1Structure And Function Study Of Protein PnpB And PnpE From PNP Degradation Pathway In Pseudomonas Sp.WBC-3 2Crystal Structure Study Of Thermoplasma Acidophilum Factor F3 As A Model For Anticancer Drug Design And Screening

p-nitrophenol (PNP) is an important raw material in the industry, which is widely used as a printing, dyeing, rubber, pharmaceutical plastics and paint industries. It is highly toxic to the enviroment and human's health. It contains a notriso in the structure that strongly attracts the electron of phenyl, thus PNP is difficult for degradation in the nature. Through the respiratory system and skin it affects the blood, liver and central nervous system in the body. It can cause dizziness, rash, itching spam, anemia and various neurological symptoms. At present, the degradation and mineralization of p-nitrophenol remains a worldwide problem. The traditional methods of PNP degradation are easy to produce secondary pollution and can not completely remove contaminants. Therefore, a number of studies have been triggered focusing on the biodegradation of PNP. It is studied that microorganisms in the environment, which are exposed to the nitro-phenol compounds long term, can degrade these pollutants. If these organisms can be used to degrade the pollution, it can remove the pollutants and can not pollute environment. This is an environment friendly method.Up to now, it is reported that a lot of bacteria can degrade p-nitrophenol. The pathways of PNP degradation have been clearly depicted. In microorganisms there are two pathways to degrade PNP. One is the hydroquinone pathway that is usually found in gram-negative bacteria. For example, in Moraxella sp, PNP monooxygenase converts PNP to hydroquinone via the potential intermediate p-benzoquinone. The other is the hydroxyquinol (1,2,4-trihydroxybenzene) pathway that is preferentially found in gram-positive bacteria, For example, in Rhodococcus opacus SAO101,PNP is converted to hydroxyquinol via 4-nitrocatechol.Pseudomonas sp. Strain WBC-3 is a PNP degradation strain, which was isolated by Shanongda Pesticides Company in Hubei, China. It can utilize methyl parathion (O,O-dimethyl O-p-nitrophenol phosphorothioate) and PNP as sole soureces of carbon, nitrogen and energy for survival. The bacterial degrade PNP through the hydroquinone pathway. Firstly, PNP is converted to p-benzoquinone by PNP 4-monooxygenase (PnpA) and is further reduced to hydroquinone by p-benzoquinone reductase (PnpB). Next, hydroquinone dioxygenase (PnpCD) converts the product toy-hydroxymuconic semialdehyde, which is then converted to maleyacetate by the NAD dependent enzyme y-hydroxymuconic semialdehyde dehydrogenase (PnpE). At last, maleylacetate reductase (PnpF) catalyzes the maleyacetate to form P-ketoadipate to enter the TCA cycle.Despite this knowledge, the structural mechanism of each reaction step in the pathway remains unclear owing to a lack of structural information of the enzymes. In order to understand the structural properties required for the activity of the enzyme and then to engineer the enzyme to improve desired properties, it is necessary to obtain and investigate the three-dimensional structures of these enzymes that catalyze the PNP degradation process. In this paper we first determined the crystal structures of . apo-PnpB, apo-PnpE, PnpB-FMN complex, and PnpE-NAD complex. We understand the whole folding model, active site structure and amino acid composition and lay a foundation for explanation the catalytic mechanism from the molecular level. The main contents include the following aspects:(1) The gene of pnpB was cloned from Pseudomonas sp.WBC-3, inserted into expression vector then transformed into expression host E.coli BL21 (DE3). PnpB highly expressed in the host and purified, which belongs to FMN and NADPH dependent family. At last the apo-PnpB and PnpB-FMN complex crystal are obtained 2.1A and 1.7A resolution diffraction data respectively.(2) The structure dctermined of PnpE-Native and PnpE-NAD complex. The gene of pnpE was cloned from Pseudomonas sp.WBC-3, inserted into expression vector then transformed into expression host E.coli BL21 (DE3). PnpE highly expressed in the host and purified, which belongs to NAD dependent ALDH (Aldehyde dehydrogenase) super family. At last the apo-PnpE and PnpE-NAD complex crystal are obtained 2.1A and 3.0A resolution diffraction data at last.(3) The structure determined of PnpB-Native and PnpB-FMN complex. We identified PnpB with???typical characteristics of the flavin domain. We research the cofactor FMN binding site and the interaction amino acids. We proposed PnpB-FMN-BQ and PnpB-FMN-NADPH model through Autodock 3.05, and identified the substrate binding site and cofactor NADPH binding site according the models. Furtherly we used site-direct mutation to confirm the Autodock results. At last we supply the catalytic mechanism of PnpB on the base of three-dimensional structure.(4) The structure determined of PnpE-Native and PnpE-NAD complex. We identified PnpE three domains:cofactor binding domain, dimerization domain, and substrate binding domain. We research the cofactor NAD binding site and the interaction amino acids. We supposed the native and the complex structure and found a key loop (L246-G252) which has the relationship with the entry of NAD, and elucidate its function in the catalytic mechanism. Next, we compared the PnpE with homologous BADH and elucidate the cofactor binding specificity. We proposed PnpE-substrate model through Autodock 3.05., and identified the substrate binding site according the models. Furtherly we used site-direct mutation to confirm the Autodock results. At last we supply the catalytic mechanism of PnpE on the base of three-dimensional structure. Cancer is a serious killer for human health. It causes the death of millions of people every year. The main reason for tumor caused human death is that malignant tumor can infiltrate and meatastasize. The key point for curing cancer is how to control the infiltration and metastasis for cancer cell.Tumor cell metastasis involves tumor cell adhesion, enzymatic degradation of the matrix, the formation of new blood vessels and a series of complex