The Study Of Two Thiamine Pyrophosphate Dependent Enzymes Of Thermotoga Maritime

Thiamine pyrophosphate (ThDP) participates in a variety of enzymatic reactions in vivo as an indispensable coenzyme. The enzymes catalyzing these sorts of ractions are therefore ThDP-dependent enzymes. Acetolactate synthase (AHAS) and 1-deoxy-D-xylulose 5-phosphate synthase (DXS) are two important ThDP-dependent enzymes involved in the central metabolic pathways in natural. AHAS is one of the key enzymes that catalyzes the first step in the biosynthesis of branched chain amino acids. It exists in bacteria, yeast and plants but lacks in animals and humans, so it could be an attractive target to screen antibiotics and herbicides. DXS is the first key enzyme in 2-methyl-D-erythritol 4-phosphate (MEP) terpenoid biosynthetic pathway which presents in bacteria, plants but not in animals and humans. Thus it is also an appealing target for developing new antibiotics and antimalarial drugs.In this thesis, the genome DNA of Thermotoga maritima was used as a template to construct pET28a-TmAHAS and pET28a-TmDXS recombinant plasmids, respectively. Then the recombinant plasmids were transformed into E. coli Rosetta (DE3) which was finally induced to express TmAHAS and TmDXS, respectively. Partially soluble expression of TmAHAS was obtained and the impacts of the pH value, reaction temperature, and reaction time on its activity were determined. Furthermore, its stability and steady-state kinetic parameters were also measured. Results showed that TmAHAS has a KmPyruwaTe of 19.9 mM and a specific activity of 13.4 U/mg. The optimal temperature, pH and reaction time were 80 ℃,7.0 and 45min, respectively. TmAHAS could be stored at -80℃ in 50 mM phosphate buffer (pH 7.0,20% glycerol) for at least four months without substantial loss of its activity. Inhibition test indicated that chlorsulfuron and chlorimuron-ethyl could inhibit TmAHAS with IC50 of 1.49 uM and 88.8nM, respectively. Furthermore, TmAHAS could also catalyze the reaction of pyruvic acid and benzaldehyde to form L-phenylacetyl methanol (L-PAC) which is the important precursor in the production of α/β-adrenaline. Therefore, TmAHAS is not only a good target for antimicrobial drug screening, but also be useful in the synthesis of chiral drugs. TmDXS was received mainly as an inclusion body protein. After denaturation and renaturation of the protein, soluble TmDXS with good activity that could be used for antibiotic screening was acqauired.In summary, the two ThDP-dependent enzymes prepared in this study could be used for antibiotic screening. Furthermore, they also possess potential usefulness in industry due to their ability of high temperature tolerance.