Báo cáo khoa học: A novel prokaryotic L-arginine:glycine amidinotransferase is involved in cylindrospermopsin biosynthesis potx

A novel prokaryotic L-arginine:glycine amidinotransferase
is involved in cylindrospermopsin biosynthesis
Julia Muenchhoff
1
, Khawar S. Siddiqui
1
, Anne Poljak
2,3
, Mark J. Raftery
2
, Kevin D. Barrow
1
and
Brett A. Neilan
1
1 School of Biotechnology and Biomolecular Sciences, University of New South Wales, Sydney, NSW, Australia
2 Bioanalytical Mass Spectrometry Facility, University of New South Wales, Sydney, NSW, Australia
3 School of Medical Sciences, University of New South Wales, Sydney, NSW, Australia
Introduction
Cyanobacterial toxins pose a serious health risk for
humans and animals when they are present at hazard-
ous levels in bodies of water used for drinking or
recreational purposes. Under eutrophic conditions,
cyanobacteria tend to form large blooms, which
drastically promote elevated toxin concentrations. The
Keywords
amidinotransferase; cyanobacterial toxin;
enzyme kinetics; protein stability; toxin
biosynthesis
Correspondence
B. A. Neilan, School of Biotechnology and

Biomolecular Sciences, University of New
South Wales, Sydney, NSW 2052, Australia
Fax: +61 2 9385 1591
Tel: +61 2 9385 3235
E-mail:
(Received 7 June 2010, revised 16 July
2010, accepted 22 July 2010)
doi:10.1111/j.1742-4658.2010.07788.x
We report the first characterization of an l-arginine:glycine amidinotrans-
ferase from a prokaryote. The enzyme, CyrA, is involved in the pathway
for biosynthesis of the polyketide-derived hepatotoxin cylindrospermopsin
from Cylindrospermopsis raciborskii AWT205. CyrA is phylogenetically dis-
tinct from other amidinotransferases, and structural alignment shows dif-
ferences between the active site residues of CyrA and the well-characterized
human l-arginine:glycine amidinotransferase (AGAT). Overexpression of
recombinant CyrA in Escherichia coli enabled biochemical characterization
of the enzyme, and we confirmed the predicted function of CyrA as an
l-arginine:glycine amidinotransferase by
1
H NMR. As compared with
AGAT, CyrA showed narrow substrate specificity when presented with
substrate analogs, and deviated from regular Michaelis–Menten kinetics in
the presence of the non-natural substrate hydroxylamine. Studies of initial
reaction velocities and product inhibition, and identification of intermediate
reaction products, were used to probe the kinetic mechanism of CyrA,
which is best described as a hybrid of ping-pong and sequential mecha-
nisms. Differences in the active site residues of CyrA and AGAT are dis-
cussed in relation to the different properties of both enzymes. The enzyme
had maximum activity and maximum stability at pH 8.5 and 6.5, respec-
tively, and an optimum temperature of 32 °C. Investigations into the stabil-

ity of the enzyme revealed that an inactivated form of this enzyme retained
an appreciable amount of secondary structure elements even on heating to
94 °C, but lost its tertiary structure at low temperature (T
max
of 44.5 °C),
resulting in a state reminiscent of a molten globule. CyrA represents
a novel group of prokaryotic amidinotransferases that utilize arginine and
glycine as substrates with a complex kinetic mechanism and substrate
specificity that differs from that of the eukaryotic l-arginine:glycine
amidinotransferases.
Abbreviations
AGAT, human
L-arginine:glycine amidinotransferase; AmtA, L-arginine:lysine amidinotransferase; ANS, 8-anilino-naphthalene-1-sulfonate; StrB,
L-arginine:inosamine phosphate amidinotransferase; StrB1, Streptomyces griseus L-arginine:inosamine phosphate amidinotransferase.
3844 FEBS Journal 277 (2010) 3844–3860 ª 2010 The Authors Journal compilation ª 2010 FEBS
problem is global, as most toxic cyanobacteria have a
worldwide distribution [1–7]. The major toxin produced
by the genus Cylindrospermopsis is cylindrospermopsin,
which was first discovered after a poisoning incident on
Palm Island (Queensland, Australia) in 1979, when 148
people, mainly children, were hospitalized with hepato-
enteritis caused by contamination of a drinking water
reservoir with Cylindrospermopsis raciborskii [8,9]. Cyl-
indrospermopsin has hepatotoxic, nephrotoxic and gen-
eral cytotoxic effects [10–12], and is a potential
carcinogen [13]. Besides C. raciborskii, five other cyano-
bacterial species have so far been shown to produce the
toxin; they are Aphanizomenon ovalisporum, Umeza-
kia natans, Rhaphdiopsis curvata, Aphanizomenon flos-
aquae and Anabaena bergii [4,14–18].

Cylindrospermopsin is a polyketide-derived alkaloid
with a central guanidino moiety and a hydroxymethyl-
uracil attached to the tricyclic carbon skeleton [19]
(Fig. 1). Putative cylindrospermopsin biosynthesis
genes have been identified in A. ovalisporum [20] and
C. raciborskii [18,21], and this led to the sequencing of
the complete gene cluster (cyr) in an Australian isolate
of C. raciborskii [22]. The cyr gene cluster spans 43 kb
and encodes 15 ORFs. On the basis of bioinformatic
analysis of the gene cluster and isotope-labeled precur-
sor feeding experiments [23], a putative biosynthetic
pathway has been proposed [22]. The first step in this
proposed pathway is the formation of guanidinoace-
tate by the amidinotransferase CyrA. The nonriboso-
mal peptide synthetase ⁄ polyketide synthase hybrid
CyrB, followed by the polyketide synthases CyrC–F,
then catalyze five successive extensions with acetate to
form the carbon backbone of cylindrospermopsin. The
biosynthesis is completed by formation of the uracil
ring (CyrG–H), and tailoring reactions, such as sulfo-
transfer (CyrJ) and hydroxylation (CyrI).
Amidinotransferases catalyze the reversible transfer
of an amidino group from a donor compound to the
amino moiety of an acceptor [24]. To date, l-argi-
nine:glycine amidinotransferases from vertebrates and
plants [25–28], an l-arginine:lysine amidinotransferase
from Pseudomonas syringae [29,30], and the l-argi-
nine:inosamine phosphate amidinotransferase (StrB)
from Streptomyces species [31] have been described.
More recently, another cyanobacterial amidinotransfer-

ase, SxtG, was discovered when the gene cluster for the
biosynthesis of the neurotoxin saxitoxin in C. racibor-
skii T3 was sequenced [32]. Amidinotransferases are a
monophyletic group of enzymes with highly conserved
sequences across distantly related organisms [33]. They
are key enzymes in the synthesis of guanidino com-
pounds, which play an important role in vertebrate
energy metabolism and in secondary metabolite produc-
tion by higher plants and prokaryotes [24,27,30,34]. The
best studied amidinotransferases are l-arginine:inos-
amine phosphate amidinotransferase (EC 2.1.4.2; StrB1)
involved in the biosynthesis of the antibiotic streptomy-
cin in the soil bacterium Streptomyces griseus [31], and
l-arginine:glycine amidinotransferase (EC 2.1.4.1)
involved in creatine biosynthesis in vertebrates [26]. In
cylindrospermopsin biosynthesis, the amidinotransfer-
ase CyrA is thought to catalyze the formation of
guanidinoacetate, which suggests transamidination
from arginine onto glycine in a manner similar to the
vertebrate l-arginine:glycine amidinotransferase. Glycine
and guanidinoacetate were confirmed as precursors in
cylindrospermopsin biosynthesis by isotope-labeled
precursor feeding experiments; however, incorporation
of labeled arginine could not be confirmed, indicating
an amidino group donor other than arginine [23]. On
the other hand, modeling of the active site of the CyrA
homolog AoaA from A. ovalisporum, based on the
crystal structure of AGAT, suggested the involvement
of arginine as a possible substrate [21]. Biochemical
characterization of the enzyme is required to resolve

