Pyridoxal kinase knockout of Dictyostelium complemented by the human homologue

The gene (pykA) encoding pyridoxal kinase which converts pyridoxal (vitamin Bs) to pyridoxal phosphate was isolated from Dictyostelium discoideum usin g insertional mutagenesis. Cells of a pykA gene knockout grew poorly in axe nic medium with low yield but growth was restored by the addition of pyrido xal phosphate. Sequencing indicated a gene, with one intron, encoding a pre dicted protein of 301 amino acids that was 42% identical in amino acid sequ ence to human pyridoxal kinase. After expression of the wild-type gene in E scherichia coli, the purified PykA protein product was shown to have pyrido xal kinase enzymatic activity with a K-m of 8.7 pM for pyridoxal. Transform ation of the Dictyostelium knockout mutant with the human pyridoxal kinase gene gave almost the same level of complementation as that seen using trans formation with the wild-type Dictyostelium gene. Phylogenetic analysis indi cated that the Dictyostelium amino acid sequence was closer to human pyrido xal kinase than to pyridoxal kinases of lower eukaryotes. (C) 2000 Federati on of European Microbiological Societies. Published by Elsevier Science B.V . All rights reserved.