AIM: To predict wild human heme oxygenase-1 (whHO-1) and hHO-1 His25Ala mutant (△hHO-1) structures, to done and express them and analyze their activities.METHODS: Swiss-PdbViewer and Antheprot 5.0 were used for the p...AIM: To predict wild human heme oxygenase-1 (whHO-1) and hHO-1 His25Ala mutant (△hHO-1) structures, to done and express them and analyze their activities.METHODS: Swiss-PdbViewer and Antheprot 5.0 were used for the prediction of structure diversity and physicalchemical changes between wild and mutant hHO-1, hHO-1 His25Ala mutant cDNA was constructed by site-directed mutagenesis in two plasmids of E.COli DH5α Expression products were purified by ammonium sulphate precipitation and Q-Sepharose Fast Flow column chromatography, and their activities were measured,RESULTS: rHO-1 had the structure of a helical fold with the heme sandwiched between heme-heme oxygenase-1 helices. Bond angle, dihedral angle and chemical bond in the active pocket changed after Ala25 was replaced by His25, but Ala25 was still contacting the surface and the electrostatic potential of the active pocket was negative. The mutated enzyme kept binding activity to heme. Two vectors pBHO-1 and pBHO-1(M) were constructed and expressed. Ammonium sulphate precipitation and column chromatography yielded 3.6-fold and 30-fold higher purities of whHO-1, respectively. The activity of △hHO-1 was reduced 91.21% after mutation comparedwith whHO-1.CONCLUSION: Proximal His25 ligand is crucial for normal hHO-1 catalytic activity. △hHO-1 is deactivated by mutation but keeps the same binding site as whHO-1. △hHO-1 might be a potential inhibitor of whHO-1 for preventing neonatal hyperbilirubinemia.展开更多
基金Supported by the National Natural Science Foundation of China,No.30170988,Shanghai Municipal Education Commission Foundation,No.2000B06,and Shanghai Jiaotong University-Shanghai Second Medical University Cooperative Foundation
文摘AIM: To predict wild human heme oxygenase-1 (whHO-1) and hHO-1 His25Ala mutant (△hHO-1) structures, to done and express them and analyze their activities.METHODS: Swiss-PdbViewer and Antheprot 5.0 were used for the prediction of structure diversity and physicalchemical changes between wild and mutant hHO-1, hHO-1 His25Ala mutant cDNA was constructed by site-directed mutagenesis in two plasmids of E.COli DH5α Expression products were purified by ammonium sulphate precipitation and Q-Sepharose Fast Flow column chromatography, and their activities were measured,RESULTS: rHO-1 had the structure of a helical fold with the heme sandwiched between heme-heme oxygenase-1 helices. Bond angle, dihedral angle and chemical bond in the active pocket changed after Ala25 was replaced by His25, but Ala25 was still contacting the surface and the electrostatic potential of the active pocket was negative. The mutated enzyme kept binding activity to heme. Two vectors pBHO-1 and pBHO-1(M) were constructed and expressed. Ammonium sulphate precipitation and column chromatography yielded 3.6-fold and 30-fold higher purities of whHO-1, respectively. The activity of △hHO-1 was reduced 91.21% after mutation comparedwith whHO-1.CONCLUSION: Proximal His25 ligand is crucial for normal hHO-1 catalytic activity. △hHO-1 is deactivated by mutation but keeps the same binding site as whHO-1. △hHO-1 might be a potential inhibitor of whHO-1 for preventing neonatal hyperbilirubinemia.