this contradiction and identify the starting compounds
for toxin production. Characterization of enzymes
from the cylindrospermopsin pathway is also necessary
to confirm the suggested mechanism for toxin produc-
tion, as none of the cylindrospermopsin-producing
organisms identified so far are amenable to genetic
modification. In this article, we describe the cloning,
purification and characterization of a novel amidino-
transferase from C. raciborskii AWT205, in order to
better understand the structure–function–stability rela-
tionship of this enzyme, which is responsible for the
first step in the biosynthesis of a cyanotoxin.
Results
CyrA is phylogenetically distinct from known
amidinotransferases
To investigate the molecular phylogeny of CyrA within
the amidinotransferase subfamily, an alignment of
CyrA with 27 sequences spanning 376 residues was
Fig. 1. Structure of cylindrospermopsin. The guanidino group
derived from guanidinoacetic acid is shown in bold.
J. Muenchhoff et al. A novel cyanobacterial amidinotransferase
FEBS Journal 277 (2010) 3844–3860 ª 2010 The Authors Journal compilation ª 2010 FEBS 3845
constructed. These sequences included representative
proteins of the amidinotransferase subfamily, as
well as uncharacterized genes annotated as ‘amidino-
transferase’ from genome sequencing projects. A
phylogenetic tree was constructed from the alignment
(Fig. 2). The amidinotransferases fell into three
major groups (groups 1–3) that were supported by
high bootstrap values. Group 3 comprised StrB

proteins from the prokaryote Streptomyces; these were
only distantly related to other amidinotransferases.
Group 2 encompassed two distinct subgroups. CyrA
and the homolog AoaA from the cylindrospermopsin
producer A. ovalisporum formed subgroup V. Sub-
group IV in group 2 consisted of several experimen-
tally uncharacterized (hypothetical) prokaryotic
amidinotransferases that have been annotated as
‘glycine amidinotransferase’ (Fig. 2). CyrA is the first
member of the phylogenetic group 2 amidinotransfe-
rases to be described experimentally.
Group 1 consisted of the eukaryotic l-arginine:gly-
cine amidinotransferase in subgroup I and two prokary-
otic enzymes in subgroup II. Subgroup III comprises
the cyanobacterial amidinotransferases (SxtG) puta-
tively involved in the biosynthesis of saxitoxin [32],
together with one uncharacterized amidinotransferase
from Beggiatoa.
Sequence analysis of CyrA reveals two active site
substitutions
A structural alignment of CyrA and StrB1 with the
well-characterized AGAT (Fig. S1) revealed that
Asp254 and His303 (numbered according to the
human protein), constituting part of the catalytic triad
in the human and Streptomyces enzymes, are con-
served in CyrA. The same applies to the active site
Cys407, which was shown to form a covalent ami-
dino–enzyme intermediate with the substrate’s amidino
Fig. 2. Phylogenetic tree of amidinotransferases. The phylogenetic tree encompasses 27 amidinotransferases, comprising both characterized
(bold) and uncharacterized enzymes. Accession numbers are given in parentheses next to the species name. Arabic numerals denote

groups, and roman numerals denote subgroups.
A novel cyanobacterial amidinotransferase J. Muenchhoff et al.
3846 FEBS Journal 277 (2010) 3844–3860 ª 2010 The Authors Journal compilation ª 2010 FEBS
group. However, Met302, involved in arginine binding
in AGAT, has been replaced by Ser247 in CyrA. A
similar substitution has been reported in the ortholog
AoaA from A. ovalisporum [21]. Furthermore, Asn300,
which contributes to the active site structure in AGAT,
is replaced by Phe245 in CyrA.
Physicochemical properties of CyrA
The native cyrA gene is 1176 bp long and codes for a
protein of 391 residues with a calculated molecular
mass of 45.68 kDa and a theoretical pI of 5.1. Recom-
binant CyrA includes the N-terminal His
6
-fusion tag
and 22 additional C-terminal vector-encoded amino
acids, which increase the calculated molecular mass to
50.12 kDa and the pI to 5.6.
Yields of purified recombinant protein varied from
10.5 to 18.5 mg per liter of culture. After purification by
immobilized metal ion affinity chromatography, recom-
binant CyrA was judged to be of >95% purity by
SDS ⁄ PAGE (Fig. S2), and had the expected molecular
mass of 50 kDa, as indicated by SDS⁄ PAGE (Fig. S2),
MALDI-TOF MS and LC-MS (Fig. S3). The presence
of the His
6
-fusion tag and the identity of the purified
protein as CyrA were confirmed by western blotting,

MS intact mass analysis and peptide mass fingerprinting
after enzymatic digestion (Table S1). The tryptic pep-
tides covered 69% of the amino acid sequence of recom-
binant CyrA, including the N-terminal and C-terminal
peptides, showing that the protein was expressed in its
complete, nontruncated form.
Purified CyrA eluted from the size exclusion chroma-
tography column in two peaks corresponding to molec-
ular masses of 185 and 98 kDa (Fig. S2). SDS ⁄ PAGE
analysis combined with activity assays confirmed that
both peaks consist exclusively of CyrA. This indicated
that CyrA is present in two forms, dimer and tetramer.
Size exclusion chromatography was repeated four times
with similar results, implying that the equilibrium
between dimeric and tetrameric forms of CyrA is stable
and reproducible under these conditions.
Amidinotransferase activity was found to be linear
over a time period of 60 min in the presence of 20 mm
l-arginine and 20 mm glycine, as well as a linear func-
tion of enzyme concentration. The plot of amidino-
transferase activity at various pH values is bell-shaped
(Fig. S4A). The highest activity of CyrA was detected
at pH 8.5. At pH 7, only 25% of the original activity
remained. For l-arginine:glycine amidinotransferases
from pig, rat and soybean, pH optima of 7.5, 7.4 and
9.5, respectively, have been reported [25,35,36]. The
optimum temperature (T
opt
) for CyrA was found to be
32 °CatpH8.At40°C, 80% of the activity relative

to T
opt
was lost (Fig. S4B). The T
opt
for soybean ami-
dinotransferase was determined to be 37 °C [25].
Analysis of end-products confirmed CyrA as an
L-arginine:glycine amidinotransferase
Isotope-labeled precursor feeding experiments con-
firmed glycine and guanidinoacetate as precursors for
cylindrospermopsin biosynthesis, but could not confirm
incorporation of ubiquitously labeled arginine into cyl-
indrospermopsin [23]. However, the transamidination
of glycine from arginine by amidinotransferase, yield-
ing guanidinoacetate, is common in vertebrates, and it
3.5 3.0 2.5 2.0 1.5 1.0
Fig. 3.
1
H-NMR spectrum of substrates and
products formed by CyrA at 600 MHz in 5%
D
2
O. The x-axis corresponds to parts per
million.
J. Muenchhoff et al. A novel cyanobacterial amidinotransferase
FEBS Journal 277 (2010) 3844–3860 ª 2010 The Authors Journal compilation ª 2010 FEBS 3847
was proposed that CyrA catalyzes the same reaction.
All characterized amidinotransferases use arginine as
the natural amidino group donor. In order to prove
that the reaction catalyzed by CyrA converted l-argi-

