摘 要: 蓝藻琥珀酸半醛脱氢酶催化琥珀酸半醛到琥珀酸的转化, 使得蓝藻的三羧酸循环变得完整。BLAST序列分析预测鱼腥藻PCC7120中all3556基因编码琥珀酸半醛脱氢酶。为了证实all3556基因编码蛋白的催化功能, 构建了pET28a-all3556表达质粒, 并在大肠杆菌BL21(DE3)菌体中进行重组蛋白诱导表达; 利用镍亲和层析方法对all3556蛋白进行了分离纯化。酶动力学测试表明all3556蛋白是一个NADP+-依赖型的琥珀酸半醛脱氢酶。生物信息学分析发现all3556蛋白和其他来源的琥珀酸半醛脱氢酶的氨基酸序列具有一定的同源性, 在催化中心的氨基酸残基高度保守。

Abstract: Succinic semialdehyde dehydrogenases (SSADHs) of cyanobacteria catalyze the oxidation of succinic semialdehyde to succinic acid and complete the cyanobacterial TCA cycle. The all3556 gene of Anabaena sp. PCC7120 was predicted to encode a succinic semialdehyde dehydrogenase by BLAST analysis. To prove the catalytic function of the protein encoded by all3556 gene, the plasmid pET28a-all3556 was constructed and transformed into E. coli BL21(DE3) competent cells. Then the all3556 protein was purified by Ni-NTA affinity chromatography. Steady-state kinetic studies showed that all3556 was a NADP+-dependent succinic semialdehyde dehydrogenase. Bioinformatic analysis indicated that all3556 protein and other succinic semialdehyde dehydrogenases had identities in amino acid sequences and that the residues in the catalytic center were highly conserved in different succinic semialdehyde dehydrogenases.