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

Applied and Environmental Microbiology
E AboulmagdAlexander Steinbüchel

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, ...Continue Reading

References

Jan 1, 1984·Annual Review of Microbiology·M M Allen
Aug 1, 1981·Biochemical Society Transactions·T AtkinsonC R Lowe
Jun 12, 1999·Phytochemistry·K Y OrabiH N el-Sohly
Jan 11, 2000·Archives of Microbiology·E AboulmagdA Steinbüchel

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Citations

Sep 17, 2008·Applied Microbiology and Biotechnology·Makoto Yagasaki, Shin-ichi Hashimoto
Aug 6, 2008·Applied Microbiology and Biotechnology·Toshinobu Arai, Kuniki Kino
Apr 2, 2011·Applied Microbiology and Biotechnology·Lars WiefelAlexander Steinbüchel
Oct 22, 2003·Journal of Molecular Biology·Detlef D LeipeL Aravind
Jun 29, 2010·Nature Reviews. Microbiology·Bernd H A Rehm
Apr 15, 2008·Applied and Environmental Microbiology·Anna SteinleAlexander Steinbüchel
Dec 30, 2009·Applied and Environmental Microbiology·Anna SteinleAlexander Steinbüchel
Sep 4, 2012·Applied and Environmental Microbiology·Steffen LütteBärbel Friedrich
Mar 28, 2002·Applied and Environmental Microbiology·Lauren L WingardA Michelle Wood
Jun 29, 2002·Applied and Environmental Microbiology·Kay M FreyAlexander Steinbüchel
Dec 8, 2005·Applied and Environmental Microbiology·Yasser ElbahloulAlexander Steinbüchel
Feb 5, 2005·Applied and Environmental Microbiology·Yasser ElbahloulAlexander Steinbüchel
Jan 20, 2016·Microbiology·Friederike KlemkeThomas Volkmer

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