| Thermostable p-nitrophenylphosphatase (Bs-TpNPPase) is the moderately thermostable enzyme from Bacillus stearothermophilus, which is an Mg2--dependent specific alkali phosphatase. Bs-TpNPPase is a member of the haloalkanoate dehalogenase (HAD) superfamily (subfamily IIA), with the substrate of p-nitrophenylphosphate (pNPP). Bs-TpNPPase has been widely used in labeling nucleic acid since it can decompose pNPP in bacteria and mammals. Bs-TpNPPase is resisitant to high temperature and organic environment, therefore it is very important in immunodetection and marking DNA.In the present study, the structure of Bs-TpNPPase has been solved by X-ray diffraction and some mutants with improved thermostability have been produced. The catalytic machenism and molecular machenism of thermostability of Bs-TpNPPase have been solved based on the structure of Bs-TpNPPase and properties of mutants. Such results could be useful in the future applications. Bs-TpNPPase has been cloned and overexpressed in Eschericaia coli. In the present study, the protein Bs-TpNPPase, a member of the haloalkanoate dehalogenase superfamily (subfamily IIA), was purified to homogeneity.To understand the structure/function relationship and investigate its mechanism for the high thermostability, assays like purificantion, crystallization and structural analysis have been performed. The main research contents and results were as follows:1. Single colonies were grown in LB medium at 37℃ containing kanamycin (50μg/ml) and ampicillin (50μg/ml) and induced by adding IPTG at a final concentration of 41mM when the absorbance of bacilli suspension reached 0.7 at 600 nm. The incubation was continued at 28℃ for another 4h. Bs-TpNPPase was purified by Ni-NTA and Sephadex G-75 column and concentrated to 25mg/ml. The purity of Bs-TpNPpase was assessed by SDS-PAGE to confirm it was higher than 95%. The optimal reaction temperature for Bs-TpNPPase is 55℃ and the activity of Bs-TpNPPase is extremely low at 0℃. Other properties were shown as follows: Tm=55℃, Km=1.7429mM, kcat= 1.04×104s-1,kcat/Km=5960mM-1s-1 is considered to be involved in Mg2+ -dependent hydrolysis of the phosphoenzyme with an optimal reaction temperature of 55℃.2. The purified protein was screened for crystallization at 20℃ by high throughput screening with crystal screen kits CS-HT(HR2-130) and INDEX-HT(HR2-134) from Hampton Research, USA. Crystals of Bs-TpNPPase were formed by the sitting-drop vapour-diffusion method, using equal volumes (1.5 μl) each of protein solution and well solution. An X-ray diffraction data set was collected to 1.5A at the Shanghai Synchrotron Radiation Facility on the beam line BL17U-MX and the diffraction data was indexed and scaled using the HKL-2000. The Bs-TpNPPase crystals indexed in space group P212121 with unit cell dimensions a=38.802A b=83.268A c= 175.799A α=β=γ=90°. The Bs-TpNPPase protein was a complex with a cap and a conserved α/β fold core domain. The two distinct domains, which belonged to the three-layer (α/β) sandwich, which were connected by two linker regions. There are 9 a-helices,16 β-sheets and 24 β-turns in the structure of Bs-TpNPPase. Such results showed that the structure of Bs-TpNPPase is similar to that of typical member of subfamily IIA. The catalytic machenism of Bs-TpNPPase is also similar to that of HAD superfamility, base on the results of active site mutation. The larger domain was the conserved core domain which comprised a six-stranded parallel β-sheet (β1-β3 and β10-β12) surrounded by seven helices (αl-α3 and α8-αll). The slightly smaller domain was the cap domain, which consisted of a three-stranded parallel P-sheet (β4-β7) surrounded by four helices (α4-α7). Two additional anti-parallel P-sheets were part of the cap domain, which covered the active site residues of the core domain.3. Structural analysis was performed in comparison with 7 HADSF members: Afp, a putative Nagd Protein from Sbacteria-thermotogae; Php, a hypothetical protein PH1952 from Pyrococcus Horikosihii; Tmp, a 4-nitrophenylphosphatase tm1742 from Thermotoga Maritima; Bsp, a putative p-nitrophenyl phosphatase from Bacillus Subtilis; Ecp, a Nagd from Escherichia coli (E.coli) K-12 Strain; Mmp, a protein from Mus musculus Mm.236127; Hup, a possible sugar phosphatase from Homo sapiens. Hydrophobic interaction, arometic clusters, K=3k-clique hydrophobic interaction clusters and cation-Pi are the important sources for Bs-TpNPPase to obtain the thermostability.4.11 mutants producing such enzymes with improved thermostability were obtained after 3 rounds of directed evolution using error-prone PCR.7 of them were single amino acid mutants (K30R, A75V, E95V, A150V, F157L, H170Y, and K175N), and the others were two double amino acid mutants (N86S/A150V, E95D/A150V, A150V/T177I, and A150V/F157L). The most thermostable mutant enzyme, A150V/F157L, contained 2 amino acid substitutions and. Its reaction temperature for maximum activity increased from 55℃ to 64℃, and catalytic efficiency was 2 fold of that of the wild-type.5. The mutation site in each mutant was located in cap domain, except K30R, in core domain. The improvement of Tm for K30R was the lowest among all the mutants indicating that the thermostability of cap domain is relatively poor. Homology modeling via SWISS-MODEL was employed to build structures of the mutants. Number of hydrogen bond, salt bridge, hydrophobic interaction and aromatic interaction were also obtained by employing PIC(Protein Interactions Calculator) webserver. The protein thermostability is considered to correlate with the composition of the protein thermostability is considered to correlate with the composition of crystal structure of wild-type and homology model of mutants. Like the Bs-TpNPPase, the increased hydrophobic interactions in cap domain are the important sources for mutants to obtain the thermostability.6. A150V and A150V/F157L were expression in M15 and purified. Crystals were formed by the sitting-drop vapour-diffusion method using equal volumes (1.5μl) each of protein solution and well solution. The X-ray diffraction data sets were collected to 1.5A and 2.0A respectively. The diffraction datas were indexed and scaled using the HKL-2000. Compariton of the crystal structures of Bs-TpNPPase, A150V and A150V/F157L, no significant difference was found in the three structures. Therefore, this phenomenon reveals that the small changes may have more contribution on the protein thermostability. The improved hydrophobic interaction and the formation of K=3k-clique hydrophobic interaction clusters contributed to the improvement of thermostability.
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