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

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 α-oxidation step in the degradation of phytenic acid in peroxisomes. We describe the x-ray crystallographic structure of PAHX to 2.5 Å 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 β-helix core, which supports three iron binding ligands (His175, Asp177, and His264); 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 (Cys191) 6.7 Å from the Fe(II) and two further histidine residues (His155 and His281) 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) (Pro173, His175, Gln176, Asp177, and His220) and 2OG binding sites (Trp193, Glu197, Ile199, Gly204, Asn269, and Arg275). PAHX may be the first of a new subfamily of coenzyme A-binding 2OG oxygenases.

In humans, the plasma level of the diet-derived isoprenoid, phytanic acid, is normally low (Ͻ30 M) (1). Significantly elevated levels of phytanic acid are found in patients with Refsum disease (RD) 3 (OMIM 266500) and to a lesser extent in patients with other peroxisomal disorders (2). Approximately 45% of reported cases of RD in the United Kingdom have been associated with defects in the function of the per-oxisomal enzyme phytanoyl-CoA 2-hydroxylase (PAHX) (3), which is initially produced as a proprotein with an N-terminal type-2 peroxisomal targeting signal (PTS2) (4). The targeting sequence is cleaved in the peroxisome, between Thr 30 and Ser 31 , to produce mature PAHX, used ubiquitously in this study and referred to hereafter as PAHX (the numbering scheme used follows that for the 338-residue pro-PAHX; gi:6093646) (5,6). Some observed cases of RD are associated with a second genetic locus recently identified as the PTS2 receptor protein, PEX7 (7,8). Symptoms of RD, for which the only current treatment is diet therapy, usually arise later in life and include retinitis pigmentosa, anosmia, deafness, peripheral polyneuropathy, cerebellar ataxia, and ichthyosis (9). 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. Most symptoms of RD are thought to develop from toxic accumulation of phytanic acid. Some symptoms, including skeletal abnormalities (10), occur during embryonic development, since they are present at birth. Phytanic acid levels would not normally be elevated at this stage, suggesting an additional role for PAHX during development. The murine form of PAHX is identical to the murine lupus nephritis-associated protein (LN1) (11), suggesting a possible role for PAHX in kidney disease that has also been observed in human RD patients (3).
Various proteins have been identified as potential PAHX interaction partners, raising the possibility that phytanoyl-CoA may not be the only substrate and that PAHX may have unidentified functions. PAHX has been reported to interact with the blood coagulation factor VIII; it has been proposed that PAHX regulates the expression of this coagulation factor (12), potentially in an oxygen-dependent manner. A further PAHX interaction partner, PAHX-associated protein 1 (PAHX-AP1), identified by a yeast two-hybrid screen (13), interacts with the brainspecific angiogenesis inhibitor 1 (10,14), suggesting a mechanism for some of the neurological symptoms of RD. The physiological significance of these PAHX interactions has not been determined, and how they occur given the apparent peroxisomal localization of PAHX is unclear.
The presence of a 3-methyl group in phytol prevents its degradation by the normal ␤-oxidation pathway for saturated fatty acids. Instead, a preliminary ␣-oxidation pathway occurs, first in the endoplasmic reticulum and subsequently in peroxisomes, to shorten the chain length of 2-hydroxyphytanoyl-CoA by one carbon, thereby enabling ␤-oxidation to proceed (Fig. 1). It has recently been shown that phytenic acid is first condensed to its coenzyme A ester prior to reduction to phytanoyl-CoA (15), which is subsequently hydroxylated to 2-hydroxyphytanoyl-CoA in the PAHX-catalyzed reaction (Fig. 2) (4 -6, 16, 17). The stereospecificity of PAHX is interesting, since it accepts both C-3 epimers as substrates but produces only the threo products (2S,3R or 2R,3S) (18,19). Thiamine pyrophosphate-dependent lyase-catalyzed fission of the C1-C2 bond of 2-hydroxyphytanoyl-CoA then occurs to produce formyl-CoA and pristanal (20 -22), the latter of which is oxidized in an NAD ϩ -dependent reaction to pristanic acid (23). After re-esterification with coenzyme A and epimerization (24 -26), metabolism via "normal" ␤-oxidation pathway, occurs, initially in peroxisomes and later in mitochondria. Recent studies have investigated the substrate specificities and activities of both pro-PAHX and mature PAHX as well as clinically observed mutants (19,27). The pro and mature forms of PAHX have a similar substrate specificity, and both accept isovaleryl-CoA as a substrate (27,28), demonstrating that in vitro a long chain fatty acid side chain is not required for binding and activity of PAHX (4). Site-directed mutagenesis studies have led to proposals for the Fe(II)-binding residues of PAHX and identified other residues likely to be involved in catalysis (27,29). Most, if not all, 2OG oxygenases bind their Fe(II) via a conserved HX(D/ E) . . . H triad of residues located on a double-stranded ␤-helix (DSBH) or jelly roll motif (30). However, significant differences exist in the way they bind their substrates and, to a lesser extent, 2OG. The only reported crystal structures for a mammalian 2OG-dependent oxygenase are for factor inhibiting hypoxia-inducible factor (FIH) (31), which, together with other 2OG-dependent oxygenases involved in the human hypoxic response, is a current target for therapeutic intervention.