nine and glycine to ornithine and guanidinoacetate,
1
H-NMR spectroscopy was used. Initially, several
attempts were made to follow the reaction progress by
NMR spectroscopy; however, the buffer component
dithiothreitol and its oxidized form obscured key reso-
nances. Therefore, the basic products (and reactants)
were isolated by anion exchange chromatography prior
to
1
H-NMR. The presence of ornithine and guani-
dinoacetate was confirmed by the appearances of reso-
nances for the a and d protons of ornithine at 3.52
and 3.02 p.p.m., respectively, and the sharp single
resonance for guanidinoacetate at 3.8 p.p.m. (Fig. 3).
These assignments were confirmed by
1
H–
13
C correla-
tion spectroscopy and
1
H–
1
H COSY spectra (data not
shown).
CyrA has narrow substrate specificity
Apart from glycine and arginine, several structurally
related compounds were tested for their ability to serve
as substrates for CyrA. l-Homoarginine, agmatine,

l-canavanine, guanidine hydrochloride, urea, c-guanid-
inobutyric acid and b-guanidinoproprionic acid were
tested as amidino group donors. l-Alanine, b-alanine,
c-aminobutyric acid, ethanolamine, taurine, l-lysine,
a-amino-oxyacetic acid and l-norvaline were used as
amidino group acceptors. The limit of detection for
the assays was 0.5 mm hydroxyguanidine and 25 lm
l-ornithine. Only incubation with hydroxylamine
resulted in the detection of product. Therefore, it was
concluded that CyrA only recognizes hydroxylamine as
an amidino group acceptor. No other compound
was an alternative substrate under these reaction
conditions.
Kinetic analyses with natural substrates suggest
a reaction mechanism different from that of other
amidinotransferases
The formation of guanidinoacetate and ornithine from
arginine and glycine obeyed regular Michaelis–Menten
kinetics. Nonlinear regression analysis revealed kinetic
constants as summarized in Table 1.
In double-reciprocal plots with arginine as the varied
substrate, the family of lines intersect to the left of the
y-axis, below the x-axis (Fig. 4). This kinetic pattern is
indicative of a random sequential mechanism, in which
both substrates bind to the enzyme in a random order
to form a compulsory ternary complex before the first
product is released. The intercept below the origin sug-
gests that binding of one ligand reduces the affinity for
the other ligand [37].
Kinetic analyses with a non-natural acceptor

reveal a complex kinetic mechanism
Initial reaction velocities for the formation of hydroxy-
guanidine and ornithine from hydroxylamine and
arginine were measured over a wide range of hydroxyl-
amine concentrations with a fixed concentration of
arginine. The substrate versus velocity plot of these
data revealed interesting features of the enzyme. First,
the plot curves downwards (Fig. S5), suggesting sub-
strate inhibition at high concentrations of hydroxyl-
amine. Second, the plot is not a rectangular hyperbola
but is sigmoidal, indicating allosteric behavior in the
presence of hydroxylamine. The Hill constant (n) of 1.6
indicated positive cooperativity, with hydroxylamine
binding to at least one peripheral site in addition to the
active site. The theoretical maximum Hill constant for
positive cooperativity is equal to the oligomeric state of
the enzyme [37], i.e. either 2 or 4 for CyrA, which is an
equilibrium of dimer and tetramer. Therefore, the Hill
constant of 1.6 indicated a considerable to moderate
cooperative effect of hydroxylamine.
Table 1. Kinetic constants of CyrA.
CyrA AGAT
a
V
max
(lmolÆmin
)1
Æmg
)1
) 1.05 ± 0.05 0.44

k
cat
(min
)1
per active site) 52.5 ± 2.5 20
K
arginine
m
(mM) 3.5 ± 1.14 2.0 ± 0.5
K
glycine
m
(mM) 6.9 ± 2.70 3.0 ± 1.0
a
The values for human L-arginine:glycine amidinotransferase are
given for comparison [55].
Fig. 4. Double reciprocal plot of initial velocity data with arginine as
the variable substrate. The glycine concentrations were 3 m
M (·),
6m
M (+), 9 mM (s), 12 mM (D), 16 mM ( ) and 20 mM ()).
A novel cyanobacterial amidinotransferase J. Muenchhoff et al.
3848 FEBS Journal 277 (2010) 3844–3860 ª 2010 The Authors Journal compilation ª 2010 FEBS
Product inhibition also suggests a random
sequential mechanism
A product inhibition study was conducted to further
diagnose and confirm the kinetic mechanism of
CyrA. Vertebrate l-arginine:glycine amidinotransferase
display strong product inhibition by ornithine, with a
K

i
of 0.25 mm [38]; hence, it was speculated that CyrA
might also be subject to inhibition by ornithine.
Unfortunately, measurement of initial velocities in the
presence of ornithine is not possible with the assay
method of Van Pilsum et al. [39], which measures the
formation of ornithine. Therefore, we measured initial
reaction velocities at a saturating level of arginine and
with varying noninhibitory concentrations of the non-
natural acceptor hydroxylamine, in the presence of sev-
eral fixed concentrations of ornithine, using the
method of Walker [40]. On a double-reciprocal plot of
the data, the lines intercept in the upper right quadrant
of the plot (Fig. 5). Such a kinetic pattern is character-
istic of partial mixed inhibition [37]. Ornithine there-
fore binds to the active site of CyrA at a binding site
distinct from the hydroxylamine-binding site. This
binding affects the rate of reaction by factor b, causing
the noncompetitive component of the mixed inhibition.
In addition, binding of ornithine to this distinct site
also alters the affinity for hydroxylamine by factor a.
This is most likely attributable to structural changes of
CyrA induced by the binding of ornithine. The loca-
tion of the common intercept in mixed-type inhibition
systems depends on the actual and relative values of a
and b. An intercept in the upper right-hand quadrant
of the double-reciprocal plot, as is the case here
(Fig. 5), indicates that b >> a [37].
The product inhibition study revealed another detail
of this highly dynamic protein. The presence of

ornithine not only has an inhibitory effect but also
affects the affinity constant of hydroxylamine, modify-
ing the allosteric behavior. The Hill constants for the
individual series of velocity measurements in the pres-
ence of different ornithine concentrations ranged from
1.6 in the absence of ornithine to 2.1 and 2 in the pres-
ence of 3 and 6 mm ornithine, respectively (Fig. S6).
Analysis of reaction products with only the
amidino group donor
In order to differentiate between a random sequential
mechanism (both arginine and glycine must bind
before ornithine is released) and a possible ping-pong
mechanism (formation of an enzyme–amidino interme-
diate and release of ornithine in the absence of
glycine), product formation by CyrA was investigated
in the presence of arginine only.
CyrA incubated with arginine was subjected to MS
and compared with CyrA that was not exposed to
arginine in order to detect a possible enzyme interme-
diate by its difference in mass resulting from the
bound amidino group (Fig. S3). CyrA samples were
also digested with trypsin, endo-AspN or endo-LysC,
and subjected to MALDI-TOF MS and LC-MS ⁄ MS
(quadrupole time-of-flight) in order to identify the pep-
tide fragment covalently linked to the amidino group
(Table S1). However, an enzyme–amidino intermediate
could not be detected.
GC-MS was employed to detect the reaction product
ornithine in enzyme preparations that were incubated
with arginine only. Ornithine was formed in the pres-

