Recombinant expression, purification and characterization of Bombyx mori (Lepidoptera: Bombycidae) pyridoxal kinase
Pyridoxal kinase (PLK; EC 2.7.1.35) is a key enzyme in the metabolism of vitamin B6 (VB6) in Bombyx mori. A fusion expressional vector pET-22b-BPLK-His was constructed using a sub-cloning technique, the recombinant B. mori PLK was then expressed in Escherichia coli, purified and characterized. Bioin...
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| Format: | Article |
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Institute of Entomology, Biology Centre, Czech Academy of Science
2011-01-01
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| Series: | European Journal of Entomology |
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| Online Access: | https://www.eje.cz/artkey/eje-201101-0003_Recombinant_expression_purification_and_characterization_of_Bombyx_mori_Lepidoptera_Bombycidae_pyridoxal_ki.php |
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| author | Shuo-Hao HUANG Wang MA Ping-Ping ZHANG Jian-Yun ZHANG Yan-Feng XIE Long-Quan HUANG |
| author_facet | Shuo-Hao HUANG Wang MA Ping-Ping ZHANG Jian-Yun ZHANG Yan-Feng XIE Long-Quan HUANG |
| author_sort | Shuo-Hao HUANG |
| collection | DOAJ |
| description | Pyridoxal kinase (PLK; EC 2.7.1.35) is a key enzyme in the metabolism of vitamin B6 (VB6) in Bombyx mori. A fusion expressional vector pET-22b-BPLK-His was constructed using a sub-cloning technique, the recombinant B. mori PLK was then expressed in Escherichia coli, purified and characterized. Bioinformatics were used to deduce the protein structure and genomic organization of this enzyme. Using Ni Sepharose affinity column chromatography, the recombinant protein was purified to very high degree (approximately 90%). The recombinant PLK exhibits a high specific enzymatic activity (1800 nmol/min/mg of protein). The maximum catalytic activity of this enzyme was recorded over a narrow pH range (5.5-6.0) and Zn2+ is the most effective cation for catalysis under saturating substrate concentrations. When only triethanolamine is present as the cation, K+ is an activator of PLK. A double reciprocal plot of initial velocity suggests that the enzyme catalyses the reaction by means of a sequential catalytic mechanism. Under optimal conditions, the Km value for the substrates of ATP and pyridoxal are 57.9 ± 5.1 and 44.1 ± 3.9 µM. B. mori's genome contains a single copy of the PLK gene, which is 7.73 kb long and contains five exons and four introns, and is located on the eighth chromosome. The PLK may be a dimer with two identical subunits under native conditions, and it is hypothesized that each monomer contains eight α-helices (α1-8), nine β-strands (β1-9) and two segments of 310 helices. |
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| institution | Directory Open Access Journal |
| issn | 1210-5759 1802-8829 |
| language | English |
| last_indexed | 2024-12-22T12:28:57Z |
| publishDate | 2011-01-01 |
| publisher | Institute of Entomology, Biology Centre, Czech Academy of Science |
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| series | European Journal of Entomology |
| spelling | doaj.art-fa74038dc7674d1f98aa94c920b2151e2022-12-21T18:25:43ZengInstitute of Entomology, Biology Centre, Czech Academy of ScienceEuropean Journal of Entomology1210-57591802-88292011-01-011081253410.14411/eje.2011.003eje-201101-0003Recombinant expression, purification and characterization of Bombyx mori (Lepidoptera: Bombycidae) pyridoxal kinaseShuo-Hao HUANG0Wang MA1Ping-Ping ZHANG2Jian-Yun ZHANG3Yan-Feng XIE4Long-Quan HUANG5Key Laboratory of Tea Biochemistry & Biotechnology of Ministry of Education and Ministry of Agriculture, Anhui Agricultural University, Hefei 230036, People's Republic of ChinaKey Laboratory of Tea Biochemistry & Biotechnology