Structural Analysis Of Hydroxyquinol 1, 2-Dioxygenase From Pseudomonas Putida DLL-E4 And Crystallographic Study On Two Pesticide Degradation Related Enzymes

Microorganisms play important roles in bioremediation of polluted environment; their degradation of pollutant is carried out by a series of enzymes in their cells. Researches on the key enzymes involved in pollutant degradation may help us understand the degradation mechamisms of pollutants, and make contributions in bioremediation. Protein crystallography utilizes protein crystallization and X-ray diffraction techniques to determine the structure of proteins, it can help us understand the structure of proteins at atomic scale, determine how the enzyme functions and clarify the key residues participating in a certain reaction process, these information would help us in directing further evolution of important enzymes, and promotes the bioremediation of environmental pollutants. This research solved the structures of Hydroxyquinol 1,2-dioxygenase (PnpC) in the p-nitrophenol degradation under two different crystallization conditions, and did crystallographic study of P-nitrophenol 4-monooxygenase (PnpA) in the P-nitrophenol degradation, and Atrazine dechlorogenase in herbicide atrazine degradation.p-Nitrophenol (PNP) is an important intermediate in chemical industry and is the metabolite of some pesticides like methyl parathion, it residues in the environment for a long time either in the soil or in the water. It is harmful to animal, plants and human beings. Hydroxyquinol 1,2-dioxygenase (PnpC) and p-nitrophenol 4-monooxygenase(PnpA) are two key enzymes involved in the degradation of P-nitrophenol by a methyl parathion degradation strain Pseudomonas putida DLL-E4. Hydroxyquinol 1,2-dioxygenase (PnpC) catalyzes ring cleavage of hydroxyquinol and convert it to maleyacetate.The PnpC expressed in E. coli BL21 (DE3) pLysS was purified by Ni-NTA and gel-filtration. Crystals in two different crystallization conditions were screened out at 293 K using sitting-drop vapor diffusion technique. In both conditions, the protein were both at the concentration of 8 mg/mL, the mother liquor components are:(Ⅰ) 0.1 M Tris-HCl pH 7.5, 1.3 M ammonium sulfate and (Ⅱ) 0.1 M Hepes pH 7.5,0.8 M tri-sodium citrate respectively. Crystals came out at almost the same time with nearly same crystal form. But they differed greatly in catalytical ability. Crystal under condition I retained activity while the latter lost when the crystals were redissolved in the buffer. We collected diffraction data of crystals from both conditions. Crystals grown from condition I diffracted to 2.3 A, with the space group C121 and unit-cell parameters a= 82.48, b=38.47, c=107.98,α=90.00°,β=118.06°,γ= 90.00°.While the latter diffracted to 1.99 A, with the space group C 121 and unit-cell parameters a=82.57, b=38.93, c=107.36,α=90.00°,β= 118.05°,γ= 90.00°. They both have one molecule in the asymmetric unit and the solvent content is 46 %. With the hydroxyquinol 1,2-dioxygenase from Nocardioides simplex 3E as search model (PDB accession number:ITMX ID 40%), we successfully calculated its phases with molecular replacement and solved their structures.PnpC is composed of 6 long a helixes and 7βsheets and strands, the structure is very similar to its search model 1TMX, their main chain almost goes the same way when they are superimposed. The model of PnpC could be devide into 2 domains, N terminal a helix domain (NHD) and C terminal catalysis domain (CCD). The four conserved aas are:TYR 160, TYR 190, HIS 218 and HIS 220. Among them, TRY 160 and HIS 218 are in the same plane which is perpendicular to the plane where TYR 194 and HIS 220 lie. By symmetry operation, the biological active dimer was constructed from the monomer. The two monomers are linked together by NHD domain (H1,H3,H4,H5,H6,H7). In the connection region, Helixl of one monomer is close to the Helix6 of the other one. Two Helix3 are antiparallel to each other. From position 27 to 46 of the Helix3 go through the channel, they in addition with Helix4 and Helix5 forms the insider part of the channel, the outer part of the channel is composed of residues from Helix3, Helix4 and Helix7, Helix 5 and Helix7 are near CCD According to the model and electron density map, we can see that PHE 28, ILE 31, LEU 35, LEU 39, PHE 42, LEU 48, ILE 57, PHE 59, LEU 60, LEU 78, LEU 82, ILE 76 and ALA 208 and their counterparts in the other molecule contribute to the hydrophobic channel. The hydrophobic tail of the phospholipids is inside the hydrophobic channel. When we compare the electron density maps of the two crystals, we found bond length distances exist among the four activity center residue. For the structure crystallized by trisodiumcitrate, the bond length is 2.46 A between TYR 160 and HIS 218 and between TYR 194 and HIS 220 is 4.23 A, while in the structure from ammonium sulfate precipitated crystals, bond length is 2.33 A between TYR 160 and HIS 218 and 4.70 A between TYR 194 and HIS 220. Besides, from the crystal crystallized by trisodium citrate, there is a benzo like molecule in the hydrophobic channel near the lipid hydrophobic tail, and is in contact with LEU 35 of both molecules.We constructed expression vectors for cutting 23,46 and 64 aas from the DHD of PnpC with pET expression system respectively, and expressed them in E. coli BL21 (DE3) pLysS, the expression product resulted in loss of activity, which means the three helixes are necessary for the activity of PnpC.P-nitrophenol 4-monooxygenase (PnpA) is a flavin adenine dinucleotide-dependent single-component PNP 4-monooxygenase which converts PNP to para-benzoquinone in the presence of NADH and FAD. We purified PnpA by Ni-NTA and gel-filtration. We screened out one crystallization condition at 293 K by sitting drop vapor diffusion technique:10 mg/mL purified PnpA, the mother liquer components are:0.1 M Hepes pH 7.0,15%PEG 4000。The crystal diffracted to 2.24 A, with the space group P212121, and unit cell parameters:a=54.47, b=77.56, c=209.17,α=90.00°,β=90.00°,γ=90.00°。We tried to solve the structure by molecular replacement, and find part of the main chain. We collected diffraction data of PnpA crystal soaked with substrate PNP,4-NC and KI, and anomalous signal was detected from the KI soaked crystal diffraction dataset.Atrazine is one of the most widely used herbicide in the world; it has caused serious pollution in soil and water. Much work has been done on the bioremediation of it, a lot of degradation strains and enzymes have been characterized. Atrazine dechlorogenase (AtzA) removes the halogen atom chloride from atrazine and convert it to hydroxyatrazine, which is less toxic to the nature. Although many enzymatic and evolution research has been carried out on AtzA, its structure is still not known.We amplified the atzA gene by PCR from total DNA of the atrazine degradation strain Pseudomonas sp. SA1, linaged it to pET vector and overexpressed it in E. coli BL21 (DE3) pLysS. AtzA was purified by ammonium sulfate precipitation, ion-exchange and gel-filtration. Stable X-ray diffraction quality crystals were got by hanging and sitting drop vapor diffusion techniques. The protein used are 10 mg/mL:0.1 M tri-Sodium citrate dihydrate pH 5.6,12% MPD and 13% PEG 4000, it diffracted to 4 A.We solved the structures of Hydroxyquinol 1,2-dioxygenase by molecular replacement from two different crystallization conditions, and found structural differences between the two structures which might be the cause of their diference in activity recovery, we cut 3 N-terminal helixs which is far from the activity center and found that the mutants have no activity, which means they should have some relationship with activity. We get crystals of P-nitrophenol 4-monooxygenase diffracted to 2.24 A and stable ctystals of Atrazine dechlorogenase which would diffract to 4A.