ence of only a single substrate, arginine, and its pro-
duction therefore does not require the presence of the
second substrate, glycine (Fig. S7). Incubation of
11 nmol of CyrA with 20 mm arginine produced only
Fig. 5. Double reciprocal plot for product
inhibition. Enzyme activity was determined
at a fixed saturating concentration of argi-
nine (50 m
M) with various concentrations of
hydroxylamine (20–150 m
M) in the presence
of several concentrations of ornithine. The
concentrations of ornithine were 0 m
M ( ),
1m
M (D), 3 mM (s), 6 mM (+) and 15 mM
(·). Inset: reaction scheme for the formation
of ornithine and hydroxyguanidine from
L-arginine and hydroxylamine as catalyzed
by CyrA.
J. Muenchhoff et al. A novel cyanobacterial amidinotransferase
FEBS Journal 277 (2010) 3844–3860 ª 2010 The Authors Journal compilation ª 2010 FEBS 3849
81 nmol of ornithine in 1 h, which is equivalent to the
slow rate of 0.0024 lmolÆmin
)1
Æmg
)1
CyrA is a thermolabile molten globule
Recombinant CyrA could be stored at – 80 °C in the
presence of 20% glycerol for at least 2 months without

significant loss of activity (>95% activity remaining).
In contrast, total loss of activity occurred within 48 h
when the enzyme was stored at 4 °C, despite the
addition of reducing agents such as dithiothreitol,
Tris(2-carboxyethyl) phosphine or b-mercaptoethanol.
Similar observations were made for recombinant
AGAT by Fritsche et al. [41]. This prompted us to
investigate the stability of CyrA in detail with the use
of far-UV CD and fluorescence spectrophotometry to
monitor the unfolding of secondary and tertiary struc-
tures, respectively. We compared fresh, active prepara-
tions of CyrA with samples that were inactive after
storage at 4 °C for 2–4 days. We monitored the integ-
rity of a -helical elements of active and inactive CyrA
at 222 nm as a function of temperature, using far-UV
CD (Fig. 6). For both active and inactive CyrA, an
appreciable degree of secondary structure was still
present after exposure to 94 °C, although active CyrA
had greater preservation of secondary structure than
inactive CyrA at all temperatures (Fig. 6A). There was
a transition from higher to lower secondary structure
for the active CyrA between 30 and 50 °C; however,
the remaining structure was stable up to 94 °C. On the
other hand, inactive CyrA did not show any transition,
and seems to exist in a stable secondary structure con-
formation that is not affected at all by the increase in
temperature. To confirm that the high ellipticity
observed here represented a-helical elements that are
stable at high temperatures, far-UV spectra of active
and inactive CyrA were recorded in the presence and

absence of urea as a denaturant. The addition of urea
caused complete loss of ellipticity, confirming that the
ellipticity was a result of secondary structure elements
(Fig. 6B). Furthermore, the far-UV spectra for active
and inactive CyrA were deconvoluted for the determi-
nation of relative amounts of a-helix and b-sheet. This
revealed a shift of a-helical elements to b-strands upon
formation of the inactive molten globule state, with a
decrease in a-helix content from 19.9% to 11.4% and
a concomitant increase in b-sheets from 27.5% to
34.2%.
As a significant degree of the secondary structure
was retained at high temperatures, the unfolding of
tertiary structure was investigated as the cause of the
loss of observed enzyme activity. 8-Anilino-naphtha-
lene-1-sulfonate (ANS) is a large hydrophobic
molecule that is commonly used as a fluorescent probe
of the hydrophobic surface exposed to solvent. The
peak intensity of ANS fluorescence corresponds to the
hydrophobic residues of a protein being maximally
exposed, and the temperature at which this occurs is
referred to as T
max
. The fluorescence melting curves of
0.1 mgÆmL
)1
active and inactive CyrA in the presence
of 25 lm ANS as a function of temperature are shown
in Fig. 7. ANS fluorescence in the presence of active
CyrA showed a low intensity between 4 and 20 °C.

This indicated a well-defined tertiary structure at low
temperatures. The active CyrA also showed a sharp
peak in intensity, with T
max
at 44.5 °C. Therefore, the
tertiary structure loses integrity when the temperature
is increased, leading to maximal exposure of the pro-
tein’s hydrophobic residues at  44 °C. In contrast,
A
B
Fig. 6. Comparison of secondary structure in active and inactive
CyrA by CD. (A) Mean residue ellipticity at 222 nm as a function of
temperature. (B) Far-UV spectra in the presence and absence of
urea.
A novel cyanobacterial amidinotransferase J. Muenchhoff et al.
3850 FEBS Journal 277 (2010) 3844–3860 ª 2010 The Authors Journal compilation ª 2010 FEBS
ANS fluorescence of inactive CyrA showed high inten-
sity around 4 °C and T
max
at  10 °C, indicating
exposure of hydrophobic residues to the solvent at
these low temperatures. This demonstrates that inac-
tive CyrA lacks a well-defined tertiary structure at any
temperature.
From the experiments described above, it was clear
that the loss of tertiary structure of CyrA stored at
4 °C is responsible for the loss of activity. CyrA rap-
idly loses its native tertiary structure when stored at
4 °C or when exposed to relatively mild temperatures
(> 35 °C), with a concomitant retention of a-helical

secondary structural elements. This state of the pro-
tein, when the tertiary structure has unfolded but the
secondary structure remains intact, is reminiscent of a
molten globule [42].
CyrA has optimum stability around neutral pH, in
contrast to its alkaline pH activity optimum
In order to minimize loss of activity of CyrA during
storage at 4 °C, we decided to investigate the stability
of CyrA at different pH values and in the presence of
NaCl, to identify conditions that would stabilize the
enzyme. The stability of CyrA under these conditions
was assessed by monitoring the unfolding of tertiary
structure with ANS fluorescence. Fresh, active CyrA
was exchanged into various buffers at 4 ° C, and the
T
max
was determined. ANS fluorescence of CyrA at
pH 6.5, 7, 7.5 and 8.5 revealed defined peaks, with
T
max
corresponding to 58, 54.5, 54 and 44.5 °C, respec-
tively (Fig. 8). There was a clear trend towards increas-
ing stability with a decrease in pH, with maximum
stability around pH 6.5. At pH 6, a defined peak in
fluorescence intensity was lacking, with maximum
intensity around 4 °C indicating that the protein had
already lost an appreciable amount of tertiary struc-
ture (data not shown). In contrast to stability, the
activity of CyrA was found to be optimal at pH 8.5
(Fig. S4A). At the stability optimum (pH 6.5), CyrA

retained only 10% of its activity as compared with
pH 8.5. Therefore, the pH optimum for activity is not
related to the stability optimum for this protein.
In the presence of 500 mm NaCl, T
max
at pH 7.5
decreased from 54 to 49 °C (Fig. S8), signifying a loss
of stability at high ionic strength. This is an important
consideration during purification procedures, as immo-
bilized metal ion affinity chromatography buffers com-
monly have high ionic strength to minimize nonspecific
interactions with the resin. We improved the purifica-
tion and storage conditions of CyrA by reducing the
NaCl concentration and lowering the pH of buffers to
7 when possible. Hence, knowledge of protein stability
afforded optimization of protein purification and
handling.
Discussion
Cyanobacterial amidinotransferases play an important
role in the biosynthesis of cyanotoxins such as
Fig. 8. Fluorescence of ANS in the presence of active CyrA at vari-
able pH values and as a function of temperature. Fluorescence was
recorded in 50 m
M Mes (pH 6.5, dashed line), 50 mM Tris ⁄ HCl
(pH 7, thin line), 50 m
M Hepes (pH 7.5, intermediate line) and
50 m
M Tris ⁄ HCl (pH 8.5, thick line).
Fig. 7. Temperature-induced unfolding of active and inactive CyrA
as observed by ANS fluorescence spectrophotometry. Fluores-

