Cyanophycin (multi-L-arginyl-poly-L-aspartate), a water-insoluble reserve polymer of cyanobacteria, is a product of nonribosomal peptide synthesis. The purification of cyanophycin synthetase of the cyanobacterium Anabaena variabilis is described. In sodium dodecylsulfate/polyacrylamide gel electrophoresis, the enzyme preparation shows one band with an apparent molecular mass of 100 kDa. The native enzyme has an apparent molecular mass of approximately 230 kDa, as determined by size-exclusion chromatography, suggesting that the active form is a homodimer. During catalysis, ATP is converted to ADP. The gene coding for cyanophycin synthetase has been identified in the sequenced genome of Synechocystis sp. PCC 6803. The C-terminal 60% of the deduced amino acid sequence of cyanophycin synthetase show sequence similarity to enzymes of the superfamily of ligases involved in the biosynthesis of murein and of folyl-poly(γ-glutamate). Cells of Escherichia coli harbouring the gene on a plasmid express active synthetase and accumulate cyanophycin-like material. The results prove that a single enzyme catalyzes the de novo synthesis of cyanophycin.Keywords : non-ribosomal peptide synthesis; cyanobacteria ; cyanophycin synthetase; purification; heterologous expression.Most cyanobacteria contain the nitrogen-rich reserve mateFrom the cyanobacterium Anabaena cylindrica, Simon [9] has enriched an enzyme that synthesizes cyanophycin in vitro rial multi-L-arginyl-poly (L-aspartic acid) (trivial name, cyanoand has studied the basic properties of the biosynthetic reaction. phycin), which is deposited in the cytoplasm in the form of gran-The substrates of the reaction are the constituent amino acids Lules [1Ϫ4]. In this polymer, nearly all β-carboxy groups of a arginine and L-aspartic acid, Mg-ATP, and cyanophycin as poly(aspartate) backbone are linked to A-amino groups of argiprimer [9]. In a strict sense, the assay measures an elongation nine residues by iso-peptide bonds, resulting in an approximate reaction. A thiol reagent such as 2-mercaptoethanol and K ϩ were 1:1 stoichiometry of aspartate/arginine [1,5,6]. The polymer is found to be required for full activity [9]. The latter properties the product of nonribosomal peptide synthesis [7]. Its molecular are, in addition to the formation of iso-peptide bonds, reminismass, as estimated by SDS/PAGE, in a given cyanobacterial specent of the requirements of the biosynthesis of glutathione [10] cies is not uniform, but ranges over about 25Ϫ100 kDa [1]. Cyaand of poly(γ-D-glutamate) [11]. nophycin is also present in filamentous cyanobacteria such asIn this communication, we describe the purification of cyaAnabaena which differentiate, in a semiregular pattern, so-called nophycin synthetase from the cyanobacterium Anabaena varheterocysts, cells specialized for fixation of N 2 under aerobic iabilis American Type Culture Collection (ATCC) 29413. It is conditions. In these species, cyanophycin may not only serve as demonstrated that a homodimer of a 100-kDa protein incorpoa reserve material,...

b ) liefert aus Athylen alle geradzahligen primaren Fettalkohole, cbenso 0). Der Vorteil von b ) gcgenuber c ) liegt in folgendem: Bei c ) sind die Reaktionsprodukte nur in der urspriinglichcn relativ flachen VerMung zu erhalten. b ) gestattet vorherige Trennung der Olefine (die leichter als bei den Alkoholen geht) und direkte Herstellung reinen Octanols, Decanols usw. oder die Vorfractionierung der Olefine etwa in C,HIs + C,HIB einerseits, andererseits. I n der Zwisohenphase der Aluminiumverbindungen kann man an C6H,,al und C,H,,al zunachst noch Athylen addieren und d a m erst oxydieren. Man erhalt dann in engerer Verteilung schlieRlich irn wesentlichen nur die Alkohole C,,, C,, und C,, (antistatistische Reaktionsfuhrung).Iler gleiohe Kunstgriff laWt sich auch siiingema5 auf die Crackolefinc nach a ) anwenden.Der stochiomet.rische A l u m i n i u m -V e r b r a u c h von 9 g A1 pro No1 Olefin bzw. Alkohol fallt bei nicht zu,geringer Molekular-grirGe nicht ins Gewieht und betragt z. B. fur Dodecanol -50 g/kz Dodecanol.Bei der Ausfiihrung ini graben licfert die Stufe der Oxydation der Alurniniumalkyle rnit Luft zugleich reinen S t i c k s t o f f als Nebenprodukt, wie er bci der Herstellung der Aluminiumalkyle in der ersten Stufe als Schutzg&s gebraucht wird.

