Institut für Molekulare Mikrobiologie und Biotechnologie, Westfälische Wilhelms-Universität Münster, Corrensstraße 3, D-48149 Münster, Germany
Correspondence
Alexander Steinbüchel
steinbu{at}uni-muenster.de
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ABSTRACT |
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Present address: Department of Molecular Microbiology, John Innes Centre, Norwich Research Park, Colney Lane, Norwich NR4 7UH, UK.
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INTRODUCTION |
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Cyanophycin is synthesized via an ATP-dependent step-by-step mechanism catalysed by a single enzyme, cyanophycin synthetase (Berg et al., 2000). Cyanobacterial cyanophycin synthetases (CphA) have been purified and characterized from Anabaena variabilis (Ziegler et al., 1998
) and the thermophilic strain Synechococcus MA19 (Hai et al., 1999
) as well as from recombinant Escherichia coli carrying cphA from the cyanobacteria Anabaena variabilis strain ATCC 29413 (Berg et al., 2000
) and Synechocystis sp. strains PCC 6803 (Ziegler et al., 1998
; Oppermann-Sanio et al., 1999
) and PCC 6308 (Aboulmagd et al., 2001a
). Cyanophycin synthetases exhibit similarities to both D-alanyl-D-alanine synthetase and Mur-ligases and most probably possess two ATP-binding sites. Polymerization of cyanophycin in vitro has been shown to be dependent on the presence of ATP, K+, Mg2+, a cyanophycin primer and a thiol reagent such as
-mercaptoethanol in the reaction mixture (Simon, 1976
; Ziegler et al., 1998
; Aboulmagd et al., 2001a
).
Recently, it was reported that several non-cyanobacterial species possess genes putatively encoding proteins with high sequence similarity to cyanophycin synthetases (Krehenbrink et al., 2002; Ziegler et al., 2002
). Moreover, low amounts of cyanophycin were accumulated in cells of an auxotrophic mutant of the non-cyanobacterium Acinetobacter calcoaceticus strain ADP1 during cultivation under phosphate limitation (Krehenbrink et al., 2002
). Although the reaction sequence and substrate specificity of cyanobacterial cyanophycin synthetases has been studied in some detail, this study, besides being the first to investigate a non-cyanobacterial cyanophycin synthetase, also delivers deeper insights into kinetic parameters of the enzyme while also taking into consideration the two different states of the cyanophycin primer.
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METHODS |
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Purification and analysis of cyanophycin-like material.
Cyanophycin-like material was isolated from E. coli DH1 cells harbouring pKOS1 by the procedure described by Simon (1976). The amino acid constituents of the isolated material were determined by HPLC as described by Aboulmagd et al. (2000)
.
One-dimensional SDS-PAGE.
One-dimensional SDS-PAGE was performed in 11·5 % (w/v) gels as described by Laemmli (1970). Proteins and cyanophycin-like material were stained with Serva Blue R.
Two-dimensional PAGE.
A. calcoaceticus ADP1 was grown in 100 ml phosphate-limited TA medium (64 µM phosphate) for 20 h, harvested by centrifugation (30 min, 2000 g, 4 °C) and washed four times in 10 ml cold washing buffer consisting of 30 mM KCl and 70 mM sodium phosphate buffer (pH 7·0). The washed cell pellet was resuspended in 120 µl cold resuspension buffer consisting of 10 mM Tris/HCl (pH 8·0), 1·5 mM MgCl2, 10 mM KCl, 0·5 mM DTT and 0·1 % (w/v) SDS per OD600 unit of the original 100 ml culture. Fifty microlitres of this suspension were then diluted with 450 µl lysis buffer consisting of 9 M urea, 1 % (w/v) DTT, 2 % (w/v) 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate and 0·8 % (w/v) Serva-Lyte pH 310. After centrifugation for 5 min (14 000 g), 330 µl of the supernatant was applied to an isoelectric focusing strip (ReadyStrip IPG strip, 17 cm, pH 310; Bio-Rad), which was left for 12 h for re-swelling. Focusing was done at 10 000 V for 5 h. After focusing, the strip was equilibrated in sample buffer consisting of 6 M urea, 2 % (w/v) SDS, 50 mM Tris/HCl (pH 8·8) and 20 % (v/v) glycerol, and the proteins were separated by 11·5 % SDS-PAGE. Staining was done with Serva Blue R.
