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  • Title: Effects of polynitrogen compounds on the activity of recombinant human HIF-1α prolyl hydroxylase 3 in E. coli.
    Author: Geng Z, Zhu J, Cao J, Geng J, Song X, Zhang Z, Bian N, Wang Z.
    Journal: J Inorg Biochem; 2011 Mar; 105(3):391-9. PubMed ID: 21421125.
    Abstract:
    Hypoxia inducible factor 1α (HIF-1α) becomes an important regulation factor within the histiocyte when it is under the hypoxia condition. Recently, prolyl hydroxylases (PHDs) have been identified to inactivation HIF-lα by hydroxylation. In this study, polynitrogen compounds were screened as HIF-1α PHD3 inhibitors. The coding region of human PHD3 DNA was optimized by using synonymous codons according to the code bias of Escherichia coli. Soluble and active human PHD3 was expressed in the E. coli with a Trx fusion tag under a lower induction temperature of 25°C. Mass spectrometry analysis of the resultant peptide product indicated a mass increase of 16 daltons, consistent with hydroxylation of the proline residue in the HIF-1α (556-574) peptide substrate. Polynitrogen compounds (1-4) inhibited the enzymatic hydroxylation of HIF-1α peptide in a concentration-dependent manner, and the apparent IC(50) values were 29.5, 16.0, 12.8 and 60.4 μM respectively. Double reciprocal (1/V versus 1/[HIF-1α peptide]) plots showed that these compounds are noncompetitive inhibitors of the hydroxylation by recombinant human PHD3 with K(i) values of 67.0, 25.3, 67.3, and 82.1 μM respectively. On the other hand, the metal complexes of these polynitrogen compounds (1-4) cannot inhibit the catalytical activity of PHD3. We hypothesized that the inhibitory mechanism of PHD3 activity by polynitrogen compounds is due to their binding to iron to form stable coordination complexes. Our results in this study indicated that polynitrogen compounds (1-4) could be potential inhibitors of PHD3 to regulate the transcriptional activity of HIF-1α.
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