The branched polypeptide multi-l-arginyl-poly-l-aspartic acid, also called cyanophycin, is a water-insoluble reserve material of cyanobacteria. The polymer is degraded by a specific hydrolytic enzyme called cyanophycinase. By heterologous expression in Escherichia coli, a gene encoding cyanophycinase has been identified in the sequenced genome of Synechocystis sp. PCC 6803. The gene, designated cphB, codes for a protein of 29.4 kDa. The high level of expression of active cyanophycinase in E. coli from the Synechocystis gene allowed for its purification to electrophoretic homogeneity. The enzyme, which appears to be specific for cyanophycin, hydrolysed the polymer to a dipeptide consisting of aspartic acid and arginine. Based on inhibitor sensitivity and primary sequence, cyanophycinase appears to be a serine-type exopeptidase related to dipeptidase E [Conlin, C

Some bacterial genomes were found to contain genes encoding putative proteins with considerable sequence homology to cyanophycin synthetase CphA of cyanobacteria. Such a gene from the Gram-positive, spore-forming anaerobe Desulfitobacterium hafniense was cloned. Expression in Escherichia coli resulted in the formation of a polydispers copolymer of aspartic acid and arginine, with a minor amount of lysine, of about 30 kDa molecular mass. In contrast to cyanophycin, this polymer was water-soluble. The structure of the polymer formed by the synthetase from Desulfitobacterium hafniense was studied by enzymatic degradation with the cyanophycin-specific hydrolase cyanophycinase, and by chemical and mass-spectroscopic analyses. Despite of the differences in solubility, indicating that both polymers cannot be completely identical, the chemical structure was found to be very similar to that of cyanophycin. The results suggest that the use of cyanophycin-like polymers as a nitrogen- rich reserve material is not restricted to cyanobacteria, and that such polymers may not necessarily be stored in granules.

Biosynthesis of the cyanobacterial nitrogen reserve cyanophycin (multi-l-arginyl-poly-l-aspartic acid) is catalysed by cyanophycin synthetase, an enzyme that consists of a single kind of polypeptide. Efficient synthesis of the polymer requires ATP, the constituent amino acids aspartic acid and arginine, and a primer like cyanophycin. Using synthetic peptide primers, the course of the biosynthetic reaction was studied. The following results were obtained: (a) sequence analysis suggests that cyanophycin synthetase has two ATP-binding sites and hence probably two active sites; (b) the enzyme catalyses the formation of cyanophycin-like polymers of 25±30 kDa apparent molecular mass in vitro; (c) primers are elongated at their C-terminus; (d) the constituent amino acids are incorporated stepwise, in the order aspartic acid followed by arginine, into the growing polymer. A mechanism for the cyanophycin synthetase reaction is proposed; (e) the specificity of the enzyme for its amino-acid substrates was also studied. Glutamic acid cannot replace aspartic acid as the acidic amino acid, whereas lysine can replace arginine but is incorporated into cyanophycin at a much lower rate.

Man we8 aus versehiedenen Reaktionen, daI3 Wasserstof f an Kohlenstoff bereits dam eine gesteigerte Reaktionsfahigkeit besitzt, wenn sich in a,P-Stellung zu seinem Treeratom auch nw eine einzige C=C-Doppelbindung befindet. So greifen Sauerstoff l ) , gewisse Chinonea), Selendioxyd3) und gelegentlich auch andere O~ydationsmittel~) bei der Reaktion mit ungesattigten Substanzen vielfach nicht an der Doppelbindung, sondern an dem der Doppelbindung nachst benachbarten Kohlenstoff unter Substitution von Wasserstoff an : A. Windous, B. 58, 491 (1920). Die Halogenkrung ungesattigter Substanzm usw. 81 Die Auflockerung derartiger Wasserstoffatome sollte auch in einer gesteigerten Reaktionsfahigkeit gegen Halogene zum Ausdruck kommen. Wie das Toluol unter geeigneten Bedingungen im Methyl, so sollten sich auch ungesattigte Verbindungen neben der Doppelbindung ,,in der Seitenkette", d. h. in der Allylstellung, halogenieren lassen. Der Verwirklichung dieses Gedankens steht die Additionsfahigkeit der Doppelbindung entgegen. Die Geschwindigkeit der Addition uberwiegt normalerweise so sehr, d d , bis vor kurzem, eke ahnliche Lenkung des Halogeniemgsverlaufs wie beim Toluol fiir homologe Athylene unbekannt war.. (Wir setzen dabei die Kernsubstitution des Toluols mit der Addition an die Doppelbindung parallel.) Isobutylen kam allerdings, wie lange bekannt istl), unter bestimmten Bedingungen recht l) Scheschukow, XC. 16, 320 (1884); vgl. auoh Pogorshelski, ebenda 86, 1129 (1904); C. 1906, 667. Die von Meisenheimer [A. 456, 142 (1927)l beobachtete Bromierung von ist vermutlich, wie Ziegler u. BiLhr [B. 62, 1696 (192911 zeigen konnten, keine direkte Substitution im Methyl. Sie durchliiuft wohl die Zwischenstufen Einen hhnliohen Meohanismus halten wir fur wahrsoheinlich bei einer kiirzlich von Wendt [B. 74, 1242, 1243, 1244 (1941)l besohriebenen Substitution iihlichen Typs, namlich dem Ubergang von @-Cyologeraniumsiiure (A) in ihr Bromderivat (D) A B C D Annalen der Chemie. 661. Band. 6 l) Das Verfahren von Groll und Hearne war uns zu Beginn unserer Arbeit (1935) ubrigens unbekannt. 2, B. 62, 51 (1919); B. 64, 476 (1921); (die letzte Arbeit mit K. J aschinowski). Neuerdings auch von Wendt [B. 74, 1243 (194l)l zitiert und obne Erfolg versucht.

