Purification of Synechocystis sp. strain PCC6308 cyanophycin synthetase and its characterization with respect to substrate and primer specificity

Abstract

Synechocystis sp. strain PCC6308 cyanophycin synthetase was purified 72-fold in three steps by anion exchange chromatography on Q Sepharose, affinity chromatography on the triazine dye matrix Procion Blue HE-RD Sepharose, and gel filtration on Superdex 200 HR from recombinant cells of Escherichia coli. The native enzyme, which catalyzed the incorporation of arginine and aspartic acid into cyanophycin, has an apparent molecular mass of 240 +/- 30 kDa and consists of identical subunits of 85 +/- 5 kDa. The K(m) values for arginine (49 microM), aspartic acid (0.45 mM), and ATP (0.20 mM) indicated that the enzyme had a high affinity towards these substrates. During in vitro cyanophycin synthesis, 1.3 +/- 0.1 mol of ATP per mol of incorporated amino acid was converted to ADP. The optima for the enzyme-catalyzed reactions were pH 8.2 and 50 degrees C, respectively. Arginine methyl ester (99.5 and 97% inhibition), argininamide (99 and 96%), S-(2-aminoethyl) cysteine (43 and 42%), beta-hydroxy aspartic acid (35 and 37%), aspartic acid beta-methyl ester (38 and 40%), norvaline (0 and 3%), citrulline (9 and 7%), and asparagine (2 and 0%) exhibited an almost equal inhibitory effect on the incorporation of both arginine and aspartic acid, respectively, when these compounds were added to the complete reaction mixture. In contrast, the incorporation of arginine was diminished to a greater extent than that of aspartic acid, respectively, with canavanine (82 and 53%), lysine (36 and 19%), agmatine (33 and 25%), D-aspartic acid (37 and 30%), L-glutamic acid (13 and 5%), and ornithine (23 and 11%). On the other hand, canavanine (45% of maximum activity) and lysine (13%) stimulated the incorporation of aspartic acid, whereas aspartic acid beta-methyl ester (53%) and asparagine (9%) stimulated the incorporation of arginine. [(3)H]lysine (15% of maximum activity) and [(3)H]canavanine (13%) were incorporated into the polymer, when they were either used instead of arginine or added to the complete reaction mixture, whereas L-glutamic acid was not incorporated. No effect on arginine incorporation was obtained by the addition of other amino acids (i.e., alanine, histidine, leucine, proline, tryptophan, and glycine). Various samples of chemically synthesized poly-alpha,beta-D,L-aspartic acid served as primers for in vitro synthesis of cyanophycin, whereas poly-alpha-L-aspartic acid was almost inactive.

FIG. 1

SDS-PAGE of cyanophycin synthetase from recombinant E. coli at different steps of purification. Lane 1, soluble cell extract (75 μg protein); lane 2, after chromatography on Q Sepharose HiLoad (55 μg); lane 3, after affinity chromatography on Procion Blue HE-RD Sepharose (30 μg); lane 4, after gel filtration on Superdex 200 HR (13 μg). The sizes of molecular mass standard proteins are provided on the left. Protein bands were visualized by staining with Serva Blue R.

FIG. 2

The activity of purified cyanophycin synthetase as a function of the concentration of (a) l-aspartic acid, (b) l-arginine, (c) ATP, and (d) cyanophycin.

FIG. 3

The influence of the incubation temperature on the cyanophycin synthetase-catalyzed incorporation of arginine. (a) Cyanophycin synthetase activity as a function of the incubation temperature. The enzyme activity was determined for 4 min after start of the reaction at the indicated temperature. (b) Time courses of the incorporation of arginine into cyanophycin at 28°C (▫) and 50°C (●). The incorporated amount of arginine was determined at the specified time points in 125 μl of complete reaction mixture, as described in Materials and Methods. Purified cyanophycin synthetase was used for these experiments.

FIG. 4

Effects produced by the addition or substitution of analogous compounds structurally related to aspartic acid and arginine on the incorporation of both amino acids. (a) Aspartic acid (▧) and arginine (░⃞) incorporation in the presence of a 5 mM concentration of the analogous compounds, which were added to the complete reaction mixture containing 5 mM l-[U-¹⁴C]aspartic acid and 0.5 mM l-[2,3,4,5-³H]arginine, as described in Materials and Methods. (b) Effect of the analogous compounds on the incorporation of arginine and aspartic acid when they were added in place of aspartic acid (▧) at a final concentration of 5 mM or in place of arginine (░⃞) at a final concentration of 0.5 mM. The activity corresponding to 100% was determined as the incorporation of 10 nmol of aspartic acid · min⁻¹ · ml⁻¹ and 11 nmol of arginine · min⁻¹ · ml⁻¹, respectively, as described in Materials and Methods using purified cyanophycin synthetase. Values were calculated on the basis of four runs (run standard deviation, ca. 10%).

FIG. 5

Structural formulas of arginine and analogous compounds tested as substrates for cyanophycin synthetase. Changes of the structures with respect to the natural substrate arginine are shaded.