To address questions regarding the structural basis of clinically observed mutations giving rise to RD (8), we have solved the crystal structure of PAHX complexed with Fe(II) and 2OG. Although the Fe(II) binding sites are well conserved between PAHX and FIH, comparison of the structures reveals significant differences in the two active sites and that many of the clinically observed mutations to PAHX are clustered around either the Fe(II) or 2OG binding sites.

EXPERIMENTAL PROCEDURES
Protein Expression and Purification-Purification of PAHX was carried out as reported (29). Briefly, Escherichia coli BL21(DE3) were transformed by pET24a/mat-pahx and grown at 37°C in 2TY medium containing 30 g/ml kanamycin. Following reduction of the temperature to 25°C, protein production was induced at an A 600 of 0.7 by the addition of isopropyl-␤-D-thiogalactopyranoside to a final concentration of 1.0 mM. In previous studies, we found that PAHX is unusually prone to site-specific Fe(II) and oxygen-mediated fragmentation. For this reason, dithiothreitol was omitted, and 1 mM EDTA was added to all buffers to prevent fragmentation during purification. PAHX was purified by carboxymethyl-Sepharose cation exchange, followed by size exclusion chromatography (Superdex S75) to yield PAHX of Ͼ95% purity by SDS-PAGE analysis. 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). The activity of purified PAHX was confirmed for 2OG turnover using the reported assay monitoring release of 14 CO 2 (32).
Crystallization-Crystallization conditions were initially sought using high throughput robotic screening methods at the Oxford Protein Production Facility (33). Optimization of the initial crystallization condition was performed using the hanging drop vapor diffusion method in VDX TM plates (Hampton Research, Aliso Viejo, CA). Hanging drops containing 2 l of 5.2 mg/ml PAHX and 2 l of well solution were suspended over 500 l of well solution containing 21% polyethylene glycol 3350, 0.3 M triammonium citrate, pH 7.1, at 18°C. Crystallization with 1 mM iron(II) sulfate and 2 mM 2OG in an anaerobic environment (Bell Technologies glove box under an argon atmosphere) produced square, plate-shaped crystals over a period of 3 weeks to a maximum size of 200 m ϫ 100 m ϫ 50 m. 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. Native data were collected at 100 K using beamline 10.1 (34) of the Synchrotron Radiation Source (SRS, Daresbury, UK) equipped with a MAR 225CCD detector. The native data were processed with MOSFLM and SCALA of the CCP4 suite version 5.0.2 (35,36) with an I222 lattice (TABLE ONE). Calculation of a Matthews coefficient of 2.1 Å 3 /dalton implied a single monomer in the asymmetric unit. Attempts at molecular replacement and isomorphous replacement were unsuccessful, so crystals were produced from SeMet-substi-tuted protein. A single SeMet crystal (120 m ϫ 70 m ϫ 5 m) was transferred to cryoprotectant (1:9 glycerol/well solution) before cryocooling in liquid nitrogen. Single wavelength anomalous dispersion (SAD) data were collected at 100 K and at the selenium peak wavelength at beamline X10A (Swiss Light Source, Villigen, Switzerland). Data were integrated with HKL2000 version 1.98.0 (37) and merged, and anomalous differences were analyzed with XPREP (38). The substructure was solved with SHELXD of the SHELX-97 suite (39) using anomalous differences to 3.5 Å in space group I222, and the sites and chirality were confirmed with SHELXE. The substructure (top seven selenium sites) was refined with SHARP version 2.0.4 (40) against the SAD data, and two additional selenium sites were identified. Phases were calculated to 3 Å using SHARP, and the combined anaerobic native and SAD data sets result in a figure of merit of 0.33. Phases were improved by density modification using RESOLVE version 2.08 (41) to 2.7 Å with a final figure of merit of 0.85, resulting in core secondary structure elements and a 7 peak in the core region attributable to the iron atom clearly visible in the electron density.
