Structure of pyridoxal kinase from sheep brain and role of the tryptophanyl residues

The primary structure of sheep brain pyridoxal kinase has been determined b y direct chemical and physical methods. The enzyme contains 312 amino acid residues with an acetylated methionine at the N-terminus, yielding a molecu lar mass of 34,861 Da. The functional role played by the two tryptophanyl r esidues in positions 52 and 244 of the polypeptide chain has been investiga ted by fluorescence spectroscopy. The tryptophanyl residues are not complet ely exposed to the rapidly relaxing solvent and they are poorly accessible to collisional quenchers. Chemical modification with NBS abolishes the cata lytic activity of the kinase. The amino acid sequence of the sheep brain en zyme shows high similarity (86.2% identity) with the human pyridoxal kinase recently reported [Hanna, Turner, and Kirkness, (1997), J. Biol. Chem. 272 , 10756-10760]. Comparison of the mammalian proteins with bacterial and yea st putative pyridoxal kinases retrieved from the Swiss-Prot data bank shows a low degree of overall similarity. In particular, the putative ATP-bindin g domain is conserved, whereas the region that appears to be crucial in the binding of the pyridoxal substrate is not. Thus, the assignment of the bac terial and yeast cDNA-deduced proteins as pyridoxal kinases should be taken with caution.