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  • ML133 HCl Three new trans P Hs AlP H MiP H and


    Three new trans-P4Hs (AlP4H, MiP4H and ScP4H) discovered by a genome mining approach in this study were all located in branch A containing DaP4H sequence. Although another three candidates from Amycolatopsis mediterranei, Bradyrhizobium sp. ORS285 and O. indicum were successfully expressed as soluble forms, no clear activities on l-proline substrate were detected. One possible explanation is that these enzymes may be other hydroxylases towards different substrates. No protein sequences have over 60% sequence identity with three new trans-P4Hs in NCBI protein databases, which further indicates that native trans-P4Hs are very rare. In conclusion, we identified three new trans-P4Hs from NCBI protein databases using genome mining and enzymatic analysis. The highest titer and productivity of trans-4Hyp were achieved by using recombinant E. coli ML133 HCl expressing AlP4H. This study provides a promising enzyme for efficient production of trans-4Hyp. The catalytic performance of AlP4H could be further improved by protein engineering technique.
    Acknowledgments This work was supported by the Key Research Program of the Chinese Academy of Sciences (Grant No. KFZD-SW-212) and the first Special Support Plan for Talents Development and High-level Innovation and Entrepreneurship Team of the Tianjin Municipal City, and Tianjin Science and Technology Program (No. 15PTCYSY00020 and No. 14ZCZDSY00058).
    Introduction Some human 2-oxoglutarate (2OG) and Fe(II) dependent hydroxylases and N-methyl group demethylases are current therapeutic targets.1, 2 Inhibition of the hypoxia inducible factor (HIF) prolyl-4-hydroxylases (PHDs) is being pursued as a treatment for anaemia and other hypoxia/ischaemia related diseases, with PHD inhibitors now in clinical trials for the treatment of anaemia in chronic kidney disease.1, 2, 3 The structurally and mechanistically related procollagen prolyl-4-hydroxylases (C-P4Hs) have also been pursued as anti-fibrotic targets.1, 4, 5,5 Other human 2OG-dependent protein hydroxylases and -methyl lysine demethylases have been identified as potential medicinal chemistry targets, including OGFOD1 (2OG and iron-dependent oxygenase domain containing protein 1), which is a prolyl-3-hydroxylase acting on ribosomal protein 23 (RPS23). 2OG dependent prolyl hydroxylases have also been identified in micro-organisms, including early eukaryotes, yeasts, bacteria, and viruses.10, 11 All identified C-3 and C-4 prolyl hydroxylases (Fig. 1) are 2OG- and Fe(II)-dependent oxygenases and likely have the conserved (but not identical) modified double stranded β-helix core fold, with associated 2OG and Fe(II) binding elements, which is characteristic of the 2OG oxygenases. The Fe(II) binding elements most commonly comprise two histidinyl-residues and an aspartyl-/glutamyl-residue. In nearly all studied cases, 2OG is observed to co-ordinate to the Fe(II) metal centre in a bidentate manner via its oxalyl group. Binding of the C-5 carboxylate of 2OG normally involves interaction with at least one basic residue, which is the case of the human PHDs, is an argininyl-residue;13, 14 however, sequence analyses imply that for the human C-P4Hs, a lysyl-residue interacts with the 2OG C5 carboxylate. Residues involved in substrate binding are more varied, though the stereoelectronic relationship of the target prolyl-residue and the proposed Fe(IV)=O hydroxylating species is likely relatively well conserved.1, 3, 12,12 Whereas some prolyl hydroxylases, such as the PHDs, are monomeric, the animal C-P4Hs are α2β2 tetramers, in which the α-subunit contains the catalytic domain and the β-subunit is identical in sequence to protein disulfide isomerase.5, 15 Few studies have been reported on the selectivity of prolyl hydroxylase inhibitors for different types of prolyl hydroxylases, including for the PHD inhibitors in clinical trials.3, 16 This is important, especially for applications in the treatment of chronic diseases, e.g. anaemia.1, 2, 3 A recent study on PHD inhibitors currently in clinical trials found that some also inhibit the prolyl-3-hydroxylase OGFOD1, but nothing has been reported on the inhibition of microbial and viral prolyl hydroxylases by these compounds. We report on the development of assays for the viral procollagen prolyl hydroxylase (vCPH) from Paramecium bursaria chlorella virus 1, a 2OG-dependent prolyl hydroxylase with greater similarity with the C-P4Hs than with the HIF PHDs,10, 11 and the application of these assays to investigate prolyl hydroxylase inhibitor selectivity.