NDLI: Purification of Synechocystis sp. Strain PCC6308 Cyanophycin Synthetase and Its Characterization with Respect to Substrate and Primer Specificity

Content Provider	PubMed Central
Author	Elsayed, Aboulmagd Oppermann-sanio, Fred B. Alexander, Steinbüchel
Copyright Year	2001
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 μM), 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°C, respectively. Arginine methyl ester (99.5 and 97% inhibition), argininamide (99 and 96%), S-(2-aminoethyl) cysteine (43 and 42%), β-hydroxy aspartic acid (35 and 37%), aspartic acid β-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 β-methyl ester (53%) and asparagine (9%) stimulated the incorporation of arginine. [3H]lysine (15% of maximum activity) and [3H]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-α,β-d,l-aspartic acid served as primers for in vitro synthesis of cyanophycin, whereas poly-α-l-aspartic acid was almost inactive.
Related Links	http://dx.doi.org/10.1128/AEM.67.5.2176-2182.2001
Starting Page	2176
File Format	PDF
ISSN	10985336
e-ISSN	10985336
Journal	Applied and Environmental Microbiology
Issue Number	5
Volume Number	67
Language	English
Publisher	American Society for Microbiology
Publisher Date	2001-05-01
Access Restriction	Open
Rights Holder	American Society for Microbiology
Subject Keyword	Research in Higher Education
Content Type	Text
Resource Type	Article
Subject	Ecology Food Science Applied Microbiology and Biotechnology Biotechnology

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