Abstract
Pyridoxal kinase (PLK; EC 2.7.1.35) is a key enzyme in the metabolism of vitamin B6 (VB6) in Bombyx mori. A fusion expressional vector pET-22b-BPLK-His was constructed using a sub-cloning technique, the recombinant B. mori PLK was then expressed in Escherichia coli, purified and characterized. Bioinformatics were used to deduce the protein structure and genomic organization of this enzyme. Using Ni Sepharose affinity column chromatography, the recombinant protein was purified to very high degree (approximately 90%). The recombinant PLK exhibits a high specific enzymatic activity (1800 nmol/min/mg of protein). The maximum catalytic activity of this enzyme was recorded over a narrow pH range (5.5-6.0) and Zn2+ is the most effective cation for catalysis under saturating substrate concentrations. When only triethanolamine is present as the cation, K+ is an activator of PLK. A double reciprocal plot of initial velocity suggests that the enzyme catalyses the reaction by means of a sequential catalytic mechanism. Under optimal conditions, the Km value for the substrates of ATP and pyridoxal are 57.9 ± 5.1 and 44.1 ± 3.9 µM. B. mori's genome contains a single copy of the PLK gene, which is 7.73 kb long and contains five exons and four introns, and is located on the eighth chromosome. The PLK may be a dimer with two identical subunits under native conditions, and it is hypothesized that each monomer contains eight α-helices (α1-8), nine β-strands (β1-9) and two segments of 310 helices.
Highlights
Vitamin B6 (VB6) exists in various forms, pyridoxal (PL), pyridoxine (PN), pyridoxamine (PM) and their phosphorylated derivatives: Pyridoxal 5'-phosphate (PLP), pyridoxine 5’-phosphate (PNP) and pyridoxamine 5’-phosphate (PMP)
Several mammalian pyridoxal kinase (PLK) 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)
DNA sequencing demonstrated that the PCR product had a B. mori PLK gene and a C-terminal hexa-histidine tag sequence
Summary
Vitamin B6 (VB6) exists in various forms, pyridoxal (PL), pyridoxine (PN), pyridoxamine (PM) and their phosphorylated derivatives: Pyridoxal 5'-phosphate (PLP), pyridoxine 5’-phosphate (PNP) and pyridoxamine 5’-phosphate (PMP). Pyridoxal kinase (PLK) (EC 2.7.1.35) catalyzes the ATP-dependent phosphorylation of PL, PM and PN to form PLP, PMP and PNP, respectively. The gene encoding PLK has been isolated from mammals (Hanna et al, 1997; Gao et al, 1998; Maras et al, 1999), microorganisms (Yang et al, 1996, 1998; Scott & Phillps, 1997) and plants (Lum et al, 2002; Wang et al, 2004). 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, 2004; Safo et al, 2004, 2006; Tang et al, 2005; Cao et al, 2006). The location and mode of substrate binding in human PLK have been deduced by analogy with the structures of sheep brain PLK in complex with various substrates (Cao et al, 2006)
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