Bacillus Fastidious Uricase Affect Mutagenesis Of The Conserved Sequences In Nature

Bacillus fastidious uricase (BF uricase) had 322 residues. Activity uricase is the same tetramer. This uricase at pH 7.4 and 37℃, its half-life about 23 days, the high thermal stability than the fungal uricase more than 10 times. Analysis of the crystal structure was found, this uricase front nine of N-terminal residues and C-terminal 296-302 residues reverse β-folding gather into dimers and maintain active center subunit interface, wherein the N terminal has basic and acidic residues and C-terminal mainly acidic residues. Destruction before the N-terminal nine residues, C terminal 301 residues are involved in electrostatic network or hydrogen bond, mutants inactive or low and the thermal stability decreased, showing the electrostatic repulsion between the subunits could decrease tetramer stability. The project is based on the structure of the conserved sequence in exploring the role of thermal stability and activity of the enzyme, and its molecular basis for the transformation.1. N-terminal sequence the impact in Bacillus fasitidious uricaseAccording to the N-terminal sequence of the recombinant expression vector AERTMFYGKGDV crystal structure, near the electrostatic interaction network analysis, design of the N-terminal mutants. Relatively N-terminal residues mutations small affect its Ki and Km and optimum temperature and pH, and the different contrast and thermal stability of activity. Unmutated uricase specific activity reached 11.5kU/g, the first three amino acid mutations ARR, QQR, RQR, VQR specific activity was 6.3kU/g,7.0kU/g,8.6kU/g,9.3kU/g,SDS-PAGE electrophoresis were found tetramer structure, RER mutant specific activity has increased for 13.5kU/g. Thermal stability and non-mutated in almost the same at 37℃ these mutants,4℃ pH 9.2 there is a slight decrease. But mutant RESRESRER, REGREGRER, SSSSSRER specific activity decreased significantly, respectively,4.26kU/g,2.71kU/g,4.83kU/g, after SDS-PAGE electrophoresis tetramer remains found, but the thermal stability at 37℃ pH 7.4 changed significantly, respectively,1.3d,0.75d,16d. Most particularly, the specific activity of the mutant Y7F retain up to 27%, but its inactivation acceleration 1000 times at 37℃.2. C-terminal sequence the impact in Bacillus fasitidious uricaseOn the C-terminal 305 residues after mutation or truncation, or connect the respective modifying groups were designed 11 mutants. Mutant D307R, no activity.Mutants cut off 314, R310E, E311R, E318R, Y319F, L322R activity decreased, respectively,3.2kU/g,4.4kU/g,3.6kU/g, 3.3kU/g,2.9kU/g,3.6kU/g, are tetrameric structure. Thermal stability at 37℃ neutral conditions decreased significantly alkaline conditions unchanged. Further design mutant L322SNSNSN, L322DSNSNSN, L322DSNSNSNHHHHHH, This demanding Bacillus uricase expression after by DEAE, after purification by preparative electrophoresis, retain 66% specific activity, but essentially the same thermal stability. This demanding Bacillus uricase whether the N-terminal or C-terminal His tag was added after the activity will decline, but stability of close with Ni2+-NTA purification effect DEAE-cellulose, but a lot of high efficiency. So L322D plus 6His tag fusion expression suitable for the study of its structure and nature of the relationship.3. Conserved region on its activity and thermal stability of bacillus fastidious uricaseAccording to the N-terminal sequence of the recombinant uricase AERTMFYGKGDV crystal structure, middle part of the near conserved sequence found the electrostatic interaction. A68L, T69V, D70L, K73E, F179L, D185N, S243A, I244F, Q245I, H246E no activity, it can be observed PAGE electrophoresis expression vector has been, F179L tetramer, but no activity. Mutant D70N, S71A, T188V, T189V, E192Q, Y249F, F298L, Q299I, F301L activity was 0.55kU/g,0.34kU/g,0.65kU/g, 0.53kU/g,1.85kU/g,4.71kU/g,1.97kU/g,0.93kU/g,2.59kU/g, is worth noting that D70N dimeric active. Thermal stability at 37 ℃ and 4 ℃, pH 7.4 and pH 9.2 decreased significantly, D70N, T188V, E192Q only for the original 0.1%. Visible these conserved sequences are involved in the catalytic process and to maintain its thermal stability4. Lonic bond on its activity and thermal stability of bacillus fastidious uncaseAccording to the results of homology modeling, observed R140, E142, E291, R293 each subunit and the other subunit to form four ionic bond, a total of eight pairs of ionic bonds.5 mutants were designed, R140E, R140Q, E142R, E291Q, E291R where in E142 is tetramer inactive, the remaining activity was 1.3kU/g,1.6kU/g,13.3kU/g, 0.1kU/g, E291Q activity increased, heat inactivated rapidly. Further design mutant N141C, P292C see if you can form a disulfide bond to improve stability. These seven mutants were ligand-induced, found by the strong affinity of oteracil potassium (final concentration of 30uM), the thermal stability of these mutants under the corresponding conditions can be hundreds of times.5. Bacillus uricase sequence and nature of the relationshipThis uricase and its mutants depolymerization inactivate inhibitors oxonic acid and xanthine and uric acid substrates can induce polymerization assembled into active tetramer. This uricase unique C-terminal and C-terminal conformation network involved in the formation of an electrostatic, hydrogen bonding between the conserved sequences are necessary for the activity and stability; tetramer subunits internal ionic bonds between adjacent also maintain active tetramer key. At low temperatures, this initial uricase very stable, but the conformational change accumulated to a certain extent the activity of rapid exponential decay, showing a clear two-stage inactivation. Overall, the uricase has excellent thermal stability, may be long-term evolution of this uricase accumulation effect caused.