Purification of cyanophycin synthetase, assay of cyanophycin synthetase activity and determination of protein concentration.
E. coli DH1(pKOS1) cells were harvested by centrifugation (20 min, 2800 g, 4 °C), washed once with TKME buffer (50 mM Tris/HCI, pH 8·2, containing 20 mM KCI, 5 mM 2-mercaptoethanol and 1 mM EDTA) and resuspended in 2 ml buffer per g fresh cell mass. The cells were disintegrated by sonication for 1 min ml1 cell suspension using a Sonoplus GM200 sonifier (Bandelin Electronic). The supernatant obtained after centrifugation for 15 min at 14 000 g was used as soluble cell fraction.
Anion-exchange chromatography on HiLoad 26/10 Q-Sepharose (Amersham Pharmacia Biotech) and gel filtration chromatography on HiLoad 26/60 Superdex 200 (Amersham Pharmacia Biotech) were performed essentially as described by Aboulmagd et al. (2000). Following gel filtration, fractions exhibiting the highest cyanophycin synthetase activity were combined and supplemented with 20 mM MgCl2 and 100 µg cyanophycin ml1. After 5 min incubation at 28 °C, the cyanophycin was pelleted by centrifugation at 14 000 g and 4 °C for 30 min, and the supernatant was carefully removed. The pellet was resuspended in 1 ml TKME buffer and incubated for another 10 min at 28 °C before being pelleted again by another round of centrifugation. The supernatant from this centrifugation contained the purified cyanophycin synthetase and was dialysed against two changes of 250 ml TKME buffer for 2 h to remove residual MgCl2.
Cyanophycin synthetase activity was measured at 30 °C employing a radiometric assay as described by Aboulmagd et al. (2000) using cyanophycin isolated from E. coli DH1(pKOS1) as primer; the apparent molecular mass of cyanophycin, as estimated by SDS-PAGE, was 2540 kDa. Briefly, enzyme activity was measured at 30 °C in 100 µl reaction volume containing 10 µl sample, a suspension of 1 mg CGP ml1, 0·5 mM arginine (0·14 nmol as L-[U-14C]arginine; 111 000 d.p.m.), 5 mM aspartate, 20 mM MgCl2, 20 mM KCl, 50 mM Tris/HCl (pH 8·2), 10 mM 2-mercaptoethanol and 4 mM ATP. The reaction time was 10 min. Although the reaction volume contained only approximately 3·5 nmol CGP primer, arginine (50 nmol) is the limiting substrate in a reaction containing aspartate and arginine. The concentration of CGP primer (CGP N-termini) remained constant throughout the reaction due to alternate addition of aspartate and arginine to the growing polymer chain. The dilution of the sample was thus adjusted to incorporate less than 10 % of the arginine present in the reaction mixture during the course of the reaction, to measure true initial velocities. In reactions using CGP-Asp as a primer and lacking aspartate, the regeneration of CGP primer N-termini does not occur. In these experiments, the reaction was stopped before 10 % of the CGP-Asp had been converted to CGP-Arg (see Results). One unit of activity was defined as 1 nmol amino acid incorporated min1. Soluble protein concentrations were determined as described by Bradford (1976)
.
Synthesis of CGP-Asp or CGP-Arg.
CGP (1 mg) isolated from E. coli DH1(pKOS1) was incubated for 12 h at 30 °C with 3 U purified cyanophycin synthetase from Synechococcus sp. MA19 (Hai et al. 1999) in 1 ml reaction buffer consisting of 50 mM Tris/HCl (pH 8·2), 20 mM KCl, 20 mM MgCl2, 10 mM 2-mercaptoethanol, 4 mM ATP, 0·5 mM arginine and 5 mM aspartate. Arginine or aspartate was omitted from the reaction buffer depending on the desired form of CGP. The resulting cyanophycin was sedimented by centrifugation (14 000 g, 10 min) and dissolved in 100 µl 20 mM HCl. This preparation was found to be free of residual cyanophycin synthetase activity.
