Crystal Structure of Pyridoxal Kinase from the Escherichia coli pdxK Gene: Implications for the Classification of Pyridoxal Kinases

Abstract

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...

Recombinant expression, purification and characterization of Bombyx mori (Lepidoptera: Bombycidae) pyridoxal kinase

“…It was not possible to detect inhibition by Zn 2+ because the substrate concentrations were so low that the initial velocity could not be measured accurately. The mechanism of phosphorylation has been elucidated for sheep and E. coli enzymes, and follows a random sequential substrate addition (Li et al, 2002(Li et al, , 2004Safo et al, 2004Safo et al, , 2006. Using PL as the variable substrate and ATP as the fixed substrate, a double reciprocal plot of initial velocity also suggests a sequential catalytic mechanism for the B. mori PLK.…”

“…Sheep brain PLK monomer contains 9 -helices, 10 -strands and 3 segments of 310 helices (Li et al, 2002). The monomer of PLK from a prokaryote cell encoded by a pdxK gene consists of eight -helices and nine ß-strands (Safo et al, 2006). It is hypothesized, based on homology modelling, that the B. mori PLK monomer contains eight -helices ( 1-8), nine -strands ( 1-9) and two segments of 310 helices.…”

“…Several mammalian PLKs have been successfully expressed in Escherichia coli and the expressed recombinant proteins exhibit the same biochemical characteristics as those of the native protein purified from various mammalian tissues (Gao et al, 1998;Lee et al, 2000;Di Salvo et al, 2004). Recently, the three-dimensional structures of PLKs from bacteria and mammals, alone and in complex with various ligands were determined, providing a better understanding of the catalytic mechanism of PLK (Li et al, 2002(Li et al, , 2004Safo et al, 2004Safo et al, , 2006Tang et al, 2005;Cao et al, 2006). The active sites are structurally very similar among members of the ribokinase superfamily .…”

“…Compared with B. mori PLK, the sequence exhibits extension of the N-terminal in plants, and increase in nonconservative residues in mammal. Secondly, the key peptide loop, which is thought to play a significant role in the functioning of PLK (Safo et al, 2006), consists of eight residues in B. mori PLK and 12 in mammals. Fig.…”

Molecular Defects of Vitamin B6 Metabolism Associated with Neonatal Epileptic Encephalopathy

“…Inhibition of PNPOx activity by the PLP product has been observed in the E. coli enzyme, with a K i of 8 µM [28]. Also, substrate inhibition of E. coli PL kinase by MgATP has been observed in the presence of either PNP or PLP [29]. A concurring mechanism for maintaining low levels of free PLP is expected to be its dephosphorylation by phosphatases.…”