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Guo, K and Newell, PC (2000) Pyridoxal kinase knockout of Dictyostelium complemented by the human homologue. FEMS Microbiol. Lett. 189:195-200

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.

PubMed

Amino Acid Sequence; Animals; Dictyostelium/genetics; Gene Expression Regulation, Enzymologic; Humans; Molecular Sequence Data; Mutagenesis, Insertional; Pyridoxal Kinase/genetics; Sequence Alignment; Sequence Homology, Amino Acid