The Use Of Site-directed Mutagenesis Studies -D- 1-deoxy-xylulose 5-phosphate Synthase Key Amino Acid Residues

Terpenoid compounds are an extensive class of natural products that have important biological functions in all living organisms. Isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DHAPP) are universal precursors for the biosynthesis of all terpenoid compounds. They are produced by2-methyl-D-erythritol4-phosphate (MEP) pathway in most bacteria, plant plastids and algaes, but synthesized by the Mevalonate (MVA) pathway in archaea, fungi, and animals.1-Deoxy-D-xylulose5-phosphate synthase (DXS) is a crucial enzyme in MEP pathway and is an attractive target for the development of antibiotics and herbicides. In order to have a deep understanding of the structural and mechanistic information of DXS, it is necessary to identify its key amino acid residues related to its activity. Site-directed mutagenesis is an important technology in protein engineering. We can change the base sequence of gene coding the protein to study the key amino acid residues in active sites.We chose six residues of DXS which are thought, in accordance to the crystal structure of E. coli DXS, to be the key amino acid residues for its activity. They are Asp427, Tyr293, Arg478, His49, His257and His299, respectively. Firstly, we got the six mutant genes though site-directed mutagenesis. Then, the mutant genes were expressed and the mutant enzymes were purified. The catalytic activity of the wild type DXS and the mutants were analyzed by paper chromatography and precolumn derivatization-HPLC method, repectively. The results showed that the mutant Y392K lost whole transketolase activity while the mutant D427K lost about60%activity. The transketolase activities of the mutants H257Q, R478A, and H299D were more or less weakened and they had to spend more time to drive the conversion of D-GAP to completion if compared to wild type DXS. H49Q have minimal impact on the transketolase activity. For the triosephosphate isomerase activities of H299D, Y392K, H49Q, H257Q, D427K and R.478A, there is no obvious influence could be observed, if compared to the wild type DXS. The steady-state kinetic parameters of all the mutants were measured by an enzymatic cascade-ultraviolet method. Compared to wiled type DXS, the mutants H49Q and D427K have higher Km values and therefore they have lower substrate affinity. The Km values of H257Q, H299D and R478A decreased, showing that their substrate affinity increased. The values of H257Q, H299D and R478A reduced, which indicated that the catalytic efficiency of these mutant enzymes shrinked. The mutants H49Q and D427K have higher kcat values and therefore they possess higher catalytic efficiency.