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Bombyx mori pyridoxal kinase cDNA cloning and enzymatic characterization
Ruijun Shi1, Jianyun Zhang, Changjun Jiang
1Key Laboratory of Tea Biochemistry & Biotechnology, Ministry of Education and Ministry of Agriculture, Anhui Agricultural University, Hefei 230036, China.
Abstract:
Pyridoxal kinase (PLK) (EC 2.7.1.35) catalyzes the ATP-dependent phosphorylation of pyridoxal, generating pyridoxal-5.-phosphate (PLP), an important cofactor for many enzymatic reactions. Bombyx mori, similar to mammals, relies on a nutritional source of vitamin B6 to synthesize PLP. This article describes how a cDNA encoding PLK was cloned from Bombyx mori using the PCR method (GenBank accession number: DQ452397). The cDNA has an 894 bp open reading frame and encodes a protein of 298 amino acid residues with a molecular mass of 33.1 kDa. The amino acid sequence shares 48.6% identity with that of human PLK, and it also contains signature conserved motifs of the PLK family. However, the protein is 10 or more amino acids shorter than the PLK from mammals and plants, and several amino acid residues conserved in the PLK from mammals and plants are changed in the protein. The cDNA cloned was expressed successfully in Escherichia coli using the T7 promoter/T7 RNA polymerase expression system, and the crude extracts containing the expressed product were found to have strong PLK enzymatic activity with a value of 30 nmol/min/mg, confirming that the cDNA encodes the functional PLK of Bombyx mori. This is the first identification of a gene encoding PLK in insects.
Insights
Researchers identified and cloned the pyridoxal kinase (PLK) gene from Bombyx mori, confirming its functional expression. This discovery marks the first identification of a PLK gene in insects, crucial for vitamin B6 metabolism.
Area of Science:
- Biochemistry
- Molecular Biology
- Genetics
Background:
- Pyridoxal kinase (PLK) is essential for synthesizing pyridoxal-5'-phosphate (PLP), a vital cofactor derived from vitamin B6.
- Bombyx mori, like mammals, requires dietary vitamin B6 for PLP synthesis, indicating a conserved metabolic pathway.
Purpose of the Study:
- To clone and characterize the cDNA encoding pyridoxal kinase (PLK) from Bombyx mori.
- To confirm the functional activity of the cloned PLK enzyme.
Main Methods:
- Polymerase Chain Reaction (PCR) was used to clone the PLK cDNA from Bombyx mori.
- The cloned cDNA was expressed in Escherichia coli using a T7 promoter system.
- Enzymatic activity assays were performed on the expressed protein.
Main Results:
- An 894 bp cDNA encoding a 298-amino acid protein (33.1 kDa) was successfully cloned.
- The Bombyx mori PLK shares 48.6% identity with human PLK but exhibits unique structural differences.
- Expressed protein demonstrated significant PLK enzymatic activity (30 nmol/min/mg).
Conclusions:
- The study successfully identified and cloned the functional PLK gene from Bombyx mori.
- This represents the first reported PLK gene identification in insects.
- The findings contribute to understanding vitamin B6 metabolism and PLK function in insects.
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