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

Abstract

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 Zn 2+ 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 -helices ( 1-8), nine -strands ( 1-9) and two segments of 310 helices.

Cloning and Characterization of Pyridoxal Kinase from Geobacillus sp. H6a

“…3a, SDS-PAGE showed single band of approximately 32 kDa in molecular weight. Molecular weight of single subunit of rGhPLK was similar to PLK reported from other organisms: 35 kDa from E. coli, 22 37 kDa from T. brucei, 23 35 kDa from A. thaliana, 8 and 33.9 kDa from B. mori, 13 The rGhPLK showed single band on Native PAGE (Fig. 3b), while gel filtration showed single peak of rGhPLK with molecular weight calculated approximately of 60 kDa (Fig.…”

“…5a). The rGhPLK was found to be active in the broad pH range when compared with PLK from A. thaliana, 8 S. typhimurium 15 and B. mori 13 which had activities in the narrow pH range of 5.5 -7.5, 6.2 -6.6 and 5.5 -6.0, respectively. The stability of rGhPLK between pH 3 and 10 was showed in Fig.…”

“…These data suggested that the rGhPLK was a dimer with two identical subunits, which was the same as other PLKs. 5,8,13,15,22,23…”

“…Mg 2+ has found to be the preferred divalent ion in PLK from S. typhimurium 15 and E. coli 9 under physiological conditions in cells. While PLK from other organisms prefers Zn 2+ such as B. mori, 13 A. thaliana 8 and human. 24…”

“…Pyridoxal kinase has been purified and characterized from Arabidopsis thaliana, 8 human, 9 Bacillus subtilis, 10 Plasmodia falciparum, 11 E. coli, 12 Bombyx mori, 13 Trypanosoma brucei, 14 Pseudomonas aeruginosa, 5 Salmonella typhimurium, 15 however, purification and characterization of PLK from thermophilic bacteria have not been reported.…”