cence in the presence of active (j) and inactive (h) CyrA as a func-
tion of temperature. Fluorescence was measured in 50 m
M
Tris ⁄ HCl (pH 8.5).
J. Muenchhoff et al. A novel cyanobacterial amidinotransferase
FEBS Journal 277 (2010) 3844–3860 ª 2010 The Authors Journal compilation ª 2010 FEBS 3851
cylindrospermopsin and saxitoxin; however, to date no
amidinotransferase from cyanobacteria has been char-
acterized. The data presented here indicate that the
amidinotransferase from C. raciborskii AWT205 differs
markedly from other known amidinotransferases with
respect to its phylogeny, substrate specificity and
kinetic mechanism. In addition, CyrA was found to be
quite thermolabile, and existed in an intermediate state
(molten globule) between its fully folded and unfolded
states.
The phylogenetic analysis of CyrA showed that
amidinotransferases fall into three different groups.
Group 1 encompasses proteins from two different
domains of life: eukaryotic enzymes involved in
primary metabolism (subgroup I), and prokaryotic
amidinotransferases involved in secondary metabolism
(subgroups II and III). Surprisingly, the prokaryotic
enzymes in groups 1 and 2 are more closely related to
the eukaryotic l-arginine:glycine amidinotransferase
(group 1) than to StrB from Streptomyces species
(group 3). This close relationship between vertebrate
l-arginine:glycine amidinotransferase and prokaryotic
amidinotransferases is also illustrated by the fact that
AGAT is regulated by end-product inhibition, a

feature that is unusual in eukaryotic enzymes but com-
mon in prokaryotic enzymes [43,44].
Two uncharacterized proteins are present in group 1.
The hypothetical protein from the enterobacterium
Photorhabdus luminescens is closely related to l-argi-
nine:lysine amidinotransferase (AmtA). Therefore, this
enzyme might utilize an amidino group acceptor other
than glycine, possibly lysine or a similar compound.
An uncharacterized protein from Beggiatoa is anno-
tated as AoaA in GenBank, but is more closely related
to the SxtG amidinotransferase than to CyrA. Conse-
quently, it seems unlikely that this enzyme represents a
bacterial l-arginine:glycine amidinotransferase such as
AoaA ⁄ CyrA.
CyrA clusters with group 2. The other amid-
inotransferases in this group are experimentally un-
characterized, but like CyrA and all other prokaryotic
amidinotransferases discovered so far, these enzymes
could also participate in secondary metabolite biosyn-
thesis. Their participation in the primary metabolism
(catabolic pathways) of arginine as a nitrogen, carbon
or energy source seems unlikely, as the major enzymes
utilized for arginine degradation in prokaryotes are
arginase, arginine deiminase, arginine succinyltransfer-
ase and arginine oxidase [45,46]. The substrate specific-
ity of CyrA could not be predicted from its phylogeny.
The vertebrate l-arginine:glycine amidinotransferase
(group 1) are not closely related to CyrA, despite their
identical substrate specificity in vivo. This might reflect
the difference in substrate use in vitro by CyrA and

AGAT, with the stringent substrate specificity of CyrA
being in stark contrast to the promiscuous behavior of
AGAT. Furthermore, CyrA and SxtG are also phylo-
genetically distant, although both are involved in sec-
ondary metabolite biosynthesis in closely related or
even the same species of cyanobacteria. Instead, SxtG
is more closely related to AmtA. SxtG presumably uti-
lizes an intermediate in saxitoxin biosynthesis as an
amidino group acceptor. This compound (4-amino-3-
oxo-guanidinoheptane) is structurally more similar to
lysine, the substrate for AmtA.
As bioinformatic analysis yielded no relevant clues
regarding the function of CyrA, we set out to bio-
chemically characterize this enzyme. Arginase activity
of overexpressed, purified CyrA was detected spectro-
photometrically by following the formation of orni-
thine upon incubation with l-arginine and glycine.
Although this indicated the utilization of l-arginine as
a substrate by CyrA, as hypothesized, the question
remained as to whether guanidinoacetate was a prod-
uct of this reaction. Therefore,
1
H-NMR analysis was
carried out, and unambiguously identified the products
of the reaction catalyzed by CyrA. This confirmed
CyrA as the first prokaryotic l-arginine:glycine
amidinotransferase to be described, and identified
l-arginine and glycine as the starting units for cylin-
drospermopsin biosynthesis. Incorporation of the gua-
nidino group of l-arginine could not be demonstrated

in previous isotope-labeled precursor feeding experi-
ments [23]. This may be because not all cyanobacteria
possess basic amino acid transporters [21,47].
CyrA shows allosteric behavior in the presence of
hydroxylamine, resulting in positive cooperativity.
Therefore, hydroxylamine might bind to a peripheral
site on the enzyme, inducing a conformational change
that causes activation by either increasing the affinity
for the substrate or enhancing catalytic performance.
Alternatively, the positive cooperativity could also be
caused by the presence of multiple hydroxylamine mole-
cules in the active site or by the oligomeric state of
CyrA, e.g. because of differences in the K
m
values of
the dimeric and tetrameric forms or cooperative binding
of substrate to a neighboring active site. However, when
the hydroxylamine concentration was increased, sub-
strate inhibition was observed. This inhibition could
either be kinetic (hydroxylamine binding to the wrong
form of the enzyme) or allosteric (hydroxylamine bind-
ing to another peripheral site, which produces a confor-
mational change that decreases activity). This allosteric
site would have a lower affinity for hydroxylamine than
the activating peripheral site, because it is occupied only
at higher substrate concentrations.
A novel cyanobacterial amidinotransferase J. Muenchhoff et al.
3852 FEBS Journal 277 (2010) 3844–3860 ª 2010 The Authors Journal compilation ª 2010 FEBS
Considering the high hydroxylamine concentrations
tested, it is not surprising that allosteric and inhibitory

effects were observed. Hydroxylamine was found to be
a poor substrate for CyrA; activity was only detectable
at concentrations above 20 mm. Substrate inhibition
occurred at concentrations higher than 150 mm.At
such high concentrations, a small polar molecule such
as hydroxylamine would be expected to bind to addi-
tional sites on the protein.
It must be noted that substrate inhibition or alloste-
ric effects were not observed with the natural
substrates l-arginine and glycine at concentrations that
exceed those in vivo (20 mm). Nevertheless, CyrA has
characteristics of allosteric enzymes, such as a dynamic
quaternary structure and ligand-induced conforma-
tional changes. Also, the flux of metabolites through
biosynthetic pathways is often regulated at the
first committed step in the pathway, which, for
cylindrospermopsin biosynthesis, is catalyzed by
CyrA. Ornithine inhibits CyrA and also affects its
allosteric behavior in the presence of hydroxylamine.
Therefore, it is possible that the activity of CyrA
could be regulated in vivo by ornithine product
inhibition.
The kinetic constants for CyrA were found to be
similar to those of AGAT (Table 1). Similarly, the K
m
values for other mammalian and plant l-arginine;
glycine amidinotransferase range from 1.8 to 9.21 mm
for l-arginine and from 0.89 to 18 mm for glycine.
Hence, the prokaryotic and eukaryotic l-arginine:gly-
cine amidinotransferases have similar performances.

