Crystal Structure of the PdxY Protein from Escherichia coli

Abstract

The crystal structure of Escherichia coli PdxY, the protein product of the pdxY gene, has been determined to a 2.2-Å resolution. PdxY is a member of the ribokinase superfamily of enzymes and has sequence homology with pyridoxal kinases that phosphorylate pyridoxal at the C-5 hydroxyl. The protein is a homodimer with an active site on each monomer composed of residues that come exclusively from each respective subunit. The active site is filled with a density that fits that of pyridoxal. In monomer A, the ligand appears to be covalently attached to Cys122 as a thiohemiacetal, while in monomer B it is not covalently attached but appears to be partially present as pyridoxal 5-phosphate. The presence of pyridoxal phosphate and pyridoxal as ligands was confirmed by the activation of aposerine hydroxymethyltransferase after release of the ligand by the denaturation of PdxY. The ligand, which appears to be covalently attached to Cys122, does not dissociate after denaturation of the protein. A detailed comparison (of functional properties, sequence homology, active site and ATPbinding-site residues, and active site flap types) of PdxY with other pyridoxal kinases as well as the ribokinase superfamily in general suggested that PdxY is a member of a new subclass of the ribokinase superfamily. The structure of PdxY also permitted an interpretation of work that was previously published about this enzyme.Pyridoxal phosphate (PLP) serves as a cofactor for many enzymes that are involved in amino acid and sugar metabolism. In many bacteria and plants, PLP is synthesized by a de novo pathway, but most cells rely on a nutritional source of vitamin B 6 , i.e., pyridoxine (PN), pyridoxal (PL), or pyridoxamine (PM) (18). All cells, however, have a salvage pathway for reutilizing the PLP that is liberated during protein turnover (21). The salvage pathway involves an ATP-dependent pyridoxal kinase that phosphorylates PL, PN, and PM. The product of PN and PM phosphorylation is converted to PLP by pyridoxine 5-phosphate oxidase, which is also expressed in most cells. Because those PL kinases that have been purified and studied show activity toward PN and PM in addition to PL, they are often referred to as PL/PN/PM kinases. For the sake of brevity, we will refer to these enzymes as PL kinases with the understanding that many of them exhibit considerable activity toward PN and PM.

Crystal Structure of Pyridoxal Kinase in Complex with Roscovitine and Derivatives

“…This effect of ATP might provide a safety mechanism for mammalian cells to prevent molecules other than vitamin B 6 from binding to the active site under the pre-reaction state, thus blocking their effects on PDXK. Our results revealed that the pyridoxal binding pocket has a surprising ability to accommodate large molecules such as roscovitine that are different from vitamin B 6 , and roscovitine analogs with N 6 modifications show promise as specific inhibitors for PDXK.…”

“…We therefore synthesized two roscovitine analogs with modifications designed so that the H-bonding possibility at N 6 would be prevented (Table II). Either methylation on N 6 or replacement of N 6 by an oxygen, leading, respectively, to N 6 -methyl-(R)-roscovitine (2) and O 6 -(R)-roscovitine (4), converted roscovitine to derivatives devoid of protein kinase inhibitory activity (Table II).…”

“…The mechanisms underlying pyridoxal kinase inhibition by several drugs have been studied (5). Recently, the crystal structure of PdxY, a PDXK homology protein of Escherichia coli was reported (6). Concurrently, the three-dimensional structures of sheep brain PDXK alone and in complex with various ligands (PDXK/ATP, PDXK/AMP-PCP/pyridoxamine, PDXK/ADP/PLP, and PDXK/ ADP) have been determined, providing a better understanding of the catalysis mechanism of PDXK (7,8).…”

“…Pyridoxal kinase (PDXK) 1 catalyzes the phosphorylation of the three forms of vitamin B 6 (pyridoxal, pyridoxamine, and pyridoxine) in the presence of ATP and Zn 2ϩ . This phosphorylation constitutes an essential step in the synthesis of pyridoxal 5Ј-phosphate (PLP), the intracellular active form of vitamin B 6 , a key cofactor for at least 140 enzymes, such as aminotransferases and decarboxylases (1)(2)(3).…”

“…This phosphorylation constitutes an essential step in the synthesis of pyridoxal 5Ј-phosphate (PLP), the intracellular active form of vitamin B 6 , a key cofactor for at least 140 enzymes, such as aminotransferases and decarboxylases (1)(2)(3). PDXK is connected to nervous system functions, because many neurotransmitters such as dopamine, norepinephrine, serotonin, and ␥-aminobutyric acid are synthesized by PLP-dependent enzymes (4).…”