The initial model was built using the program COOT version 0.0.31 (42) into composite omit maps calculated with RESOLVE. Before refinement commenced, 5% of the data were flagged for calculation of a free R-factor (R free ). Initially, simulated annealing was performed in CNS version 1.1 using combined phases calculated from the model and phase probabilities from SHARP. Iterative refinement using CNS version 1.1 and model building using COOT continued until R free was below 30%. At this stage, restrained TLS refinement was performed using REFMAC5 (43) with only the model phases. Iterative rounds of manual refitting and crystallographic refinement using the programs COOT and REFMAC5 continued until R free was no longer improved. Statistics are given in TABLE ONE.

RESULTS AND DISCUSSION
The PAHX Structure-PAHX crystallizes as a monomer in space group I222 with one molecule per asymmetric unit. The solvent content of the crystals is 40%, and the crystals contain large solvent channels that coincide with access to the active site. Three disordered loops that border the active site face this solvent channel and include residues 165-172, 223-233, and 303-318. Other disordered regions are near the N terminus and comprise residues 31-42 and 51 and 52. The most extensive contact between monomers in the lattice, 841 Å 2 per monomer, is a ϳ18-Å-long extended pair of antiparallel ␤-strands (␤-3 and ␤-4) in which ␤-4 is antiparallel to ␤-4 of a symmetry-related molecule along a crystallographic 2-fold axis. Another significant lattice contact involves loop 240 -244, which packs against Lys 321 of the C-terminal helix, and residues 288 -291 of one symmetry-related molecule and residue 94 of another.
The PAHX fold is a mixed ␣-␤ structure composed of a major and a minor ␤-sheet surrounded by five ␣-helices and four 3 10 helices (Fig. 3). As in other 2OG oxygenases for which structures are available (30, 31, 44 -49), the core of the protein consists of a DSBH fold composed of eight ␤-strands ( Fig. 4 and supplemental Fig. 2S). However, in PAHX, only seven ␤-strands of the DSBH are apparent: ␤-6, ␤-8, ␤-9, ␤-10, ␤-11, ␤-12, and ␤-13, here defined as ␤-strands I, III, IV, V, VI, VII, and VIII, respectively. In PAHX, the residues that correspond to DSBH ␤-strand II (␤-7) (residues 165-172) in other 2OG oxygenases are disordered and left out of the model. This strand is probably involved in substrate binding (see below). Four additional ␤-strands (␤-1, ␤-2, ␤-5, and ␤-15) hydrogen-bond antiparallel to the core DSBH, resulting in the eight-stranded major ␤-sheet (Fig. 3). It is interesting to note that both ␤-strands ␤-5 and ␤-15 are paired antiparallel to DSBH ␤-strand I (␤-6), a unique feature among the 2OG oxygenase family. Fe(II) Binding Site-The Fe(II)-containing active site is located between one end of the ␤-sheets that form the DSBH core in a manner similar to other 2OG oxygenases (30, 31, 44 -49) (Fig. 4). The experimental electron density clearly shows the Fe(II) coordinated in an approximately octahedral manner by two oxygens from 2OG, a water molecule (B-factor 44.6 Å 2 ), and the side chains of His 175 , Asp 177 , and His 264 (Fig. 5, A-C). The HXD motif is located just after the sequence that is typically DSBH ␤-strand II (residues 165-172), and the second Fe(II)-binding histidine (His 264 ) is located on DSBH ␤-strand VII (␤-12). The observation of Fe(II) coordination by these three residues confirms their provisional assignment by mutation analyses coupled to iron-binding assays (27). The two Fe(II)-binding histidines adopt similar conformations to those observed in other 2OG oxygenases. The aspartate is in a similar conformation to that observed in deacetoxycephalosporin synthase (DAOCS) and proline-3-hydroxylase (30,48) but differs from that observed in FIH and clavaminic acid synthase (CAS) (Fig. 4) (31, 46). However, the structures of FIH and CAS may reflect unusual cases. In addition to binding Fe(II), the aspartate of FIH forms a hydrogen bond to a backbone amide of the peptide substrate, and in the trifunctional CAS the Fe(II) binding carboxylate is derived from a glutamate rather than an aspartate residue.