However, the kinetic mechanism of CyrA in the pres-
ence of l-arginine and glycine as substrates differs
from the well-established ping-pong mechanism of
l-arginine:glycine amidinotransferase, as shown for the
porcine [35] and human [41] enzymes. Initial velocity
studies indicated a random sequential mechanism, and
the noncompetitive inhibition of ornithine with respect
to hydroxylamine confirmed this. Furthermore, the
initial velocity study suggested that binding of one
substrate reduces the affinity for the other. Similarly,
the product inhibition study implied that binding of
ornithine causes conformational changes that affect
the binding of hydroxylamine, and therefore confirms
the proposal that binding of one substrate ⁄ product
affects binding of the other. Such ligand-induced,
structural changes have been described for AGAT in
the form of a ‘lid’ structure that opens and closes,
regulating access to the active site. Binding of the
large s ubstrate ⁄ product (l-arginine ⁄ ornithine) to AGAT
induces the open conformation of the lid, whereas
binding of the small substrate ⁄ product (glycine ⁄ guani-
dinoacetate) induces the closed conformation [48].
In the classical random sequential mechanism, reac-
tion products are not formed in the presence of only one
substrate; however, here, the reaction product ornithine
was formed in the presence of only one substrate, l-argi-
nine, albeit in very low amounts and without the
detection of an enzyme–amidino intermediate. Two
explanations can reconcile these contradictory results.
Water could act as the second substrate instead of gly-

cine to accept the amidino group and produce ornithine.
Although water is a weak nucleophile and CyrA has
extremely stringent substrate specificity, this possibility
cannot be excluded completely if one considers the high
concentration of water (55 m), which will cause the reac-
tion equilibrium to shift towards the formation of orni-
thine. Alternatively, an enzyme intermediate might have
formed, as in a ping-pong mechanism, but be unstable,
so that it decays to free enzyme and urea. For example,
AGAT forms a covalent enzyme–amidino intermediate
that is only stable at low pH [49,50]. Instability of the
intermediate would make detection very difficult. The
formation of product, ornithine, in the presence of only
one substrate suggests that the reaction mechanism of
CyrA is neither a classical sequential nor a ping-pong
mechanism, but a hybrid of these two systems, in
which an enzyme intermediate may be formed, but is
not compulsory.
Many examples of enzymes that do not fall into the
strict classification of sequential or ping-pong mecha-
nisms, but lie somewhere in between these two systems,
have been reported [51–57]. These studies show that it
can be misleading to diagnose a kinetic mechanism on
the basis of only initial velocity patterns, and recom-
mend including additional experiments to confirm the
mechanism. A hybrid ping-pong–random sequential
mechanism fits all the data in this study, and helps to
explain other features of CyrA, including its stringent
substrate specificity. In such a mechanism, both sub-
strates can bind to the enzyme simultaneously, but a

partial reaction can still occur via formation of an
enzyme intermediate. Therefore, the ternary complex of
enzyme and both substrates is able to form but is not a
requirement, as the reaction can also proceed as two
partial reactions; hence the formation of product in the
presence of only one substrate. Depending on condi-
tions such as substrate concentration, the system will
behave either like a rapid equilibrium random system
or like a rapid equilibrium ping-pong system. There-
fore, the system might appear as either a sequential or a
ping-pong mechanism in initial velocity studies [37].
A hybrid ping-pong–random sequential mechanism
also helps to explain the observed stringent substrate
specificity of CyrA, because it postulates that there are
distinct binding sites for each substrate. If the amidino
J. Muenchhoff et al. A novel cyanobacterial amidinotransferase
FEBS Journal 277 (2010) 3844–3860 ª 2010 The Authors Journal compilation ª 2010 FEBS 3853
group acceptor binds at the same site as the first sub-
strate ⁄ product (l-arginine ⁄ ornithine), CyrA should be
able to accept other compounds smaller than ornithine
as amidino group acceptors. This is the case in
l-arginine:glycine amidinotransferase which only have
one substrate-binding site, giving rise to the classical
ping-pong mechanism and acceptance of a wide range
of substrates besides their physiological substrates
[36,58]. However, CyrA does not accept l-alanine,
b-alanine or ethanolamine as substrate. Even the small-
est possible structures, such as hydroxylamine, are poor
substrates for this enzyme. We therefore suggest that
CyrA possesses a separate, specific binding site for the

amidino group acceptor glycine. The most obvious dif-
ference between glycine, hydroxylamine and ethanol-
amine at physiologically relevant pH is the negative
charge of glycine’s carboxyl group. It is likely that bind-
ing of glycine in CyrA’s active site is enhanced through
ionic interaction with a charged residue of the enzyme.
To support this kinetic model, the level of conserva-
tion of residues involved in substrate binding and cataly-
sis between AGAT and CyrA was investigated by
structural alignment. The amino acids constituting the
catalytic triad as identified in the crystal structure of
AGAT (Asp254, His303 and Cys407) are conserved in
CyrA (Asp197, His248 and Cys356). However, other
amino acids located in the active site are substituted in
CyrA, namely Asn300 and Met302 (AGAT numbering),
which are replaced by Phe245 and Ser247 in CyrA. In
particular, the replacement of a polar, hydrophilic
amino acid (Asn300) with a large, nonpolar hydropho-
bic amino acid (Phe245) might explain the inability of
CyrA to accept larger substrates. In StrB1, the Asn300
and Asn302 are replaced by the smaller amino acids ala-
nine and threonine. This results in a much larger active
site than in AGAT, and allows for binding of inosamine
phosphate, the substrate of StrB1 [31].
CyrA was observed to be unstable at 4 °C. The low
values for T
max
and the activity optimum are character-
istic of cold-adapted, psychrophilic enzymes [59], in con-
trast to the host organism, which prefers growth at

mesophilic temperatures of 25–28 °C [60]. Investigations
into the unfolding of the secondary and tertiary struc-
tures of the enzyme revealed a rapid loss of tertiary
structure, whereas a substantial part of secondary struc-
ture persists in a native-like state upon storage at a tem-
perature of 4 °C or higher. The inactive CyrA shows
higher ANS fluorescence than the fully folded active
CyrA. This is characteristic of the molten globule state,
a protein folding intermediate that has less compact ter-
tiary structure but substantial secondary structure
[61,62]. Besides being an intermediate in protein folding,
the molten globule state, with its increased structural
flexibility, can also have biological functions. There are
examples of molten globules that have an important role
in the insertion of proteins into membranes and in bind-
ing of DNA or receptor molecules [63–65]. We cannot
state whether CyrA exists as a molten globule state
in vivo and has a particular function, or whether the
molten globule is only observed as an in vitro folding
intermediate. CyrA in the molten globule state is cata-
lytically inactive, and CyrA is also not likely to bind to
membranes or participate in molecular recognition.
In summary, CyrA represents a novel group of pro-
karyotic amidinotransferases that utilize arginine and
glycine as native substrates, similarly to the vertebrate
group of l-arginine:glycine amidinotransferases. The
complex kinetic mechanism and substrate specificity of
CyrA differ from those of the eukaryotic enzyme, and
specific amino acid changes were identified that are
likely to cause these differences. Further experiments

to probe the mechanism and substrate binding of
CyrA are underway, as are attempts to crystallize this
enzyme for X-ray studies.
Experimental procedures
Computational analysis
The amino acid sequence of native and recombinant CyrA
(UniProt ID B0LI36) was submitted to the online tool
protparam [66], accessed via the expasy proteomics server
( for calculation
of the molecular mass, theoretical pI and molar extinction
coefficient.
For phylogenetic analysis, amino acid sequences were
aligned using the multiple sequence alignment tool
clustalx 1.8 [67]. The alignment was manually edited and
submitted to the multiphyl webserver modelgenerator
for substitution model selection [68,69]. Phylogenetic trees
were constructed according to the maximum likelihood
approach, using the LG+I+G+F substitution model [70]
on the phyml webserver [71], with a bootstrap number of
100. The consensus tree was reproduced using the freeware
figtree ( Refer-
ence sequences were obtained from the Entrez Protein data-
base, BRENDA and via psi-blast searches.
In addition, a structural alignment of CyrA and StrB1
(from Streptomyces griseus) against the crystal structure of
AGAT was prepared using the online server fugue [72].
Cyanobacterial culture conditions
C. raciborskii AWT205 [5] was grown in Jaworski medium
[73] in static batch culture at 26 °C under continuous
illumination (10 lmolÆm

