Effects of polynitrogen compounds on the activity of recombinant human Hypoxia inducible factor ‐1αprolyl hydroxylase 3 in Escherichia coli

Hypoxia inducible factor 1α (HIF‐1α) becomes an important regulation factor within the histiocyte when it is under the hypoxia condition. Prolyl hydroxylases have been identified to inactivation HIF‐lα by hydroxylation. In this study, polynitrogen compounds were screened as HIF‐1α PHD3 inhibitors. Soluble and active human PHD3 was expressed in the Escherichia coli with a Trx fusion tag under a lower induction temperature of 25 °C. Polynitrogen compounds (1–4) inhibited the enzymatic hydroxylation of HIF‐1α peptide in a concentration‐dependent manner, and the apparent IC 50 values were 0.81, 2.09, 5.27 and 2.5 μM respectively. Global non‐linear regression of Michaelis‐Menten equation and double reciprocal (1/V vs 1/[HIF‐1α peptide]) plots distinguished type of enzyme inhibition of these compounds. The K i values of non‐competitive and competitive inhibitors of the hydroxylation by recombinant human PHD3 were 7.86, 3.69 and 1.59, 2.92 μM respectively. We hypothesized that inhibitory mechanism of PHD3 activity by polynitrogen compounds due to their binding to iron, and structure determined way of iron coordination leading to distinct type of enzyme inhibition. 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. This work is supported by the National Natural Science Foundation of China (90813020).