacterial arginine-dependent acid resistance system
( 18). These proteins are
homologous to the histidine decarboxylase from Lactobacillus sp. that
also functions in acid resistance
( 36). This class of proteins
forms trimers of cleaved protomers, each with an αββα
sandwich fold. Two adjacent protomers contribute amino acid residues to each
enzyme active site ( 37).
Finally, the Class 5 enzymes described here evolved in the crenarchaea from an
AdoMetDC scaffold. The bacterial and archaeal AdoMetDC proteins are dimers of
two cleaved protomers that form a four-layer αββα
sandwich fold ( 29,
30). Although gel filtration
experiments indicated that the SSO0585 AdoMetDC is a dimer, the SSO0536 ArgDC
appeared to be a tetramer. It is not clear how the quaternary structures of
the proteins affect substrate specificity. Hyperthermophiles contain relatively few PLP-dependent decarboxylases
compared with mesophiles, although they have a full complement of
PLP-dependent aminotransferases. M. jannaschii (optimum growth at 83
°C) has a PLP-dependent tyrosine decarboxylase for methanofuran coenzyme
biosynthesis ( 38) and a
PLP-dependent diaminopimelate decarboxylase for lysine biosynthesis
( 39). Crenarchaea that grow at
even higher temperatures, such as Pyrobaculum aerophilum (100 °C
growth optimum), appear to have no PLP-dependent decarboxylases (they use an
α-aminoadipate pathway for lysine biosynthesis instead of a
diaminopimelate pathway). At high temperatures, the selectivity of these
enzymes for decarboxylation may decrease, leading to inactivating side
reactions ( 40). Further
studies on the temperature dependence of these side reactions are needed to
compare the pyruvoyl and PLP-dependent decarboxylases. We thank Drs. Marvin Hackert, Jon Robertus, Ian Molineux, and Gisela Kramer
for helpful discussions. We are grateful to Dr. Patrick Woster for the
generous gift of DFMO. The acquisition of mass spectra by LC-ESI-MS and the
resulting determinations of the molecular weights of proteins were done by Dr.
Herng-Hsiang Lo in the CRED Analytical Instrumentation Facility Core at the
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