Aboulmagd E, Oppermann-Sanio F B, Steinbüchel A
Institut für Mikrobiologie, Westfälische Wilhelms-Universität, Correnstrasse 3, D-48149 Münster, Germany.
Appl Environ Microbiol. 2001 May;67(5):2176-82. doi: 10.1128/AEM.67.5.2176-2182.2001.
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.
聚球藻属PCC6308藻青素合成酶通过三步从大肠杆菌重组细胞中纯化了72倍，这三步分别为：在Q Sepharose上进行阴离子交换色谱、在三嗪染料基质Procion Blue HE-RD Sepharose上进行亲和色谱以及在Superdex 200 HR上进行凝胶过滤。该天然酶催化精氨酸和天冬氨酸掺入藻青素，其表观分子量为240±30 kDa，由85±5 kDa的相同亚基组成。精氨酸（49 μM）、天冬氨酸（0.45 mM）和ATP（0.20 mM）的K(m)值表明该酶对这些底物具有高亲和力。在体外藻青素合成过程中，每掺入1摩尔氨基酸会将1.3±0.1摩尔ATP转化为ADP。该酶催化反应的最适条件分别为pH 8.2和50℃。当将精氨酸甲酯（抑制率分别为99.5%和97%）、精氨酰胺（99%和96%）、S-(2-氨基乙基)半胱氨酸（43%和42%）、β-羟基天冬氨酸（35%和37%）、天冬氨酸β-甲酯（38%和40%）、正缬氨酸（0%和3%）、瓜氨酸（9%和7%）以及天冬酰胺（2%和0%）添加到完整反应混合物中时，它们分别对精氨酸和天冬氨酸的掺入表现出几乎相同的抑制作用。相比之下，刀豆氨酸（抑制率分别为82%和53%）、赖氨酸（36%和19%）、胍丁胺（33%和25%）、D-天冬氨酸（37%和30%）、L-谷氨酸（13%和5%）以及鸟氨酸（23%和11%）分别使精氨酸的掺入比天冬氨酸的掺入减少得更多。另一方面，刀豆氨酸（最大活性的45%）和赖氨酸（13%）刺激天冬氨酸的掺入，而天冬氨酸β-甲酯（53%）和天冬酰胺（9%）刺激精氨酸的掺入。当[(3)H]赖氨酸（最大活性的15%）和[(3)H]刀豆氨酸（13%）分别替代精氨酸使用或添加到完整反应混合物中时，它们会掺入聚合物中，而L-谷氨酸不掺入。添加其他氨基酸（即丙氨酸、组氨酸、亮氨酸、脯氨酸、色氨酸和甘氨酸）对精氨酸掺入没有影响。各种化学合成的聚-α,β-D,L-天冬氨酸样品可作为体外合成藻青素的引物，而聚-α-L-天冬氨酸几乎无活性。
