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
Trang 1is involved in cylindrospermopsin biosynthesis
Julia Muenchhoff1, Khawar S Siddiqui1, Anne Poljak2,3, Mark J Raftery2, Kevin D Barrow1and Brett A Neilan1
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: b.neilan@unsw.edu.au
(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 1H 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 32C 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
94C, but lost its tertiary structure at low temperature (Tmax of 44.5C), 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.
Trang 2problem 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-aquaeand 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-skiiT3 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.
Trang 3constructed 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 Sub-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.
Trang 4group 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 His6-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 His6-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 concentrafunc-tion 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 (Topt) for CyrA was found to be
32C at pH 8 At 40 C, 80% of the activity relative
to Topt was lost (Fig S4B) The Toptfor soybean ami-dinotransferase was determined to be 37C [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
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.
Trang 5was 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,
1H-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 1H-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 1H–13C
correla-tion spectroscopy and 1H–1H 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
Vmax(lmolÆmin)1Æmg)1) 1.05 ± 0.05 0.44
kcat(min)1per active site) 52.5 ± 2.5 20
K arginine
m (m M ) 3.5 ± 1.14 2.0 ± 0.5
K glycine
m (m M ) 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 (·),
6 m M (+), 9 m M (s), 12 m M (D), 16 m M ( ) and 20 m M ()).
Trang 6Product 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
Kiof 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 inhibiloca-tion
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 ( ),
1 m M (D), 3 m M (s), 6 m M (+) and 15 m M
(·) Inset: reaction scheme for the formation
of ornithine and hydroxyguanidine from
L -arginine and hydroxylamine as catalyzed
by CyrA.
Trang 781 nmol of ornithine in 1 h, which is equivalent to the
slow rate of 0.0024 lmolÆmin)1Æmg)1
CyrA is a thermolabile molten globule
Recombinant CyrA could be stored at – 80C 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 4C, 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 4C 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 94C, 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 50C; however,
the remaining structure was stable up to 94C 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 Tmax 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 Tmax at 44.5C 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.
Trang 8ANS fluorescence of inactive CyrA showed high
inten-sity around 4C and Tmax 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
4C is responsible for the loss of activity CyrA
rap-idly loses its native tertiary structure when stored at
4C or when exposed to relatively mild temperatures
(> 35C), 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 4C, 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 4C, and the
Tmax was determined ANS fluorescence of CyrA at
pH 6.5, 7, 7.5 and 8.5 revealed defined peaks, with
Tmaxcorresponding to 58, 54.5, 54 and 44.5C, 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 4C 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, Tmax at pH 7.5 decreased from 54 to 49C (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 condipurifica-tions 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 m M 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).
Trang 9cylindrospermopsin 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 oxidsuccinyltransfer-ase [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, 1H-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 Km 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
Trang 10Considering 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 Km
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 substrate⁄ 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 formareac-tion 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