[Cloning and characterization of a new antibacterial target, 3,4-dihydroxy-2-butanone-4-phosphate synthase]

Riboflavin, called generally vitamin B12, is the precursor of cofactor flavin adenine dinucleotide (FAD) and flavin mononucleotide, which plays crucial roles in the biosynthesis of organisms. Bacteria need to synthesize riboflavin to maintain their survival and proliferation if they cannot obtain flavin from the surroundings. 3,4-Dihydroxy-2-butanone4-phosphate synthase (DHBPs) is one of the key enzymes in biosynthesis of riboflavin. In the presence of Mg2+, DHBPs can degrade ribulose-5-phosphate (Ru5P) into formate and 3,4-dihydroxy-2-butanone-4-phosphate (DHBP). Potentially, these enzymes related to biosynthesis and metabolism of riboflavin, including DHBPs, may serve as the target for new antibacterial drug. In order to determine the three-dimensional structure and screen small molecule of inhibitor of DHBPs, we expressed and purified DHBPs from Streptococcus pneumonia (S. pn), and characterized the activity of this enzyme.

DHBPs gene was amplified by PCR, and over-expression plasmid pW28-DHBPs was constructed. pW28-DHBPs was transformed into Escherichia coli BL21 (DE3) to express DHBPs; the recombinant protein was purified by nickel column and ion-exchange column. The enzymatic activity was tested using spectroscopy.

The plasmid of pW28-DHBPs was verified by restrictive enzyme digestion and sequencing. Soluble DHBPs was expressed in E. coli BL21, and purified with 95% purity. The result of size exclusion chromatography indicates that DHBPs was dimer in solution. Additionally, the recombinant protein has activity to hydrolyze Ru5P into formate and DHBP in the conditions of pH 7.5 and 25 degrees C and in the presence of Mg2+.

DHBPs could be highly expressed in soluble form in E. coli BL21 strain, and the recombinant protein has activity to hydrolyze Ru5P.