)2
Æs
)1
).
A novel cyanobacterial amidinotransferase J. Muenchhoff et al.
3854 FEBS Journal 277 (2010) 3844–3860 ª 2010 The Authors Journal compilation ª 2010 FEBS
Cloning, protein expression and purification
Cells from a 100 mL culture of C. raciborskii AWT205
were harvested by centrifugation (5000 g, 20 min), and
genomic DNA was extracted from the cell pellet with the
Ultra Clean Plant DNA Isolation Kit (MoBio, Carlsbad,
CA, USA), according to the manufacturer’s instructions.
PCR amplification of cyrA was performed using primers
cyrA-F (5¢-CATATGCAAACAGAATTGTAAATAGCT-
3¢) and cyrA-R (5¢-CTCGAGAATAATGATGAAGCGA-
GAAAC-3¢), which incorporated NdeI and XhoI restriction
sites, respectively. The cyrA PCR product was cloned into
the expression vector pET30a (Novagen, Madison, WI,
USA) via pGEM-T Easy (Promega, Madison, WI, USA),
verified by sequence analysis, and transformed into the Esc-
herichia coli expression strain BL21(DE3) for expression as a
C-terminal His
6
-tagged fusion protein. However, despite
considerable efforts, the amidinotransferase could not be
expressed in its soluble form. Therefore, cyrA was excised
from pET30a and cloned into pET15b for expression with
an N-terminal His
6
-tag. This resulted in the addition of

22 vector-encoded amino acids to the C-terminus of
recombinant CyrA. For expression of recombinant
His
6
-tagged CyrA, 1 L of MagicMedium (Invitrogen,
Carlsbad, CA, USA) supplemented with 100 lgÆmL
)1
ampicillin was inoculated with a 2.5% overnight starter
culture of E. coli BL21(DE3) harboring pET15-cyrA.
Cultures were grown for 18–24 h at 30 °C before harvesting
by centrifugation (8000 g, 15 min).
Cell pellets were subjected to one freeze–thaw cycle
before suspension in one volume (w ⁄ v) of chilled lysis buf-
fer (50 mm Hepes, pH 7, 500 mm NaCl, 5% glycerol,
0.5 mm dithiothreitol, 0.5 mm phenylmethanesulfonyl fluo-
ride, 30 mm imidazole). Cells were lysed by sonication in
the presence of lysozyme and DNase, and the lysate was
cleared by centrifugation (16 000 g, 30 min, 4 °C). The
lysate was then loaded onto a 1 mL HiTrap Chelating Col-
umn (GE Healthcare, Waukesha, WI, USA) charged with
Ni
2+
for purification of the recombinant protein by com-
petitive elution with imidazole. The effluent collected from
the column was subjected to SDS ⁄ PAGE [74] to identify
fractions that contained pure recombinant protein. Such
fractions were pooled, desalted with an Amicon Filtration
Unit (10 kDa cut-off; Millipore, Billerica, MA, USA) and
diluted in storage buffer (50 mm Hepes, pH 7, 5% glycerol,
10 mm dithiothreitol). Purified recombinant CyrA prepara-

tions were either used immediately or snap frozen in liquid
nitrogen in the presence of 20% glycerol for storage at
)80 °C. The protein concentration of purified CyrA prepa-
rations was determined with a spectrophotometer (Nano-
Drop Technologies, Wilmington, DE, USA), using the
following relationship: A
280 nm
of 1.285 = 1 mgÆmL
)1
.
Western blot analysis on total protein extract and
on fractions of purified protein was performed with a
nickel–nitrilotriacetic acid–alkaline phosphatase conjugate
(Qiagen, Hilden, Germany), using 5-bromo-4-chloro-3-indolyl
phosphate ⁄ Nitro Blue tetrazolium chloride (Sigma-Aldrich,
St Louis, MO, USA). The protein band corresponding to
50 kDa on SDS ⁄ PAGE was excised, destained and
extracted. The protein was analyzed by MALDI-TOF MS
to determine the intact mass [75,76]. CyrA was
then digested with trypsin and analyzed by MALDI-
TOF MS [76–78]. The resulting data were analyzed with
the mascot search engine ( />home.html).
Size exclusion chromatography for determination
of native molecular mass
Size exclusion chromatography was performed on a Super-
dex 200 10 ⁄ 300 GL column (GE Healthcare) fitted to an
A
¨
KTA Basic 900 series Fast Protein Liquid Chromatogra-
phy System. The system was controlled by unicorn soft-

ware, and the column was equilibrated in 50 mm Hepes
(pH 7.5) and 112 mm NaCl at a flow rate of 0.4 mLÆmin
)1
,
using a 0.2 mL sample loop. The column was calibrated
with protein standards of known molecular mass, and
approximately 100 lg of CyrA was applied. Eluted proteins
were detected at 280 nm.
1
H-NMR spectroscopy
1
H-NMR spectroscopy was employed to identify the prod-
ucts of CyrA upon incubation with arginine and glycine
as substrates.
1
H-NMR spectroscopy was performed at
600 MHz on a Bruker DMX 600 spectrometer (Bruker,
Billerica, MA, USA) with a 5 mm QNP probe. Samples
were dissolved in 5% D
2
O and run with the standard
Bruker water suppression program.
To obtain sufficient amounts of product for analysis, a
preparation of 10 mml-arginine and 10 mm glycine in
100 mm Tris ⁄ HCl (pH 8.5), containing 1 mm dithiothreitol,
was incubated overnight at 30 °C in the presence of 2 mg
of CyrA in a total reaction volume of 10 mL. The reaction
mixture was then applied to a Dowex 50 column (1 ·
10 cm) in the H
+

form. The column was washed with
water (100 mL), and the basic products and reactants were
eluted with 2 m NH
3
(50 mL). After evaporation, the sam-
ple was dissolved in 5% D
2
O (500 lL) for NMR spectros-
copy. Standards of l-arginine, glycine, ornithine and
guanidinoacetate were run under identical conditions.
Amidinotransferase activity assays
The colorimetric assay for amidinotransferase activity was
based on the specific reaction of ninhydrin with ornithine
at low pH [39]. Assays were carried out as previously
described [39], with the following modifications: the
total assay volume of 300 lL contained 50 mm Tris⁄ HCl
J. Muenchhoff et al. A novel cyanobacterial amidinotransferase
FEBS Journal 277 (2010) 3844–3860 ª 2010 The Authors Journal compilation ª 2010 FEBS 3855
(pH 8.5), 20 mml-arginine (dono r), 20 mm glycine (acceptor)
and 10–20 lg of enzyme. The reaction was carried out in
1.5 mL microcentrifuge tubes at 30 °C for 30 min. The
absorbance at 505 nm was measured with a Cary 100 UV
spectrophotometer (Varian, Palo Alto, CA, USA). Appro-
priate controls were included, consisting of reaction mix-
tures that were either stopped without incubation,
contained one or no substrate, or contained boiled enzyme
preparations. For accuracy, all assays were carried out in
triplicate.
Various structurally related compounds were tested as
potential amidino group acceptors instead of glycine, such

