Structure of Human Phytanoyl-CoA 2-Hydroxylase Identifies Molecular Mechanisms of Refsum Disease*

Michael A Mcdonough,Kathryn L Kavanagh,Danica Butler,Timothy Searls,Udo Oppermann,Christopher J Schofield

doi:10.1074/jbc.m507528200

Michael A Mcdonough, Kathryn L Kavanagh + Show 4 more

Open Access

https://doi.org/10.1074/jbc.m507528200

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Abstract

Refsum disease (RD), a neurological syndrome characterized by adult onset retinitis pigmentosa, anosmia, sensory neuropathy, and phytanic acidaemia, is caused by elevated levels of phytanic acid. Many cases of RD are associated with mutations in phytanoyl-CoA 2-hydroxylase (PAHX), an Fe(II) and 2-oxoglutarate (2OG)-dependent oxygenase that catalyzes the initial alpha-oxidation step in the degradation of phytenic acid in peroxisomes. We describe the x-ray crystallographic structure of PAHX to 2.5 A resolution complexed with Fe(II) and 2OG and predict the molecular consequences of mutations causing RD. Like other 2OG oxygenases, PAHX possesses a double-stranded beta-helix core, which supports three iron binding ligands (His(175), Asp(177), and His(264)); the 2-oxoacid group of 2OG binds to the Fe(II) in a bidentate manner. The manner in which PAHX binds to Fe(II) and 2OG together with the presence of a cysteine residue (Cys(191)) 6.7 A from the Fe(II) and two further histidine residues (His(155) and His(281)) at its active site distinguishes it from that of the other human 2OG oxygenase for which structures are available, factor inhibiting hypoxia-inducible factor. Of the 15 PAHX residues observed to be mutated in RD patients, 11 cluster in two distinct groups around the Fe(II) (Pro(173), His(175), Gln(176), Asp(177), and His(220)) and 2OG binding sites (Trp(193), Glu(197), Ile(199), Gly(204), Asn(269), and Arg(275)). PAHX may be the first of a new subfamily of coenzyme A-binding 2OG oxygenases.

Highlights

In humans, the plasma level of the diet-derived isoprenoid, phytanic acid, is normally low (Ͻ30 ␮M) [1]
We describe the x-ray crystallographic structure of phytanoyl-CoA 2-hydroxylase (PAHX) to 2.5 Aresolution complexed with Fe(II) and 2OG and predict the molecular consequences of mutations causing Refsum disease (RD)
Symptoms of RD can be subtle in the early stages, making diagnosis difficult, and it has been proposed that the disease is more widespread than clinical data suggest

Summary

EXPERIMENTAL PROCEDURES

Protein Expression and Purification—Purification of PAHX was carried out as reported [29]. Electrospray ionization mass spectrometry revealed that the mass of the purified PAHX was consistent with loss of the N-terminal methionine from the predicted amino acid sequence (observed, 35,436 Da; calculated without N-terminal methionine, 35,435 Da). Amino acid sequence analysis by Edman degradation confirmed the identity of the protein and loss of the N-terminal methionine (observed, STGISS). Optimization of the initial crystallization condition was performed using the hanging drop vapor diffusion method in VDXTM plates (Hampton Research, Aliso Viejo, CA). SeMet PAHX crystals were grown aerobically using the same conditions, except that iron(II) sulfate and 2OG were substituted with zinc(II) chloride and N-oxalylglycine. Crystallographic Data Collection and Structure Solution—A single crystal, anaerobically grown (200 ϫ 100 ϫ 50 ␮m) was transferred to cryoprotectant (1:7 glycerol/well solution) and immediately cryocooled in liquid nitrogen. Attempts at molecular replacement and isomorphous replacement were unsuccessful, so crystals were produced from SeMet-substi-

TABLE ONE

Ramachandran statistics

RESULTS AND DISCUSSION

TABLE TWO