Expression, Purification And Crystal Structure Of Deaminase Amne

Microorganisms play important roles in bioremediation of polluted envirorunent. The 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 eontributions 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, and these informations would help us in modifying and transforming of key enzymes.Nitrobenzene is a major chemical industrial raw material and chemical intermediate, it residues in the environment for a long time either in the water or in the soil, which is harmful for human. In this study, deaminase AmnE is the research object which comes from Pseudomonas sp. AP-3. The AmnE proteins expressed in Escherichia coli BL21 (DE3) were purified by Ni-NTA affinity chromatography and Resource Q anion exchange chromatography. The hanging-drop vapour-diffusion method and commercial crystal-ization kits were used for the screening of crystallization conditions. Diffraction-quality crystals were obtained with mother liquor consisting of 0.2 M potassium chloride,23% (w/v) polyethylene glycol 3350 and 0.2 M bis-tris pH 7.5. The crystals were diffracted to a resolution of 2.09 A and belonged to the orthorhombic, space group C2221, with unit-cell parameters a= 63.23 A, b= 88.93 A, c= 137.83 A. We successfully solved the structure with the putative translation initiation inhibitor ph0845 as the molecular replacement model. There are three protein molecules per asymmetric unit and each of them is made up by three a-helices and five P-sheets with linking loop. We also did the crystallization experiment of the compound of AmnE with its substrate analogue. But we did not get crystals with good resolution and the crystals need optimization deeply.The recently report of deaminase from the biodegradation pathway of nitrobenzene is limited at the research of heredity character and activity determination. This work may fill the gaps in the protein structure research and help us understand the biological function. Moreover, it may support an important guiding for modifying of the deaminase. Finally, it would be used for constructing the gene engineering strain of deamination.