of Ministry of Education and Ministry of Agriculture, Anhui Agricultural University, Hefei 230036, People's Republic of ChinaCollege of Life Science, Anhui Agricultural University, Hefei 230036, People's Republic of ChinaCollege of Life Science, Anhui Agricultural University, Hefei 230036, People's Republic of ChinaKey Laboratory of Tea Biochemistry & Biotechnology of Ministry of Education and Ministry of Agriculture, Anhui Agricultural University, Hefei 230036, People's Republic of ChinaKey Laboratory of Tea Biochemistry & Biotechnology of Ministry of Education and Ministry of Agriculture, Anhui Agricultural University, Hefei 230036, People's Republic of ChinaPyridoxal kinase (PLK; EC 2.7.1.35) is a key enzyme in the metabolism of vitamin B6 (VB6) in Bombyx mori. A fusion expressional vector pET-22b-BPLK-His was constructed using a sub-cloning technique, the recombinant B. mori PLK was then expressed in Escherichia coli, purified and characterized. Bioinformatics were used to deduce the protein structure and genomic organization of this enzyme. Using Ni Sepharose affinity column chromatography, the recombinant protein was purified to very high degree (approximately 90%). The recombinant PLK exhibits a high specific enzymatic activity (1800 nmol/min/mg of protein). The maximum catalytic activity of this enzyme was recorded over a narrow pH range (5.5-6.0) and Zn2+ is the most effective cation for catalysis under saturating substrate concentrations. When only triethanolamine is present as the cation, K+ is an activator of PLK. A double reciprocal plot of initial velocity suggests that the enzyme catalyses the reaction by means of a sequential catalytic mechanism. Under optimal conditions, the Km value for the substrates of ATP and pyridoxal are 57.9 ± 5.1 and 44.1 ± 3.9 µM. B. mori's genome contains a single copy of the PLK gene, which is 7.73 kb long and contains five exons and four introns, and is located on the eighth chromosome. The PLK may be a dimer with two identical subunits under native conditions, and it is hypothesized that each monomer contains eight α-helices (α1-8), nine β-strands (β1-9) and two segments of 310 helices.https://www.eje.cz/artkey/eje-201101-0003_Recombinant_expression_purification_and_characterization_of_Bombyx_mori_Lepidoptera_Bombycidae_pyridoxal_ki.phpbombyx moripyridoxal kinaserecombinant expressionpurificationcharacterization |
| spellingShingle | Shuo-Hao HUANG Wang MA Ping-Ping ZHANG Jian-Yun ZHANG Yan-Feng XIE Long-Quan HUANG Recombinant expression, purification and characterization of Bombyx mori (Lepidoptera: Bombycidae) pyridoxal kinase European Journal of Entomology bombyx mori pyridoxal kinase recombinant expression purification characterization |
| title | Recombinant expression, purification and characterization of Bombyx mori (Lepidoptera: Bombycidae) pyridoxal kinase |
| title_full | Recombinant expression, purification and characterization of Bombyx mori (Lepidoptera: Bombycidae) pyridoxal kinase |
| title_fullStr | Recombinant expression, purification and characterization of Bombyx mori (Lepidoptera: Bombycidae) pyridoxal kinase |
| title_full_unstemmed | Recombinant expression, purification and characterization of Bombyx mori (Lepidoptera: Bombycidae) pyridoxal kinase |
| title_short | Recombinant expression, purification and characterization of Bombyx mori (Lepidoptera: Bombycidae) pyridoxal kinase |
| title_sort | recombinant expression purification and characterization of bombyx mori lepidoptera bombycidae pyridoxal kinase |
| topic | bombyx mori pyridoxal kinase recombinant expression purification characterization |
| url | https://www.eje.cz/artkey/eje-201101-0003_Recombinant_expression_purification_and_characterization_of_Bombyx_mori_Lepidoptera_Bombycidae_pyridoxal_ki.php |
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