Team:UESTC-China/part
Basic parts
| Part number | Type | Composition | Length |
|---|---|---|---|
| BBa_K2286000 | Coding | Halohydrin dehalogenase mutant W249P | 762bp |
| BBa_K2286002 | Coding | Ascorbate oxidase signal peptide and haloalkane dehalogenase mutant DhaA31 | 981bp |
| BBa_K2286003 | Coding | Epoxide hydrolase | 882bp |
| BBa_K2286004 | Coding | Cytokinin-degrading Cytokinin Oxidase 3 | 1569bp |
| BBa_K2286005 | Regulatory | A seedling and root specific promoter | 1448bp |
| BBa_K2286007 | Coding | Halohydrin dehalogenase mutant F12Q-F186L | 765bp |
| BBa_K2286009 | Coding | Halohydrin dehalogenase mutant F12Q-W249P | 765bp |
| BBa_K2286010 | Coding | Halohydrin dehalogenase mutant F186L-W249P | 765bp |
| BBa_K2286011 | Coding | Halohydrin dehalogenase mutant F12Q-F186L-W249P | 765bp |
| BBa_K2286012 | Coding | Halohydrin dehalogenase wild type | 765bp |
Parts contribution
This year, we conducted a series of determinations and improvements for the two basic parts, BBa_K1199043 and BBa_K2013002. As for BBa_K1199043, we have provided information about its structure, reaction mechanism and kinetic constants. In addition, we also explored the effect of pH and temperature on the enzyme, and obtained the optimum pH and temperature. Further, we validated that DhaA31 was able to work in tobacco. For BBa_K2013002, we improved its structural information and reaction mechanism, and measured its kinetic constants.
| Part number | Source |
|---|---|
| BBa_K1199043 | iGEM2013 |
| BBa_K2013002 | iGEM2016 |
Improve a previous part
Halogen dehalogenase HheC is one of the key enzymes for bacterial degradation of organic halides, which are important pollutants in the environment. It is involved in the biodegradation of many halogen-containing environmental pollutants and has potential value in the control of environmental pollution. Although there are many highly active mutants towards 1,3-DCP. However, the activity of mutants for 2,3-DCP did not change much. Based on this, this year, We, UESTC-China, used the method of saturation mutation to modify BBa_K1199044 (HheC-W249P), the HheC mutant with high activity towards 1,3-DCP, and obtained two mutants, the HheC-P84A and the HheC-P84A-W249P with higher activity for 2,3-DCP degradation. The activity of HheC-P84A to 2,3-DCP was 2.42 times than that of HheC-W249P and the activity of HheC-P84A-W249P was 1.15 times than that of HheC-W249P.
| Part number | Type | Composition | Length |
|---|---|---|---|
| BBa_K2286006 | Coding | Halohydrin dehalogenase mutant P84A | 765bp |
| BBa_K2286008 | Coding | Halohydrin dehalogenase mutant P84A-W249P | 765bp |
Molecular dynamics simulation
It is reported that halohydrin dehalogenase HheC is a symmetric tetramer that can be thought of as a dimer. The four active centers of HheC tetramer are centrosymmetric. The active sites include substrate binding sites and halide binding sites, surrounded by four Loop regions, Loop1 (F12-Q87), Loop2 (A133 (373), Loop3 (P175-F188) and Loop4 (F243-P253), while the Ser132-Tyr145-Arg149 catalytic triad is contained in these four Loops. There is a synergistic effect between the amino acid residues on the four Loop regions, which are involved in the change of the active site conformation during the enzymatic catalysis.
As the current information on 2,3-DCP halogen alcohol dehalogenase transformation is minimal, there is little information on the crystal structure of halide dehalogenase HheC and 2,3-DCP complexes. Therefore, in order to find the amino acid residues associated with HheC degradation of 2,3-DCP, we need to obtain a more desirable initial structure by molecular docking. Through the screening of PDB database, we found a compound structure of HheC numbered 1pwz and (R)-styrene oxide.