Studies on the binding of cyanophycin synthetase protein to cyanophycin.
Purified cyanophycin synthetase was incubated for 5 min at 28 °C with 100 µg cyanophycin suspension in a total volume of 100 µl binding buffer consisting of 50 mM Tris/HCl (pH 8·2), 20 mM KCl, 20 mM MgCl2 and 10 mM 2-mercaptoethanol. Cyanophycin was pelleted by centrifugation (14 000 g, 15 min) and the supernatant carefully removed by aspiration. The cyanophycin pellet was then resuspended in 100 µl reaction buffer [0·5 mM arginine with 0·14 nmol as L-[U-14C]arginine; 111 000 d.p.m., 5 mM aspartate, 20 mM MgCl2, 20 mM KCl, 50 mM Tris/HCl (pH 8·2), 10 mM 2-mercaptoethanol]. The reaction was started by the addition of 4 mM ATP.
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RESULTS |
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Substrate specificity of the purified enzyme
In contrast to the cyanobacterial cyanophycin synthetases studied so far, it was shown previously that the enzyme from A. calcoaceticus ADP1 did not incorporate lysine into the polymer when expressed heterologously in E. coli (Krehenbrink et al., 2002). It was also shown in the previous study that the in vitro incorporation of lysine using crude cell extracts from recombinant E. coli occurred at only about 0·04 % of the rate of arginine and was therefore exceptionally low (Krehenbrink et al., 2002
). Using the purified enzyme, the low incorporation of lysine was confirmed. Glutamate, although reported to occur in cyanophycin isolated from nitrogen-limited cells of cyanobacterium Synechocystis sp. PCC 6308 (Merritt et al., 1994
), was not incorporated in place of either aspartate or arginine at any significant rate. The continuous reaction was shown to be dependent on the presence of aspartate, arginine, a cyanophycin primer, ATP and Mg2+ ions in the reaction mixture (Table 3
), reflecting the characteristics of the cyanobacterial enzymes studied so far (Simon, 1976
; Aboulmagd et al., 2000
; Ziegler et al., 1998
). The relatively high apparent activity with arginine or aspartic acid as sole substrate probably resulted from single covalent attachments of these substrates to the cyanophycin primer.
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To determine the Km value for ATP of the site involved in the addition of arginine [(Arg)ATP site], CGP-Asp was used as a primer in a reaction mixture lacking aspartate, at varying ATP concentrations (0·012 mM) and fixed concentrations of 20 mM Mg2+ and 0·5 mM arginine (0·14 nmol as L-[U-14C]arginine; 111 000 d.p.m.). The reaction time was 10 min. The reaction was limited by the amount of CGP-Asp (3·5 nmol in the reaction volume). In a reaction containing both amino acid substrates (arginine and aspartate), the amino acid acceptor is continuously regenerated by alternate addition of arginine or aspartate to the CGP primer; the concentration of primer thus remains constant. To measure true initial velocities and to reduce distortions caused by depletion of the arginine acceptor CGP-Asp, the reaction was stopped before 10 % of the CGP-Asp present was converted to CGP-Arg. In this way, the apparent Km value of the (Arg)ATP site was determined to be 38 µM. The maximum volume activity (Vmax) was calculated to be 3·79 U ml1; 35 U ml1 (complete reaction) was used.
The apparent Km value for ATP for the complete reaction was determined in a similar way, but using unmodified cyanophycin as a primer and varying the ATP concentration over a wider range (0·18 mM) in the presence of 5 mM aspartate. The apparent Km value was found to be 278 µM at a Vmax of 35·41 U ml1; 35 U ml1 (complete reaction) was used.
A reaction lacking arginine is subject to the same CGP primer limitation as a reaction lacking aspartate; therefore, the same initial velocity conditions (i.e. less than 10 % conversion of CGP-Arg) apply. Due to this and the much higher Km value for aspartate compared to that for arginine, the Km value for ATP for the site involved in the addition of aspartate [(Asp)ATP site] could not be measured directly. The concentration of aspartate used in the assay is ten times higher than that of arginine (5 mM as opposed to 0·5 mM). The high concentrations of non-radioactive aspartate that needed to be added to saturate the enzyme resulted in a dilution of the radioactive aspartate below the detection limit. Thus, the Km value for the (Asp)ATP site was instead calculated from the Km value for ATP for the (Arg)ATP site and the reaction velocities of the complete reaction at varying ATP concentrations.
