Biochemical Characterization and Structural Analysis of N-acetylornithine Transaminase from Synechocystis sp. PCC6803

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

Abstract

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

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.

Figures/Tables 8

Table 1 PCR primer sequences

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

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

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)

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

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

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

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

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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