Figure 1. 3D model of HheC
Based on this complex, we used AutoDock to rigidly couple HheC with 2,3-DCP. In the results of the docking, we chose a more reasonable initial structure as the next step molecular simulation model. After obtaining the initial structure of the substrate and HheC, we used Visual Molecular Dynamics and Pymol to analyze and simulate it. In the process, we found that there were other active sites around the substrate 2,3-DCP in addition to the catalytic triad. Accordingly, we analyzed the RMSD and RMSF values of the amino acids used throughout the reaction system. The results showed that there were larger fluctuations in 80-99Loop, 175-188Loop and 130-140Loop regions of the HheC. In order to obtain the active site amino acid associated with 2,3-DCP degradation accurately, we also decomposed the side chain energy of the amino acid around the active pouch of substrate.
Figure 2. HheC and 2,3-DCP system energy decomposition
From the figure we found that the activity of F186 site was very high. In addition to that, these sites, 12,132,145,187, were also actively involved in the whole reaction. And both the 132 and 145 sites are derived from the catalytic triplets, suggesting that the HheC and 2,3-DCP models obtained by molecular docking are reasonable and effective.
Based on the above predicted sites, removing the sites in the catalytic triad, we selected the site of F12 and F186 in Loop2 and Loop3 which were around the active sites. Using the same method, we screened the P84, a high activity site in the other two loops. Finally, we selected F12, P84, F186, N176 these four sites for site-directed mutagenesis, exploring the catalytic activity of them to 2,3-DCP.
Construction and Screening of Saturated Mutants
According to the results of molecular simulation, we constructed mutants by the method of saturation mutation, and superimposed the mutation on the basis of W249P. We obtained the monoclones randomly picked out by a toothpick and placed in a 96-well culture plate containing Amp and LB liquid medium for overnight culture. The obtained cells were broken and detected by colorimetric method. After a large number of screenings, we obtained two active mutants of HheC-P84A and HheC-P84A-W249P.
Catalytic activity determination of benign mutants
Halogen halide dehalogenation releases halide ions when catalyzing the conversion of O-halohydrin to epoxides. The interaction of the halide ions with Hg^(2+) allows Hg^(2+) to be separated from SCN^-, then forming a colored composite with Fe^(3+). This colored composite can be determined by absorption at 460 nm. Therefore, based on the preliminary screening, we used the halide ion assay to quantify the activity of the two active mutants to determine the activity level.
➤ Catalytic activity towards 2,3-DCP
Figure 3. The catalytic activity of mutants on 2,3-DCP
Data were measured in 50mM Tris-H2SO4 at pH 8.5 and 37℃
The results shows that the catalytic activity of mutant P84A to 2,3-DCP is 2.42 times that of W249P, and the catalytic activity of P84A-W249P to 2,3-DCP is 1.15 times that of W249P at 37℃ and pH 8.5.
➤ Catalytic activity towards CPD
Haloalcohol dehalogenase HheC can catalyze o-halide into epoxides and hydrogen halides through intramolecular nucleophilic substitution mechanisms, which are involved in the catalytic degradation of many halogen compounds and have a wide range of catalytic substrates. Therefore, in order to clarify whether the high activity of the mutants is suitable for other substrates, we selected 3-chloropropane-1,2-diol (CPD) as the substrate for further detection. The results showed that the activity of P84A toward CPD increased a little.
Figure 4. The catalytic activity of mutants on CPD
Data were measured in 50mM Tris-H2SO4 at pH 8.5 and 37℃
The above experimental results show that amino acid lie in 84 of HheC has an important effect on its catalytic activity towards the 2,3-DCP and CPD. Compared with W249P, the mutant P84A-W249P successfully improves its catalytic activity towards 2,3-DCP. While the mutant P84A not only improves its catalytic activity toward 2,3-DCP, but also maintains its high activity for CPD. Thus, based on BBa_K1199044, we successfully obtain mutants with improved catalytic activity to 2,3-DCP by molecular simulation and saturation mutagenesis.
