Crystal structure and catalytic mechanism of pyridoxal kinase from Pseudomonas aeruginosa

Abstract

Pyridoxal kinase is a ubiquitous enzyme essential for pyridoxal 5′-phosphate (PLP) homeostasis since PLP is required for the catalytic activity of a variety of PLP-dependent enzymes involved in amino acid, lipid, and sugar metabolism as well as neurotransmitter biosynthesis. Previously, two catalytic mechanisms were proposed with regard to Pdx kinases, in which either the aspartate or the cysteine residue is involved as a catalytic residue. Because the Pdx kinase of Pseudomonas aeruginosa (PaPdxK) contains both residues, the catalytic mechanism of PaPdxK remains elusive. To elucidate the substrate-recognition and catalytic mechanisms of PaPdxK, the crystal structure of PaPdxK was determined at a 2.0 Å resolution. The PaPdxK structure possesses a channel that can accommodate substrates and a metallic cofactor. Our structure-based biochemical and mutational analyses in combination with modeling studies suggest that PaPdxK catalysis is mediated by an acid-base mechanism through the catalytic acid Asp225 and a helical dipole moment.