2OG Binding-The 2OG co-substrate coordinates the Fe(II) in a bidentate manner with its 2-keto group trans to Asp 177 O␦1 and the oxygen of the 1-carboxylate trans to His 264 N⑀2 (Fig. 5B). The position of the 2OG 2-keto oxygen relative to the three Fe(II)-binding residues is observed to be conserved in all relevant crystal structures for 2OG oxygenases. However, the relative position of the 1-carboxylate of 2OG varies between being trans to His 264 (or its equivalent), as observed in PAHX, or trans to His 175 (or its equivalent) (Fig. 5B). In the case of CAS, the relative position of the 1-carboxylate was observed to change upon binding the dioxygen analogue, nitric oxide (50), from being trans to His 144 (His 175 in PAHX) to being trans to His 279 (His 264 in PAHX). Binding of the substrate has been observed to displace a water molecule from the Fe(II), thus enabling binding of oxygen (51). Thus, the coordination position of the 2OG 1-carboxylate group in PAHX may vary from its observed position upon (co-)substrate binding or under noncrystalline conditions. It is possible that the observed coordination position of the C1Ј-carboxylate in PAHX reflects a protected form of the enzyme, preventing oxygen binding to avoid the generation of potentially damaging reactive oxidizing intermediates in the absence of substrate. This may be particularly important in the oxidizing environment of the peroxisomes.
The 1-carboxylate oxygen of 2OG not ligated to the Fe(II) is in position to form a hydrogen bond (3.2 Å) with N⑀ of Lys 120 located on ␤-strand ␤-5 (Fig. 5A). For some 2OG oxygenases, the side chain of a basic or polar residue is also positioned close to the C1Ј-carboxylate of 2OG (e.g. Arg 297 of CAS, Arg 160/162 of DAOCS, and Arg 95/97/122 of proline-3-hydroxylase). However, PAHX is the first case where the basic residue, Lys 120 , is actually close enough to hydrogen-bond (3.2 Å) with the 2OG C1Ј-carboxylate (Fig. 5A). It is possible either that substrate binding weakens the hydrogen bond, enabling a rearrangement of the FIGURE 3. Stereoview from the crystal structure of the PAHX-Fe(II)-2OG complex as a ribbon representation. The conserved eight stranded DSBH core found in all Fe(II) and 2OG-dependent oxygenases is colored yellow. Only seven strands (␤-6, ␤-13, ␤-8, and ␤-11 of the major sheet and ␤-9, ␤-10, and ␤-12 of the minor sheet) are observed in PAHX, since the residues making up the DSBH ␤-strand II are disordered. Additional ␤-strands attached to the major ␤-sheet are colored slate blue. The Fe(II)-binding residues and 2OG C5Ј-carboxylate interacting arginine of PAHX along with the 2OG co-substrate and Fe(II) cofactor are shown as a stick representation. ␣and 3 10helices are shown in red.  DECEMBER 9, 2005 • VOLUME 280 • NUMBER 49 ligation position of the 2OG C1Ј-carboxylate, or that after oxidative decarboxylation of 2OG the side chain of Lys 120 is involved in the release of carbon dioxide and/or succinate from the catalytic site.

Structure of Human Phytanoyl-CoA 2-Hydroxylase
In 2OG oxygenases, the C5Ј-carboxylate of 2OG is typically bound by an arginine or lysine and by a hydroxyl group (30, 31, 44 -49). For the DAOCS subfamily, the basic arginine and hydroxyl originate from an RXS motif located on DSBH ␤-strand VIII (␤-13) (30). In FIH, Lys 214 (from DSBH ␤-strand IV) and Thr 196 (from DSBH ␤-strand II) interact with the C5Ј-carboxylate of 2OG. In PAHX, Arg 275 , located on DSBH ␤-strand VIII (␤-13) at the back of the 2OG binding pocket, is in position to form an electrostatic interaction with the C5Ј-carboxylate of 2OG similar to that observed in DAOCS. Based upon the weaker electron density and higher temperature factors, the C5Ј carboxylate appears less ordered than the 2-oxoacid portion of 2OG. This is probably due to the disordered state of DSBH ␤-strand II discussed below. There is no RXS motif in PAHX, since the serine is substituted by an alanine. However, O␥ of Ser 266 , two residues from the His 264 of the HXD . . . H Fe(II) binding triad on ␤-12 (DSBH ␤-strand VII), is in position to hydrogen-bond (2.7 Å) to the C5Ј-carboxylate of 2OG. It is notable that this serine is conserved in close homologues of PAHX from Mus musculus, Xenopus laevis, Danio rerio, Caenorhabditis elegans, the more distant human homologue PHYHD1, and two homologues from the thienamycin antibiotic biosynthetic gene cluster encoded for by thnG and thnQ (see supplemental information for sequence alignment) (53,54).
