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: 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 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 [...]... folding intermediate CyrA in the molten globule state is catalytically inactive, and CyrA is also not likely to bind to membranes or participate in molecular recognition In summary, CyrA represents a novel group of prokaryotic amidinotransferases that utilize arginine and glycine as native substrates, similarly to the vertebrate group of l-arginine:glycine amidinotransferases The complex kinetic mechanism.. .A novel cyanobacterial amidinotransferase J Muenchhoff et al group acceptor binds at the same site as the first substrate ⁄ 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... hydrophilic amino acid (Asn300) with a large, nonpolar hydrophobic 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 alanine 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... instructions PCR amplification of cyrA was performed using primers cyrA-F (5¢-CATATGCAAACAGAATTGTAAATAGCT3¢) and cyrA-R (5¢-CTCGAGAATAATGATGAAGCGAGAAAC-3¢), which incorporated NdeI and XhoI restriction sites, respectively The cyrA PCR product was cloned into the expression vector pET3 0a (Novagen, Madison, WI, USA) via pGEM-T Easy (Promega, Madison, WI, USA), verified by sequence analysis, and transformed into the... 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... Hernandez-Guzman G & Alvarez-Morales A (2001) Isolation and characterization of the gene coding for the amidinotransferase involved inthe biosynthesis of phaseolotoxin in Pseudomonas syringae pv phaseolicola MPMI 14, 545–554 30 Maerkisch U & Reuter G (1990) Biosynthesis of homoarginine and ornithine as precursors of the phytoeffector phaseolotoxin by the amidinotransfer from arginine to lysine catalysed by an amidinotransferase. .. cylindrospermopsin Mutat Res 472, 155–161 Li R, Carmichael WW, Brittain S, Eaglesham GK, Shaw GR, Mahakhant A, Noparatnaraporn N, Yongmanitchai W, Kaya K & Watanabe MM (2001) Isolation and identification of the cyanotoxin cylindrospermopsin and deoxy -cylindrospermopsin from a Thailand strain of Cylindrospermopsis raciborskii (Cyanobacteria) Toxicon 39, 973–980 Preussel K, Stuken A, Wiedner C, Chorus I & Fastner... glycine, hydroxylamine and ethanolamine at physiologically relevant pH is the negative charge of glycine’s carboxyl group It is likely that binding 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 conservation of residues involved in substrate binding and catalysis between AGAT and CyrA was investigated... on cylindrospermopsin producing Aphanizomenon flos-aquae (Cyanobacteria) isolated from two German lakes Toxicon 47, 156–162 Banker R, Carmeli S, Hadas O, Teltsch B, Porat R & Sukenik A (1997) Identification of cylindrospermopsin in Aphanizomenon ovalisporum (Cyanophycaeae) isolated from lake Kinneret, Israel J Phycol 33, 613–616 Li R, Carmichael WW, Brittain S, Eaglesham GK, Shaw GR, Liu Y & Watanabe... 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, . eukaryotic l-arginine:glycine amidinotransferases. Abbreviations AGAT, human L-arginine:glycine amidinotransferase; AmtA, L-arginine:lysine amidinotransferase; . performed using primers cyrA-F (5¢-CATATGCAAACAGAATTGTAAATAGCT- 3¢) and cyrA-R (5¢-CTCGAGAATAATGATGAAGCGA- GAAAC-3¢), which incorporated NdeI and XhoI restriction sites,