集胞藻PCC6803中N-乙酰鸟氨酸转氨酶的生化表征及结构分析

doi:10.13560/j.cnki.biotech.bull.1985.2020-1149

摘要/Abstract

摘要：

旨在对集胞藻PCC6803中slr1022基因编码的N-乙酰鸟氨酸转氨酶进行生化表征及结构分析,为进一步研究该酶的催化功能及机制奠定理论基础。以集胞藻PCC6803基因组为模板,通过PCR扩增获得slr1022基因,将其连接到表达载体pET-28a上,转化大肠杆菌BL21（DE3）感受态。经IPTG诱导,Ni-NTA亲和层析纯化后获得重组Slr1022蛋白,然后通过紫外分光光度法对Slr1022蛋白的催化功能进行表征,并运用生物信息学软件对该蛋白进行结构分析。成功构建pET28a-slr1022重组表达质粒,并诱导表达重组Slr1022蛋白,SDS-PAGE电泳鉴定该蛋白分子量约为50 kD,与理论大小相符。Slr1022蛋白与底物N-乙酰鸟氨酸的结合常数K_m和最大反应速度V_max分别是0.12 mmol/L和0.60 μmol/（L·s）,并且Slr1022蛋白与另一底物α-酮戊二酸的K_m和V_max分别是0.039 mmol/L和0.65 μmol/（L·s）。Slr1022蛋白在pH 8.5时催化活性最强。Slr1022蛋白和其他来源的N-乙酰鸟氨酸转氨酶氨基酸序列有一定的同源性,而且活性位点的氨基酸残基高度保守。成功克隆并表达纯化出Slr1022蛋白,酶学性质和生物信息学研究表明Slr1022蛋白为N-乙酰鸟氨酸转氨酶。

关键词: N-乙酰鸟氨酸转氨酶, 原核表达, 生化表征, 结构分析, 集胞藻PCC6803

Abstract:

The aim of this study is to characterize the biochemical and structural properties of N-acetylornithine transaminase encoded by slr1022 gene from Synechocystis sp. PCC6803,for laying theoretical foundation in further study on the catalytic function and mechanism of the enzyme. The slr1022 gene was obtained through PCR amplification using the genomic DNA of Synechocystis sp. PCC6803 as template,and then was linked to the expression vector pET-28a. The constructed plasmid was transformed into Escherichia coli strain BL21（DE3）. The recombinant Slr1022 protein was purified by Ni-NTA affinity chromatography after the expressing strain was induced by IPTG. The catalytic function of the Slr1022 protein was characterized preliminarily through UV spectrophotometric methods,and the structure of the Slr1022 protein was analyzed by bioinformatics software. The recombinant expressing plasmid of the pET28a-slr1022 was constructed and the Slr1022 protein was expressed successfully. The molecular weight of the protein by SDS-PAGE was about 50 kD,which was consistent with the theoretical size. The binding constant K_m and maximum reaction velocity V_max of the Slr1022 protein with substrate N-acetylornithine were 0.12 mmol/L and 0.60 μmol/（L·s）,respectively,and the K_m and V_max of Slr1022 protein with α-ketoglutarate were 0.039 mmol/L and 0.65 μmol/（L·s）,respectively. Slr1022 protein had the highest catalytic activity at pH 8.5. The amino acid sequence of Slr1022 protein and other N-acetylornithine transaminase from different sources shared certain homology,and the amino acid residues of the active site were highly conserved. In conclusion,the slr1022 gene is successfully cloned and the Slr1022 protein is expressed and purified,enzymatic properties and bioinformatics analysis demonstrates that Slr1022 protein is N-acetylornithine transaminase.

Key words: N-acetylornithine transaminase, prokaryotic expression, biochemical characterization, structural analysis, Synechocystis sp. PCC6803

白福美, 李至敏, 王小琴, 胡紫微, 鲍玲玲, 李志敏. 集胞藻PCC6803中N-乙酰鸟氨酸转氨酶的生化表征及结构分析[J]. 生物技术通报, 2021, 37(5): 98-107.

BAI Fu-mei, LI Zhi-min, WANG Xiao-qin, HU Zi-wei, BAO Ling-ling, LI Zhi-min. Biochemical Characterization and Structural Analysis of N-acetylornithine Transaminase from Synechocystis sp. PCC6803[J]. Biotechnology Bulletin, 2021, 37(5): 98-107.

图/表 8

表1 PCR引物序列

Table 1 PCR primer sequences

Primer	Primer sequence（5'-3'）	Restriction enzyme
slr1022-F	GGAATTCCATATGACCTATTCC-CCTGTTGTTGAATC	Nde I
slr1022-R	CCGCTCGAGTCAAACCAAAGT-GGCGATCGCCTGAC	Xho I

图1 集胞藻PCC6803中slr1022基因克隆及pET28a-slr1022重组质粒的构建 A：slr1022基因PCR扩增产物。M：DNA分子标准;1-2：PCR扩增产物。B：slr1022基因和pET-28a载体双酶切产物。M：DNA分子标准;1-2：分别为pET-28a载体和slr1022基因双酶切产物。C：pET28a-slr1022重组质粒鉴定。M：DNA分子标准;1：正对照;2-8：菌落PCR产物

