Structure prediction and activity analysis of human heme oxygenase-1 and its mutant.
OBJECTIVE
To predict wild human heme oxygenase-1 (whHO-1) and hHO-1 His25Ala mutant (delta hHO-1) structures, to clone and express them and analyze their activities.


METHODS
Swiss-PdbViewer and Antheprot 5.0 were used for the prediction of structure diversity and physical-chemical changes between wild and mutant hHO-1. hHO-1 His25Ala mutant cDNA was constructed by site-directed mutagenesis in two plasmids of E. coli DH5alpha. 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 delta hHO-1 was reduced 91.21% after mutation compared with whHO-1.


CONCLUSIONS
Proximal His25 ligand is crucial for normal hHO-1 catalytic activity. delta hHO-1 is deactivated by mutation but keeps the same binding site as whHO-1. delta hHO-1 might be a potential inhibitor of whHO-1 for preventing neonatal hyperbilirubinemia.
Department of Pediatrics, Rui Jin Hospital, Shanghai Second Medical University, 197 Rui Jin Er Road, Shanghai 200025, China. xzw63@hotmail.com
Zhen-Wei
ZW
Xia
Wen-Pu
WP
Zhou
Wen-Jun
WJ
Cui
Xue-Hong
XH
Zhang
Qing-Xiang
QX
Shen
Yun-Zhu
YZ
Li
Shan-Chang
SC
Yu
D002850:Chromatography, Gel; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D006419:Heme Oxygenase (Decyclizing) / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D051547:Heme Oxygenase-1; D006801:Humans; D007700:Kinetics; D008565:Membrane Proteins; D008958:Models, Molecular; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed; D010957:Plasmids / Q000235:genetics; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D015183:Restriction Mapping
D008565:Membrane Proteins; D011994:Recombinant Proteins; C496541:HMOX1 protein, human; D006419:Heme Oxygenase (Decyclizing); D051547:Heme Oxygenase-1