Unusual Features of the PAHX Active Site and Substrate Binding-In addition to the Fe(II)-and 2OG-binding residues, there are a number of other polar residues in the PAHX active site including two histidines, His 155 and His 281 (Fig. 5A). The side chain of His 281 is close to Asp 177 (which binds to Fe(II)) and is also conserved in the close homologues of PAHX from M. musculus, X. laevis, D. rerio, and C. elegans. These residues are not conserved in FIH, and their presence may explain why PAHX appears particularly susceptible to inhibition by Ni(II) (27,29,55), although competition at the Fe(II) binding site is also important in this regard. Cys 191 is conserved in the PAHX homologues from M. musculus, X. laevis, D. rerio, and C. elegans. The observation that the sulfydryl of Cys 191 is 6.7 Å from the Fe(II) and only 4.6 Å from the water molecule ligated to Fe(II) suggests that it may be of mechanistic significance (Fig. 5A). Assuming that oxygen binds in the position occupied by the water molecule, the sulfur of Cys 191 might be within interacting distance of the oxygen. Another mechanistic possibility is that Cys 191 is reversibly acylated by the acyl-CoA substrates for PAHX. However, the relatively inaccessible position of Cys 191 , at least when 2OG is bound, suggests that acylation is unlikely.
Taking into account the size of the PAHX substrate, phytanoyl-CoA, it is likely that the disordered loops that surround the active site play a role in substrate binding and may only become ordered in the presence of phytanoyl-CoA or in the presence of a protein that "presents" the substrate to the enzyme, such as sterol carrier protein-2 (28). As noted previously, residues equivalent to 165-172 have ␤-strand secondary structure and form DSBH ␤-strand II in other reported structures for 2OG oxygenases. The region between residues 223 and 233 appears to extend from a long loop between DSBH ␤-strands IV and V (␤-9 and ␤-10) and probably serves to enclose the active site. The region between residues 303 and 318 is located prior to the C-terminal helix, which for some 2OG oxygenase family members has been shown to play a role in substrate recognition (56); in FIH, the C-terminal helices are involved in dimerization in addition to substrate recognition (57). Assuming the structure of PAHX as observed represents an "open" conformation with its disordered loops, a large groove leading to the active site is clearly visible on its surface. The groove has dimensions of ϳ10 ϫ 40 Å, the appropriate size for phytanoyl-CoA to bind (Fig. 5D). The co-crystallization of PAHX with substrate would provide a greater understanding of the substrate selectivity of PAHX. Soluble substrate analogues such as isovaleryl-CoA are an alternative for co-crystallization studies, since phytanoyl-CoA is relatively insoluble. Co-crystallization attempts with isovaleryl-CoA have not yet been successful.
Clinically Observed and Other Mutants-A total of 16 clinically observed missense mutations in PAHX have been identified (TABLE  TWO) (8,58). Analysis of the PAHX structure reveals the majority of these mutations to be clustered in two regions: one around the Fe(II) (five mutants) and the other around the 2OG binding pocket (six mutants) (Fig. 6). The enzymatic activity of P29S, Q176K, G204S, N269H, R275W, and R275Q clinical mutations have been assayed using recombinant enzyme (29). The P29S mutation, located in the PTS2, has been proposed to effect targeting of pro-PAHX to the peroxisome (29,59).
