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. 2014 Jul;76(7):1021-7.
doi: 10.1292/jvms.13-0623. Epub 2014 Apr 16.

Characterization of a functionally active recombinant 1-deoxy-D-xylulose-5-phosphate synthase from Babesia bovis

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Characterization of a functionally active recombinant 1-deoxy-D-xylulose-5-phosphate synthase from Babesia bovis

Jing Wang et al. J Vet Med Sci. 2014 Jul.

Abstract

The 1-deoxy-D-xylulose-5-phosphate synthase (DXS) enzyme has been characterized in other species, but not in the genus Babesia, which causes major losses in the livestock industries worldwide. Therefore, we isolated, cloned and expressed the wild-type B. bovis dxs cDNA in Escherichia coli and evaluated its enzymatic activity in vitro. DNA sequence analysis revealed an open reading frame of 2061 bp capable of encoding a polypeptide of 686 amino acid residues with a calculated isoelectric point of pH 6.93 and a molecular mass of 75 kDa. The expressed soluble recombinant fusion DXS protein was approximately 78 kDa, which is similar to the native enzyme identified from the parasite merozoite using anti-rDXS serum. The recombinant fusion DXS enzyme exhibited Km values of 380 ± 46 µM and 790 ± 52 µM for D,L-glyceraldehyde 3-phosphate and pyruvate, respectively. In this work, we present the first cloning, expression and characterization of DXS enzyme from B. bovis.

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Figures

Fig. 1.
Fig. 1.
Bioinformatics analysis of translated BbDXS polypeptide. (A) The predicted functional domains of BbDXS are shown by BLASTp. It consisted of TPP-binding module, PYR binding domain and a transketolase C-terminal domain. (B) DXS amino acid sequences were aligned with using the CLUSTAL Omega program. Arabidopsis thaliana (GenBank: NP_566686.2), Escherichia coli (GenBank: WP_001583256.1) and Theileria annulata (GenBank: XP_954253.1).
Fig. 2.
Fig. 2.
Molecular characterization of native BbDXS enzyme. (A) Lane M, molecular size marker. Lane 1, purified recombinant BbDXS fused with his-tag after analysis by SDS-PAGE; (B) Lane M, molecular size marker. Lane 2 and Lane 4, Western blot analysis of the purified rBbDXS; Lane 3 and Lane 5, Western blot analysis of the lysates of B.bovis; Lane 6 and Lane 7, Western blot analysis of the erythrocytes and leukocyte of health bovine, respectively. The first antibody of Lane 2 and Lane 3 was preimmune sera of rabbit;the first antibody of Lane 4 to Lane 7 was rabbit anti-rBbDXS anti-serum.
Fig. 3.
Fig. 3.
Separation of the reaction mixture with different substrate by TLC. After induction of E. coli BL21 (DE3)/pET −30a-DXS with IPTG, purified rBbDXS was incubated with Na-pyruvate and D,L-glyceraldehyde 3-phosphate or Dihydroxyacetone (DHAP). In the product mixture separation by TLC, the product DOXP was detected under 365 nm UV either pyruvate and DL-GAP (lane3) or pyruvate and DHAP (lane4) as substrates. When TPP was omitted (lane 2), no DOXP could be detected. Lane 1 corresponds to a standard of chemically synthesized D-1-deoxyxylulose 5-phosphate (DOXP)(Sigma).
Fig. 4.
Fig. 4.
Effects of substrate concentration on the activities of purified DXS for pyruvate (A) and D,L-GAP (B). DXS activities were determined in the presence of a fixed concentration of the second substrate by using a coupled assay method.

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