Structure and Mechanism of a Viral Collagen Prolyl Hydroxylase.

James E Longbotham,Nigel S Scrutton,Linus O Johannissen,Emily Flashman,Hanna Tarhonskaya,Christoph Loenarz,Colin Levy,Shuo Jiang,Christopher J Schofield,Sam Hay

doi:10.1021/acs.biochem.5b00789

James E Longbotham, Nigel S Scrutton + Show 8 more

Open Access

https://doi.org/10.1021/acs.biochem.5b00789

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Abstract

The Fe(II)- and 2-oxoglutarate (2-OG)-dependent dioxygenases comprise a large and diverse enzyme superfamily the members of which have multiple physiological roles. Despite this diversity, these enzymes share a common chemical mechanism and a core structural fold, a double-stranded β-helix (DSBH), as well as conserved active site residues. The prolyl hydroxylases are members of this large superfamily. Prolyl hydroxylases are involved in collagen biosynthesis and oxygen sensing in mammalian cells. Structural–mechanistic studies with prolyl hydroxylases have broader implications for understanding mechanisms in the Fe(II)- and 2-OG-dependent dioxygenase superfamily. Here, we describe crystal structures of an N-terminally truncated viral collagen prolyl hydroxylase (vCPH). The crystal structure shows that vCPH contains the conserved DSBH motif and iron binding active site residues of 2-OG oxygenases. Molecular dynamics simulations are used to delineate structural changes in vCPH upon binding its substrate. Kinetic investigations are used to report on reaction cycle intermediates and compare them to the closest homologues of vCPH. The study highlights the utility of vCPH as a model enzyme for broader mechanistic analysis of Fe(II)- and 2-OG-dependent dioxygenases, including those of biomedical interest.

Highlights

Prolyl hydroxylases are members of the large and diverse superfamily of enzymes, the Fe(II)- and 2-oxoglutarate (2OG)-dependent dioxygenases.[1]
There are three PHD enzymes (PHD1−3) that modulate the stability of isoforms of the hypoxia inducible factor (HIF), a transcription factor responsible for regulation of genes involved in the hypoxic response.[14,15]
The crystal structure of a viral collagen prolyl hydroxylase has been determined for the first time (Figure 2). vCPH contains many of the structural features conserved in other Fe(II)- and 2-OG-dependent dioxygenases, including the DSBH motif and iron binding residues[12] (Figure 2A)

Summary

Introduction

Prolyl hydroxylases are members of the large and diverse superfamily of enzymes, the Fe(II)- and 2-oxoglutarate (2OG)-dependent dioxygenases.[1] Enzymes from the 2-OG oxygenase superfamily catalyze a large and diverse range of reactions and have a variety of physiological roles.[2−4] Examples include antibiotic synthesis in microorganisms,[5] histone modifications,[6] and DNA repair/RNA modification.[7,8] Despite this diversity of roles, these enzymes share a common reaction mechanism (Figure 1).[1] They utilize Fe(II) and the cosubstrate 2-OG in the active site to activate oxygen, resulting in the twoelectron oxidation of the substrate This is coupled to the decarboxylation of 2-OG to produce carbon dioxide and succinate (Figure 1). Stopped-flow spectroscopy was used to monitor the formation and/or decay of a metal−ligand charge-transfer (MLCT) complex and formation of a hydroxylated product, providing information about the kinetics of the reaction cycle and information about the spectroscopic properties of chemical reaction intermediates (Figure 1)

Results

Discussion

Conclusion