The pdxK and pdxY genes have been found to code for pyridoxal kinases, enzymes involved in the pyridoxal phosphate salvage pathway. Two pyridoxal kinase structures have recently been published, including Escherichia coli pyridoxal kinase 2 (ePL kinase 2) and sheep pyridoxal kinase, products of the pdxY and pdxK genes, respectively. We now report the crystal structure of E. coli pyridoxal kinase 1 (ePL kinase 1), encoded by a pdxK gene, and an isoform of ePL kinase 2. The structures were determined in the unliganded and binary complexes with either MgATP or pyridoxal to 2.1-, 2.6-, and 3.2-Å resolutions, respectively. The active site of ePL kinase 1 does not show significant conformational change upon binding of either pyridoxal or MgATP. Like sheep PL kinase, ePL kinase 1 exhibits a sequential random mechanism. Unlike sheep pyridoxal kinase, ePL kinase 1 may not tolerate wide variation in the size and chemical nature of the 4 substituent on the substrate. This is the result of differences in a key residue at position 59 on a loop (loop II) that partially forms the active site. Residue 59, which is His in ePL kinase 1, interacts with the formyl group at C-4 of pyridoxal and may also determine if residues from another loop (loop I) can fill the active site in the absence of the substrate. Both loop I and loop II are suggested to play significant roles in the functions of PL kinases.Pyridoxal 5Ј-phosphate (PLP) serves as a cofactor for many enzymes 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), and pyridoxamine (PM) (24). Most cell types have a salvage pathway for reutilizing the PL liberated during protein turnover (27). The salvage pathway involves an ATP-dependent pyridoxal kinase that phosphorylates PL, PN, and PM by transferring the terminal phosphate of ATP to the 5Ј-hydroxyl group of these substrates. The product of PN and PM phosphorylation is converted to PLP by pyridoxine 5Ј-phosphate oxidase, which is also expressed in most cells. The PL kinases that have been purified and studied show activity with PN and PM, in addition to PL, and are often referred to as PL/PN/PM kinases (14, 15). For brevity, we will refer to these enzymes as PL kinases with the understanding that many of them exhibit considerable activity with PN and PM.PL kinase has been purified from bacterial, plant, and mammalian sources, and evidence suggests that most organisms contain a single PL kinase, coded by a pdxK gene. However, studies with Escherichia coli mutants that were blocked in the de novo biosynthetic pathway and with an inactivated pdxK gene, which codes for E. coli pyridoxal kinase 1 (ePL kinase 1), were still able to grow on pyridoxal (26). A search for a gene that expressed a protein that had PL kinase activity in E. coli led to the discovery of the pdxY gene (26). The E. coli protein ePL kinase 2, expressed by this gene, was shown to have some PL kinase act...
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.
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