as l-alanine, b-alanine, c-aminobutyric acid, ethanolamine,
taurine, l-lysine, a-amino-oxyacetic acid and l-norvaline,
by measuring the amount of l-ornithine generated, as
described above.
However, in order to test various amidino group donor
analogs, a different colorimetric assay needed to be
employed that relies on the ability of an amidinotransferase
to amidinate the artificial substrate hydroxylamine to form
hydroxyguanidine, which was measured after its reaction
with pentacyanoaminoferrate [40]. The donor analogs tested
were l-homoarginine, agmatine, l-canavanine, guanidine
hydrochloride, urea, c-guanidinobutyric acid and b-guanidi-
noproprionic acid. The reaction mixture (45 lL) contained
100 mm Tris ⁄ HCl (pH 8.5), 90 mm test substrate, 200 mm
hydroxylamine and 15 lg of purified CyrA. Assays were
carried out for 45 min at 30 °C, as described previously
[40]. A standard curve for hydroxyguanidine–pentacyanoa-
minoferrate complex was constructed, and the absorbance
at 490 nm was determined with a SpectraMax 340 microtit-
er plate reader (Molecular Devices, Sunnyvale, CA, USA).
For this purpose, hydroxyguanidine was chemically synthe-
sized as previously described [40]. Purity of the synthesized
compound was confirmed by spectral analysis from 400 to
560 nm.
The pH optimum of CyrA was determined by measuring
the amount of ornithine formed at 30 °C at a pH range
from 5.5 to 10 in the presence of 100 mm buffer (pH 5.5–
6.5, Mes ⁄ NaOH; pH 7–7.4, K
2
HPO

4
⁄ KH
2
PO
4
; pH 8–9,
Tris ⁄ HCl; and pH 9.5–10, Ches ⁄ NaOH). The temperature
optimum for CyrA was determined at pH 8 by measuring
the amount of hydroxyguanidine formed at various temper-
atures, ranging from 15 to 61 °C.
Kinetic analysis
In order to investigate the kinetic mechanism of CyrA, ini-
tial reaction rates (formation of ornithine) were measured
over a range of l-arginine concentrations (2–24 mm)at
fixed concentrations of glycine (3, 6, 9, 12, 16 and 20 mm).
Kinetic constants were obtained by nonlinear regression
analysis with the enzyme kinetics module 1.1 linked to
sigmaplot 8.02. All kinetic analyses were repeated at least
three times, with reproducible results.
Product inhibition study
Initial reaction rates were measured in the presence of dif-
ferent concentrations of ornithine (0, 1, 3, 6 and 15 mm)at
a saturating level of l-arginine (50 mm), with hydroxyl-
amine as the varied substrate (20–150 mm), using the
method of Walker [40]. Assays were performed in 45 lL
with 25 lg of CyrA for 30 min at 30 °C in the presence of
100 mm Tris ⁄ HCl (pH 8).
MS analysis of the CyrA reaction sequence
CyrA (270 lg) was incubated in the presence of 18 mm
l-arginine at 30 °C. After 2 h, the protein was separated

from small molecular mass substrate and ⁄ or product with a
Microcon YM-10 centrifugal filter device (Millipore). CyrA
preparations treated in the same way but without the addi-
tion of l-arginine were used as the background control.
The filtrate was subjected to electron capture negative ioni-
zation GC-MS for ornithine analysis, with an adaptation of
a published method [79]. A detailed description of this
method can be found in Doc. S1.
The protein fraction was subjected to MALDI-TOF MS
and ESI LC-MS for intact mass analysis. MALDI-
TOF MS and LC-MS ⁄ MS were carried out after digestion
with trypsin, endo-LysC or endo-AspN to obtain maximal
sequence coverage. MS was performed according to previ-
ously described procedures [75–78], and further details can
be found in Doc. S1.
CD
Far-UV CD spectra in the range 190–260 nm and thermal
unfolding curves at 222 nm in 100 mm Ches (pH 8.5) were
generated with active CyrA and CyrA that showed no activ-
ity upon storage at 4 °C for  48 h. Spectra were recorded
with a JASCO J-810CD Spectropolarimeter (Jasco, Easton,
MD, USA) under constant nitrogen flow, connected to a
Peltier temperature controller. The temperature was varied
from 4 to 94 ° C at a rate of 0.6 °CÆmin
)1
to monitor unfold-
ing at 222 nm. Far-UV spectra in the presence and absence
of urea were recorded at 14 °C. The path length was 1 cm,
and the protein concentration was 0.5 mgÆmL
)1

. Spectra
were corrected for buffer signal. Data are expressed as the
mean residue ellipticity, [h] (degÆcm
2
Ædmol
)1
). The relative
amounts of a-helix and b-sheet were deconvoluted from far-
UV spectra for active and inactive CyrA with the online tool
k2d2 [80].
Fluorescence spectrophotometry
Fluorescence spectra were collected using a Perkin Elmer
LS50B Luminescence Spectrometer equipped with a Perkin
Elmer PTP1 Peltier temperature programmer (Perkin
A novel cyanobacterial amidinotransferase J. Muenchhoff et al.
3856 FEBS Journal 277 (2010) 3844–3860 ª 2010 The Authors Journal compilation ª 2010 FEBS
Elmer, Waltham, MA, USA). In order to obtain thermal
unfolding curves, the temperature was varied from 4 to
80 °C at a rate of 0.6 °CÆmin
)1
(identical to the CD scan
rate). The path length was 1 cm, and the protein concentra-
tion was kept at 0.1 mgÆmL
)1
in all experiments in which
active and inactive CyrA were compared, to validate the
comparison of fluorescence intensity between samples. The
fluorescence of 25 lm ANS in the presence of active and
inactive CyrA was monitored at 480 nm, with an excitation
wavelength of 380 nm, in 50 mm Tris ⁄ HCl (pH 8.5). Addi-

tional thermal unfolding curves for active CyrA were
obtained at pH 6 and 6.5 (50 mm Mes), pH 7 (50 mm
Tris ⁄ HCl) and pH 7.5 (50 mm Hepes), as well as in the
presence of 500 mm NaCl at pH 7.5. All spectra were cor-
rected for buffer signal.
Acknowledgements
The authors would like to acknowledge O. Pilak for
help with size exclusion chromatography and S. Mur-
ray for advice on phylogenetic analysis. This work was
supported by The Australian Research Council. MS
results were obtained at the Bioanalytical Mass Spec-
trometry Facility within the Analytical Centre of the
University of New South Wales. This work was under-
taken using infrastructure provided by NSW Govern-
ment co-investment in the National Collaborative
Research Infrastructure Scheme (NCRIS).
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Supporting information
The following supplementary material is available:
Fig. S1. Structural alignment of amidinotransferases.
Fig. S2. Size exclusion chromatogram and SDS ⁄ PAGE
of purified CyrA.
Fig. S3. MALDI-TOF MS and ESI LC-MS of CyrA
(intact mass) with and without arginine.

Fig. S4. Activity of CyrA as a function of pH and
temperature.
Fig. S5. Initial velocity data with arginine and hydrox-
ylamine as substrates.
Fig. S6. Hill plots of initial velocities in the presence
of different concentrations of ornithine as a product
inhibitor.
Fig. S7. GC-MS for qualitative detection of ornithine
in samples of CyrA that were incubated with arginine
only.
Fig. S8. Fluorescence of ANS in the presence of active
CyrA at 0 and 500 mm NaCl as a function of tempera-
ture.
Table S1. Summary of all data obtained by MALDI
and ESI LC-MS ⁄ MS on CyrA peptides (with and
without arginine) generated by three proteolytic
enzymes of varying specificity (endo-AspN, trypsin and
endo-LysC).
Doc. S1. MS analysis of CyrA reaction sequence.
This supplementary material can be found in the
online version of this article.
Please note: As a service to our authors and readers,
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should be addressed to the authors.
A novel cyanobacterial amidinotransferase J. Muenchhoff et al.
3860 FEBS Journal 277 (2010) 3844–3860 ª 2010 The Authors Journal compilation ª 2010 FEBS