Assuming complete independence of both ATP-binding sites, the saturated fraction (sf) of these sites would be sf(Arg)=[ATP]/(Km(Arg)+[ATP]) and sf(Asp)=[ATP]/(Km(Asp)+[ATP]), respectively. The fraction of enzyme molecules with both sites saturated is the product of these two terms: sf(both)=sf(Arg)xsf(Asp). Only this fraction contributes to the continuous reaction involving both amino acids, the reaction is thus rate-limited primarily by the site with lower affinity for ATP. Normalizing the measurements from the Km determination of the reaction lacking aspartate to Vmax=1 gives the saturated fraction of the (Arg)ATP binding site under MichaelisMenten assumptions. sf(Asp) can then be calculated by sf(Asp)=sf(both)/sf(Arg). From the corrected data, the apparent Km value for the (Asp)ATP binding site was calculated to be 210 µM. Fig. 3 summarizes the theoretical saturation of the two binding sites and the complete enzyme at different ATP concentrations and shows the experimental data.
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To determine the relevance of the two possible forms of cyanophycin inside the cell, the whole-cell proteins of A. calcoaceticus ADP1 cells cultivated under phosphate-limited conditions were electrophoretically separated by 2D-PAGE. CGP-Asp and CGP-Arg both focused at the expected positions in the gel and were easily distinguished from each other. No form seemed to be more prevalent than the other, and the amounts, as well as the molecular mass range, of the molecules of both forms appeared to be almost identical as indicated by similar intensities of the spots in the electrophoretogram (Fig. 6a). The distribution of the various cyanophycin molecules in the real 2D-gel matched almost perfectly the distribution of cyanophycin molecules in a virtual 2D-gel, which was drawn on the basis of the theoretical values calculated for the isoelectric points (pI) of CGP-Asp and CGP-Arg molecules of defined molecular masses as occurring in A. calcoaceticus ADP1 (Fig. 6b
). The theoretical pI values were estimated according to Sillero & Ribeiro (1989)
. Depending on the molecular masses of the cyanophycin molecules, the pI values of CGP-Arg and CGP-Asp differed by about 0·70 to 0·85.
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DISCUSSION |
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The lack of incorporation of lysine into CGP in A. calcoaceticus ADP1 and in recombinant E. coli harbouring cphA from A. calcoaceticus (Krehenbrink et al., 2002) was confirmed by the detection of only negligible in vitro activity of the purified enzyme with this alternative substrate. This restricted substrate range clearly distinguishes the cyanophycin synthetase of A. calcoaceticus ADP1 from all other cyanophycin synthetases investigated so far. Interestingly, lysine could never be detected in cyanophycin isolated from cyanobacterial cells, although all cloned cyanobacterial cyanophycin synthetases, such as the enzymes from Anabaena variabilis (Berg et al., 2000
), Synechococcus sp. MA19 (Hai et al., 1999
), Synechocystis sp. strain PCC 6308 (Aboulmagd et al., 2001b
) and Synechocystis sp. strain PCC 6803 (Ziegler et al., 1998
), synthesized cyanophycin in which arginine was partially replaced by lysine when expressed heterologously in E. coli. In vitro experiments also demonstrated that these cyanobacterial cyanophycin synthetases incorporated substantial amounts of lysine into the polymer. The discrepancy between the in vivo activities of cyanobacterial cyanophycin synthetases in their natural hosts and in E. coli may be due to kinetic reasons. For the cyanophycin synthetase of Anabaena cylindrica, Berg et al. (2000)
found similar Km values for arginine and lysine (about 15 µM), but an about 30-fold higher maximal incorporation rate for arginine compared to lysine. Due to the overexpression of the enzyme in E. coli and its higher specific activity, the cells accumulate cyanophycin at a high rate and may become deprived of arginine, thus subsequently favouring the incorporation of lysine (Berg et al., 2000
). Like all cyanobacterial cyanophycin synthetases investigated in detail so far, the enzyme from A. calcoaceticus also did not accept glutamic acid as a substrate in place of either aspartic acid or arginine. The reported replacement of arginine by glutamic acid in a cyanophycin sample isolated from Synechocystis sp. strain PCC 6308 (Merritt et al., 1994
) therefore remains an enigma.
