The Active Site of an Algal Prolyl 4-Hydroxylase Has a Large Structural Plasticity

M Kristian Koski,Reija Hieta,Claudia Böllner,Kari I Kivirikko,Johanna Myllyharju,Rik K Wierenga

doi:10.1074/jbc.m706554200

M Kristian Koski, Reija Hieta + Show 4 more

Open Access

https://doi.org/10.1074/jbc.m706554200

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Abstract

Prolyl 4-hydroxylases (P4Hs) are 2-oxoglutarate dioxygenases that catalyze the hydroxylation of peptidyl prolines. They play an important role in collagen synthesis, oxygen homeostasis, and plant cell wall formation. We describe four structures of a P4H from the green alga Chlamydomonas reinhardtii, two of the apoenzyme at 1.93 and 2.90 A resolution, one complexed with the competitive inhibitor Zn2+, and one with Zn2+ and pyridine 2,4-dicarboxylate (which is an analogue of 2-oxoglutarate) at 1.85 A resolution. The structures reveal the double-stranded beta-helix core fold (jellyroll motif), typical for 2-oxoglutarate dioxygenases. The catalytic site is at the center of an extended shallow groove lined by two flexible loops. Mutagenesis studies together with the crystallographic data indicate that this groove participates in the binding of the proline-rich peptide-substrates. It is discussed that the algal P4H and the catalytic domain of collagen P4Hs have notable structural similarities, suggesting that these enzymes form a separate structural subgroup of P4Hs different from the hypoxia-inducible factor P4Hs. Key structural differences between these two subgroups are described. These studies provide first insight into the structure-function relationships of the collagen P4Hs, which unlike the hypoxia-inducible factor P4Hs use proline-rich peptides as their substrates.

Highlights

Tide linkages, requiring Fe2ϩ, 2-oxoglutarate, O2, and ascorbate [1,2,3] (Fig. 1)
The animal Prolyl 4-hydroxylases (P4Hs) have been grouped in two categories; collagen P4Hs (C-P4Hs), which have a central role in the synthesis of all collagens [2, 4], and hypoxia-inducible factor (HIF) P4Hs, which play a key role in the response of cells to hypoxia [5,6]
We report here on the determination of several apo and liganded structures of an algal P4H of C. reinhardtii (Cr-P4H-1) [8], which shows 26% sequence identity to the catalytic domain of C-P4H-I, and which, like the C-P4Hs, uses proline-rich peptide-substrates [8]

Summary

Introduction

Tide linkages, requiring Fe2ϩ, 2-oxoglutarate, O2, and ascorbate [1,2,3] (Fig. 1). Plant P4Hs have been cloned from Arabidopsis thaliana [17, 18], Nicotiana tabacum [19], and the green alga Chlamydomonas reinhardtii [8] Their physiological substrates are proline-rich cell-wall proteins, they can hydroxylate collagenous X-Pro-Gly repeats. Prolyl 4-Hydroxylase Structure that of the peptide-substrate binding domain of the human C-P4H-I ␣ subunit has been solved by NMR and x-ray crystallography [23, 24]. We report here on the determination of several apo and liganded structures of an algal P4H of C. reinhardtii (Cr-P4H-1) [8], which shows 26% sequence identity to the catalytic domain of C-P4H-I (starting from Cr-P4H-1 Val-29, Fig. 2), and which, like the C-P4Hs, uses proline-rich peptide-substrates [8]. Mutagenesis studies and crystallographic data on the crystal contact interactions of loops of neighboring molecules in this groove suggest that it is important for peptide-substrate binding

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