Pyridoxal kinase knockout of Dictyostelium complemented by the human homologue |
| |
Authors: | Guo K Newell P C |
| |
Institution: | Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, UK. |
| |
Abstract: | The gene (pykA) encoding pyridoxal kinase which converts pyridoxal (vitamin B(6)) to pyridoxal phosphate was isolated from Dictyostelium discoideum using insertional mutagenesis. Cells of a pykA gene knockout grew poorly in axenic medium with low yield but growth was restored by the addition of pyridoxal phosphate. Sequencing indicated a gene, with one intron, encoding a predicted protein of 301 amino acids that was 42% identical in amino acid sequence to human pyridoxal kinase. After expression of the wild-type gene in Escherichia coli, the purified PykA protein product was shown to have pyridoxal kinase enzymatic activity with a K(m) of 8.7 microM for pyridoxal. Transformation of the Dictyostelium knockout mutant with the human pyridoxal kinase gene gave almost the same level of complementation as that seen using transformation with the wild-type Dictyostelium gene. Phylogenetic analysis indicated that the Dictyostelium amino acid sequence was closer to human pyridoxal kinase than to pyridoxal kinases of lower eukaryotes. |
| |
Keywords: | Pyridoxal kinase Vitamin B6 Dictyostelium |
本文献已被 PubMed 等数据库收录! |
|