process. For these reasons, the interaction between the protein degradation enzyme with the surrounding enviroment plays a key role in the tumor deterioration. These degradation enzymes include matrix metalloproteinase enzyme (MMPs), plasminogen and aminopeptidase (AP). Aminopeptidase N (APN) is one of the family, which is associated with malignancy degrading enzyme. APN exists widely in animal cells, which participates in the invasion of tumor cells to basement membrane, matrix, as well as the penetration of the blood vessel wall.Human APN (hAPN) is the type II membrane bound glycoprotein, it is composed of 976 amino acids and its molecular weight is 150 KD. hAPN belongs to zinc-dependent metalloproteinases and M1 family aminopeptidase subfamily Gluzincins. APN becomes an important target for anticancer drug research since it plays an important role in the invasion and metastasis of cancer cells. It causes a lot of attention for using APN as a target for anticancer drugs. In 1987, Bestatin was marked and used as an immune enhancer in Japan. Recently, many compounds which have structure similarity with Bestatin have been designed, such as Probestatin, Actinomin, etc. However, the three dimensional structure of human aminopeptidase has not been resolved, which stands in the way of further research for anticancer drug design. In this case the structure of its homologous protein would lead to unexpected findings.Currently, a lot of hAPN homologous have been resolved. For example,the structure of E.coli APN and its complex structure with Bestatin. The APN crystal structure from Neisseria meningitides and the structure of Trion interaction F3 factor from Thermoplasma acidophilum. These results lay a good foundation for the inhibitors design of aminopeptidase N. Now, the inhibitor design of APN is based on the homologous structure and complex structure. The problem is that the active site of these bacterial homologous structures have differences with human APN. This causes the inevitable bias for drug design to a certain extent and weak the drug's effect.Thermoplasma acidophilum F3 factor is the most identity homologous protein with human APN. So, we modified the active site of F3 factor to make it the same with human APN by site-directed mutagenesis. We solved the complex structure of mutant factor F3 with compound D24 in the paper. Such a holo-form experimental structure helpfully insinuates a more bulky pocket than Bestatin-bound E. coli APN. This evidence discloses that compound D24 targetting the structure of E. coli APN cannot bind to the activity cleft of factor F3 with high affinity. Thus, there is a potential risk of inefficiency to design hAPN targeting drug while using E.coli APN as the target model. We do propose here now that engineered factor F3 can be employed as a reasonable alternative of hAPN for drug design and development.In the paper, the activity site of Thermoplasma acidophilum factor F3 was mutated by site-directed mutagenesis. The 101E A was mutated into Q and the 261N was mutated into T, then the mutated gene was inserted into the pET21b expression vector. The plasmid contained the mutated gene was transformed into expression host E.coli BL21(DE3). The mutated F3 factor highly expressed in the host, cultured the complex crystal of F3 factor with D24 and obtained the high resolution diffraction data at last. According to the complex structure, we analysis the structure of its acitivity site and compared with E.coli's APN -Bestatin complex structure.(1) Structure based sequence alignment shows the factor F3 of T. acidophilum has only two different amino acids in active site from the hAPN. One is E101 substituted by Q in hAPN, and the other is N261 substituted by T. Site-directed mutations result in an engineered protein with active site identical to hAPN.(2) We got four kinds crystals of mutanted F3 factor with four kinds inhibitors(Dsh39, Bestatin, D24, Dsh27). At last we only resolved F3-D24 complex crystal structure. And there is no compounds'electron density in other three crystals. It is said that these three compounds (Bestatin, Dsh39, Dsh27) cannot form stable complex.(3) The zinc-coordinating atoms form a close to a perfect tetrahedral coordination sphere with the zinc ion in the center. In our model, the zinc ion is coordinated by H265, H269, E288 and compound D24. In the compound D24 complex structure, there are eight amino acids interacting with the inhibitor:Q101, A229, A231, E233, E288, T292, R316 and G352. The inhibitor is stabilized mainly by the combination of hydrophobic interaction and several hydrogen bonds.(4) In the two native structures, E101 adopts distinctive conformations. In lzlw, it adopts an outside orientation while in 1z5h it is oriented to the inside but not quite extended due to the same charge repulsion with E233 that stand by. In our structure, the side chain of Q101 is buried inside deeply due to the loose of negative charge and stabilized by the inhibitor. The mutation of N261T does not cause conformational changes except that it increases the hydrophobicity of its surrounding environment.(5) We compared E.coli APN with Bestatin and factor F3 mutant with compound D24 demonstrated that the former has a slighter cleft than the factor F3 mutants. That's the reason that the inhibitor designed based on E.coli coordinate cannot securely buckle up the F3 mutant. Therefore, engineered factor F3 can be functioned as a satisfactory alternation of hAPN for drug design and screening. In conclusion, our structure would like to provide a brand new start point for the development of potent anti-cancer leads targeting hAPN.(6) We performed biochemical characterization of this enzyme. The experiments returned the Km value of 62.453?m which was higher than that of native factor F3 (34.7?m). The engineered protein shows the maximum enzyme activity at 75?and pH5.5 using Leu-NA as substrate. Our biochemical data proved the feasibility of the substitution of hAPN by the engineered F3 protein.Recombinant T. acidophilum factor F3 and the substrate of Leu-p-nitroanilide were used to evaluate the IC50 of the synthesized compound D24. It showed the IC50 value of 30?M for factor F3 mutants.