Significance Of Cysteine Residues For Functions Of Aspergillus Flavus Urate Oxidase

Urate oxidase (uricase; EC 1.7.3.3; or UOX) belongs to the purine degradation pathway and catalyzes in the presence of molecular oxygen the hydroxylation of uric acid into a metabolic product identified as the 5-hydroxyisourate(5-HIU). Urate oxidase is present in many species, but is absent in human and higher apes. This emphasizes an evolutionary advantage: it has been suggested that uric acid is a powerful antioxidant, so humans would have less free radicals and thus less cancer as a result of aging. However, congenital or acquired factors increased production of uric acid in the body or blocked excretion, can cause hyperuricemia, arising a variety of related diseases such as gout, kidney, etc. In addition, the rapid decomposition of cells can lead to hyperuricemia in the process of chemotherapy of tumors, resulting in tumor lysis syndrome (TLS). Uricase was thought of as the standard treatment for the TLS, and is becoming the potential, effective way for the hyperuriceamia.Use of urate oxidase treatment of these diseases is an effective way.Aspergillus flavus urate oxidase is a member of T-fold family of proteins. The presently known T-fold proteins share a high structural similarity although they only exhibit a low amino acid sequence identity, they all showed a similar oligomerization pattern. X-ray diffraction analysis of the Aspergillus flavus urate oxidase showed that the asymmetric unit contains one monomer (34kDa) of 301 amino-acids and the whole homo-tetramer (135kDa) is built using the two-fold axes of the current I222 crystal symmetry. Each monomer is associated with one active site located at a dimmer interface. Three non-conservative cysteine amino acid (Cys35, Cys103, Cys290) present in the Aspergillus flavus urate oxidase sequence and they do not form a subunit disulfide bonds within and between subunits. However, studies have shown that sulfur atoms of three cystines are likely to affect the activity of urate oxidase because of their orientation, oxidation, or connection to the adducts.To investigate the function of the cystine residues in the enzyme, mutants of the C35A, C103A, C290A, C35A/C103A, C35A/C290A, C103A/C290A, C35A/C103A/ C290A were expressed in E.coil expression system. First, the mutant gene was constructed by fusion PCR method. The PCR products were cloned into pMD-T vector for nucleotide sequence analysis. The obtained sequences were compared with the original sequence by Vector NTI software to verify the expected mutant gene. Then, the mutant gene was digested by Nde I/Hind III,extracted and subcloned into the corresponding restriction sites of the expression vector pET 42a (+) to construct the mutant expression plasmid respectively. Finally, expression plasmid was transformed into the E.coli BL21 (DE3) competent cells to obtain engineered strains by ampicillin selection on the LB plate. Randomly selected mutant strains were inoculated into LB culture medium until the OD600 reached about 0.6-1.0, and then 0.6mM of IPTG was added for induction for 5h at 37℃. The induced cells were analyzed by SDS-PAGE. The results showed that the mutant with expected size were high-level expressed successfully in a complete soluble form. The productions of the mutant amount to 30-50% of the total bacterial soluble proteins.Based on the previous study and considering the characterizations of the mutant, the purified mutants were prepared. First, the mutant proteins were captured by Phenyl sepharose FF. Next, the mutants were further purified by DEAE sepharose FF to reduce host cell protein, nucleic acid and endotoxin. At last, the mutants were polished and reconcentrated by Phenyl sephrose HP. After three steps purification, the mutants were purified up to 95% purity.The physical and chemical characterizations of the purified mutants were further evaluated. SDS-PAGE showed the mutants were similar to the commercial product Rasburicase. Western blot showed that the mutants can specifically react to the rabbit anti -Rauburicase serum. Non-reduced SDS-PAGE showed that dimmer of the mutants amount to 0%-29%,respectively. HPLC showed that the mutants performed the same reserve time to the original protein. From the HPLC result,it is induced that the mutants existed in the tetramer form under the normal circumstance. The controversial results between the SDS-PAGE and HPLC can be explained by the process of SDS-PAGE. In the process of the SDS-PAGE, the tetramer was disassociated, cystine residues were exposed, neighbouring cystine residues were oxidized into disulfide bond and further developed dimmer.The bioactivity analysis in vitro was done under the reduced condition, the mutants showed 41.8%-134.1% activity compared to the original urate oxidase. Amount o f which, the mutant C103A performed highest acivity with 134.1%, the mutant C35A/C103A/C290A performed the lowest activity of 41.8%. The difference between the mutants and wild type urate oxidase can be explained by the different sites cystines residues have different action on the maintenance of the active centre conformation. Whereas in the reduced condition, the bioactivity of the mutants and wild-type urate oxidase changed obviously, amount of which the mutant C290A enhance up to 64.4%, the mutant C35A/C103A/C290A reduce up to 15.5. The reason can be related to the removal of the adduct connected to the cystine residues. In the process of the large-scale preparation, the bioactivity was influenced by the above reasons. Given the above results, the cystine residues have indirect action on the maintenance of the active centre conformation though the bioactivity of the urate oxidase was reduced because of oxidation of the cystines residues. The action of the three cystines was connected each other. If all the three cystines were removed, the bioactivity would reduce greatly. But if the Cys103 was replaced with ala alone, the bioactivity of the urate oxidase improved highly. The activity analysis in vivo was further evaluated on the mice hyperuricemia model. The result showed that the mutant C35A/C103A/C290A showed lowest activity of reducing uric acid, the mutants with single mutation showed similar activity to the wild type urate oxidase.