Fig. 1 Cloning of slr1022 gene from Synechocystis sp. PCC6803 and construction of its recombinant plasmid pET28a-slr1022 A: PCR amplification product of slr1022 gene. M: DNA marker. Lane 1-2: PCR product. B: Double enzymatic digestion products of slr1022 gene and pET-28a vector. M: DNA marker. Lane 1-2: Double enzymatic digestion product of pET-28a vector and slr1022 gene, respectively. C: Identification of recombinant plasmid pET28a-slr1022. M: DNA marker. Lane 1: Positive control. Lane 2-8: Colony PCR product

图2 重组Slr1022蛋白的诱导表达 M：蛋白质标准;1-3：pET-28a空载体的表达（分别为细胞破碎液,离心后上清液和离心后沉淀重悬液）;4-6：pET28a-slr1022重组质粒的表达（分别为细胞破碎液,离心后上清液和离心后沉淀重悬液）

Fig. 2 Expression of recombinant Slr1022 protein M: Protein marker. Lane 1-3: Expression of pET-28a plasmid (cell lysate, supernatant and pellet, respectively). Lane 4-6: Expression of pET28a-slr1022 recombinant plasmid (cell lysate, supernatant and pellet, respectively)

图3 重组Slr1022蛋白的亲和层析纯化结果电泳图 A：重组Slr1022蛋白的纯化。M：蛋白质标准;1-3：分别为细胞破碎液,离心后上清液和流穿液;4-8：分别为20、40、60、80和100 mmol/L 咪唑洗脱液;9-14：200 mmol/L 咪唑洗脱液。B：纯化后的Slr1022蛋白。M：蛋白质标准;1：纯化后的Slr1022蛋白

Fig. 3 SDS-PAGE of recombinant Slr1022 protein A: Purification of recombinant Slr1022 protein. M: Protein marker. Lane 1-3: Cell lysate, supernatant and flow through. Lane 4-8: 20, 40, 60, 80, 100 mmol/L imidazole elution. 9-14: 200 mmol/L imidazole elution. B: The purified Slr1022 protein. M: Protein marker. Lane 1: The purified Slr1022 protein

图4 重组Slr1022蛋白的酶动力学参数 A：初始反应速度随N-乙酰鸟氨酸浓度的变化曲线,固定α-酮戊二酸浓度为1 mmol/L;B：初始反应速度随α-酮戊二酸浓度的变化曲线,固定N-乙酰鸟氨酸浓度为2 mmol/L。数据以平均值 ± 标准差表示,n=3

Fig. 4 Kinetic profiles of recombinant Slr1022 protein A: Plot of the initial velocities as function of N-acetylornithine concentrations, the concentration of α-ketoglutarate was fixed at 1 mmol/L. B: Plot of the initial velocities as function of α-ketoglutarate concentrations, the concentration of N-acetylornithine was fixed at 2 mmol/L. Data are expressed as the mean ± standard deviation, n = 3

图5 pH对重组Slr1022蛋白催化活性的影响数据以平均值 ± 标准差表示,n=3

Fig. 5 Effects of pH on the catalytic activity of recombinant Slr1022 protein Data are expressed as the mean ± standard deviation, n = 3

图6 不同来源的N-乙酰鸟氨酸转氨酶的氨基酸序列比对 slr1022、2E54、6W7X、2EH6和2PB0分别为来源于集胞藻PCC6803、海栖热袍菌、嗜麦芽窄单胞菌、超嗜热菌和鼠伤寒沙门氏菌的N-乙酰鸟氨酸转氨酶。▲表示与PLP结合的氨基酸残基;★表示与PLP形成希夫碱的氨基酸残基;◆表示来自另一单体和PLP结合的氨基酸残基

Fig. 6 Protein sequences alignment of N-acetylornithine aminotransferases from different sources slr1022, 2E54, 6W7X, 2EH6 and 2PB0 were N-acetylornithine aminotransferases from Synechocystis PCC6803, Thermotoga maritima, Stenotrophomonas maltophilia, Aquifex aeolicus and Salmonella typhimurium respectively. Residues interacting with PLP are marked by▲, and★ indicates the residues forming Schiff base with PLP and ◆ indicates the residues from the other subunit which interact with PLP

图7 Slr1022蛋白分子同源建模结构 A：Slr1022蛋白的卡通示意图。B：活性中心放大示意图。C：活性中心保守氨基酸残基。α-螺旋、β-折叠及无规则卷曲的颜色分别为红色、黄色和绿色。辅因子PLP与保守氨基酸残基为棍棒结构,其碳原子颜色分别为浅蓝色与绿色。黄色虚线为与PLP相互作用的氢键。该模拟结构由SWISS-MODEL软件基于海栖热袍菌中N-乙酰鸟氨酸转氨酶晶体结构（PDB ID：2E54）绘制

Fig. 7 Homologous modeling structure of Slr1022 protein A: Cartoon structure of Slr1022 protein. B: Enlarged schematic diagram of active site. C: The conserved amino acid residues in the active site. The colors of α-helix, β-strands and loops are depicted as red, yellow and green, respectively. The cofactor PLP and the conserved amino acid residues are shown as sticks, and their carbon atoms are light blue and green, respectively. The yellow dotted lines indicate the hydrogen bonds interacting with PLP. The modeled structure is depicted by SWISS-MODEL based on the structure of N-acetylornithine aminotransferase of Thermotoga maritima (PBD: 2E54)

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