Isolation and characterization of the 5′-flanking region of the human PDXK gene

Pyridoxal kinase is a key enzyme for the biosynthesis of pyridoxal 5'-phosphate. Pyridoxal 5'-phosphate is the catalytically active form of vitamin B-6, and acts as a cofactor in > 140 different enzyme reactions. It is still unknown how the kinase synthesis is regulated in the cells, and nothing has been reported about the gene promoter. In the present study, based on the bioinformatics analysis of the 5'-flanking region of the human PDXK gene, we cloned the promoter region by PCR. Through the construction of a series of luciferase expression vectors containing the human PDXK promoter region, we characterized the promoter in terms of its structure and function. The transcription start site is at 198 bp upstream of the ATG translation initiation site. An important regulatory region is located at - 665/ - 433 bp upstream of the transcription start site. The promoter lacks the canonical TATA box, but contains three GC-boxes and one E-box. A deletion and mutation experiment revealed that the transcription factor Sp1 binding site C (- 553/ - 543) is critical in maintaining the robust promoter activity. Knockdown of Sp1 by RNA interference and chromatin immunoprecipitation analysis further proved that the Sp1 is involved in the regulation of the PDXK gene expression.