The affinities of the cyanophycin synthetase of A. calcoaceticus ADP1 for aspartic acid and arginine resembled those of cyanobacterial cyanophycin synthetases. The apparent Km values for arginine of all cyanophycin synthetases investigated so far are generally much lower than the Km values for aspartic acid (Table 4). It may be assumed that the intracellular concentrations of arginine are much lower than those of aspartic acid due to the long arginine biosynthesis pathway. Therefore, cyanophycin synthetases with a high affinity towards arginine may have been selected during evolution, enabling effective synthesis and accumulation of cyanophycin. The problem of provision of cyanophycin synthetases with arginine was discussed above in connection with the incorporation of lysine instead of arginine. The availability of arginine is a limiting factor for cyanophycin synthetase. For biotechnological production of cyanophycin, this bottleneck must be overcome by metabolic engineering. Although cyanobacteria are accessible to metabolic engineering (Koksharova & Wolk, 2002
), heterotrophic bacteria like A. calcoaceticus or recombinant strains expressing cyanobacterial cyanophycin synthetases are better candidates for the industrial production of cyanophycin or related biopolymers for several reasons (Frey et al., 2002
).
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Analysis of the binding of the cyanophycin synthetase protein to cyanophycin revealed that the state of the cyanophycin molecules played a major role in the binding mechanism. Generally, Mg2+-independent binding to CGP-Arg and Mg2+-dependent binding to CGP-Asp can be differentiated. The enzyme bound equally well to both forms of CGP in the presence of Mg2+. This, together with the 2D-PAGE data showing both forms of CGP to be equally abundant in cells, suggests that the affinities of the enzyme towards the amino acid substrates are balanced to allow for roughly equal rates of incorporation under the conditions present in the cell. The differing requirement of Mg2+ in the binding of the enzyme to CGP also offers an explanation of the behaviour of CGP binding observed upon increasing the concentration of Mg2+. While at very low concentrations of Mg2+ only CGP-Arg is able to bind to the enzyme, the amount of bound enzyme is drastically increased at higher Mg2+ concentrations, when binding to CGP-Asp also becomes possible. The sharp increase in the amount of bound enzyme at concentrations greater than 2 mM MgCl2 also suggests a certain cooperativity of the Mg2+-dependent binding process.
Whether there is only one binding site for cyanophycin on the cyanophycin synthetase molecule or whether there are two, one for CGP-Arg and the other for CGP-Asp, could not be elucidated by these experiments. It may be possible that divalent Mg2+ ions also bind to the carboxylic group of aspartate, thus mimicking the arginine residue by converting a negative into a positive charge and allowing binding of the enzyme also to CGP-Asp and not only to CGP-Arg. However, from a steric point of view, this is rather unlikely considering the much larger size of an arginine residue in comparison to Mg2+. Therefore, it appears more likely that the enzyme molecule possesses two binding sites, one for CGP-Arg and one for CGP-Asp. Furthermore, the role of magnesium in the reaction is a dual one, as it is not only crucial for the binding of the enzyme to CGP-Asp but also for the reaction, i.e. the addition of aspartic acid to CGP-Arg. While the enzyme bound very well to CGP-Arg in the absence of magnesium, the reaction never proceeded beyond binding. As many ligases involve Mg2+ in nucleotide binding (Shi & Walsh, 1995; Bertrand et al., 1999
; Mol et al., 2003
), this may also be the case at least for the (Asp)ATP site. As the enzyme does not bind to CGP-Asp in the absence of Mg2+, the role of Mg2+ in the catalytic cycle during the addition of arginine still has to be established.
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ACKNOWLEDGEMENTS |
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Received 5 March 2004;
revised 10 May 2004;
accepted 13 May 2004.
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