SummaryThe production of biodegradable polymers in transgenic plants in order to replace petrochemical compounds is an important challenge for plant biotechnology. Polyaspartate, a biodegradable substitute for polycarboxylates, is the backbone of the cyanobacterial storage material cyanophycin. Cyanophycin, a copolymer of L -aspartic acid and L -arginine, is produced via non-ribosomal polypeptide biosynthesis by the enzyme cyanophycin synthetase. A gene from Thermosynechococcus elongatus BP-1 encoding cyanophycin synthetase has been expressed constitutively in tobacco and potato. The presence of the transgene-encoded messenger RNA (mRNA) correlated with changes in leaf morphology and decelerated growth. Such transgenic plants were found to produce up to 1.1% dry weight of a polymer with cyanophycin-like properties. Aggregated material, able to bind a specific cyanophycin antibody, was detected in the cytoplasm and the nucleus of the transgenic plants.

Cyanophycin (multi-L-arginyl-poly-L-aspartic acid) is a nitrogen storage polymer found in most cyanobacteria and some heterotrophic bacteria. The cyanobacterium Synechocystis sp. strain PCC 6803 accumulates cyanophycin following a transition from nitrogen-limited to nitrogen-excess conditions. Here we show that the accumulation of cyanophycin depends on the activation of the key enzyme of arginine biosynthesis, N-acetyl-L-glutamate kinase, by signal transduction protein P II .Cyanophycin (multi-L-arginyl-poly-L-aspartic acid) is a nitrogen-rich reserve polymer present in most cyanobacteria (reviewed in references 4, 5, 34, and 43) as well as in some heterotrophic bacteria (27,49). It consists of a poly-␣-aspartic acid backbone, with arginine linked to the ␤-carboxyl group of almost every aspartyl residue via isopeptide bonds (44). Cyanophycin is synthesized by a single enzyme, cyanophycin synthetase, from aspartate and arginine in an ATP-dependent reaction using a stillunidentified primer (1,2,8,17,42,48). The amount of cyanophycin in cyanobacteria varies considerably with growth conditions. Its content is usually less than 1% of dry weight in rapidly growing cultures but is high (up to 18%) in stationaryphase cultures and under conditions of unbalanced growth such as sulfate or phosphate limitation (6,30,40,45). When nitrogenstarved cyanobacterial cultures were provided with combined nitrogen sources, a rapid but transient accumulation of cyanophycin occurred (3). The cyanophycin contents of Anabaena cylindrica and Synechocystis sp. strain PCC 6803 increased severalfold when translation was inhibited by chloramphenicol (6, 41), indicating that rapid synthesis of the polymer did not depend on de novo synthesis of cyanophycin synthetase and that consumption of amino acids by protein synthesis may compete with the accumulation of cyanophycin. Furthermore, no correlation was found between the extractable activity of cyanophycin synthetase and the rate of polymer accumulation (31). These and several similar studies could not, so far, elucidate the mechanism(s) by which cyanophycin accumulation is regulated. Recently, it was shown that the genes for cyanophycin metabolism are under nitrogen control in the diazotrophic strain Anabaena sp. strain PCC 7120 (35). Furthermore, an involvement of the signal transduction protein P II in the control of cyanophycin synthesis was suggested (19, 29) (see below).The cyanobacterial P II protein is a member of the large family of P II signal transduction proteins, which play pervasive roles in nitrogen control in bacteria, plants, and some archaea (for recent reviews, see references 7 and 12). Similar to its Escherichia coli counterpart, P II from the cyanobacterium Synechococcus elongatus PCC 7942 binds ATP and 2-oxoglutarate in a synergistic manner (13,24). In the presence of increased 2-oxoglutarate levels, corresponding to nitrogen-limited conditions, P II is phosphorylated at seryl residue 49 (14). Dephosphorylation of P II -P in Synechocystis sp. strain PCC 6803 is catalyzed ...