Clinical mutations that affect iron binding include those involving His 175 (H175R) and Asp 177 (D177G), which directly ligate the iron, and Gln 176 (Q176K), located between the two Fe(II)-binding residues. Mutation of His 175 or Asp 177 to alanine ablates PAHX activity and causes impaired Fe(II) binding in vitro (27), and the H175R and D177G clinical mutants are likely to have a similar consequence. The side chain of Gln 176 is in position to make two hydrogen bonds with the backbone atoms Ser 216 oxygen and Lys 218 nitrogen located on a stretch of random coil leading to a disordered loop (residues 223-233), which is probably involved in substrate binding as mentioned above. The Q176K clinical mutation is likely to interrupt this hydrogen bond network and cause changes in the backbone conformation for both the Fe(II)-binding residues and the potential substrate binding loop 223-233. In vitro, both the Q176K and Q176A mutations show uncoupling of 2OG oxidation and hydroxylation of phytanoyl-CoA (27,29).
Two other clinical mutations, H220Y and P173S, are also predicted to affect Fe(II) binding. His 220 is located on the sequence approaching the disordered loop (223-233), predicted to be involved in substrate binding, and its N⑀1 atom is in position to hydrogen-bond with the backbone amide of Gln 176 . Mutation of His 220 to a bulky hydrophobic tyrosine may ablate the hydrogen bond and destabilize or modify the conformation of the Fe(II)-binding residues on either side of Gln 176 . Region 165-172 is disordered, representing a high level of conformational flexibility. Pro 173 introduces a conformationally rigid anchor point that prevents the flexibility of the sequence just prior from being transferred to the subsequent Fe(II)-binding residues His 175 and Asp 177 . The clinical mutation, P173S, removes this rigid anchor and probably destabilizes Fe(II) binding.
Clinical mutations that affect, or probably affect, 2OG binding include Arg 275

Structure-function relationships of observed clinical and nonclinical mutations to PAHX
Relationships were clinically observed (58), except N83Y and H175R (8) and R275W (6) and nonclinical mutations of PAHX (NC) and how they relate to the structure. The numbers in the homo/heterozygous column refer to the number of times the particular homo/heterozygous mutation was identified in the study.

Structure of Human Phytanoyl-CoA 2-Hydroxylase
disrupts these interactions due to increased steric bulk. The backbone oxygen of Gly 204 is in position to hydrogen-bond with the side chain of Arg 275 (2.9 Å); the addition of a hydroxymethylene side chain in the clinical mutation G204S could destabilize the interaction due to steric constraints. Asn 269 is in position to make four hydrogen bonds involved in maintaining the structure of the turn immediately prior to Arg 275 . These hydrogen bonds include Asn 269 O␦1 to Thr 271 nitrogen (3.2 Å) and O␥1 (2.7 Å), and Asn 269 N␦2 to Gly 273 oxygen (2.8 Å) and Lys 161 oxygen (2.9 Å). Four clinical mutations (including one in the PTS2) are not obviously involved in Fe(II) or 2OG binding. Phe 257 is located on the outer face of the major ␤-sheet contacting helix ␣-4 and is buried in a hydrophobic pocket made up of residues Tyr 61 , Leu 67 , Ile 69 , Leu 72 , Ile 139 , Tyr 142 , Phe 146 , Val 190 , Ala 192 , and Thr 255 . The F275S mutation will unfavorably place a polar side chain in a hydrophobic pocket and possibly interfere with the overall structure or impair protein folding. Two clinical mutations, R245Q and N83Y, are located on the surface of the enzyme and far from the active site. These mutations may result in disruption of protein-protein interactions proposed to involve PAHX, such as that with sterol carrier protein-2, proposed to be responsible for solubilization and presentation of phytanoyl-CoA (as opposed to phytanic acid, pristanic acid, or pristanoyl-CoA (61)) to PAHX (62,63).
In studies aimed at defining the Fe(II) binding ligands of PAHX, various other histidine mutations have been made and assayed. The H264A mutation has been shown to disrupt Fe(II) binding, an observation supported by the structure. His 259 is located at the i position of a Type I ␤-turn between strands ␤-11 and ␤-12 (the sixth and seventh ␤-strands of the DSBH core) and makes three hydrogen bonds: His 259 oxygen to Leu 262 nitrogen at the i ϩ 3 position (3.0 Å), His 259 N␦1 to Ile 261 nitrogen at the i ϩ 2 position (2.9 Å), and His 259 N⑀2 to Asp 187 O␦1 (3.2 Å). The H259A mutation removes two of the hydrogen bonds and probably destabilizes the ␤-turn, which in turn destabilizes the core DSBH. His 213 is located on a small stretch of 3 10 -helix (residues 212-214) and is positioned to interact with the C-terminal end of helix ␣-1 and probably stabilizes the helix dipole through electrostatic interactions. The H213A mutation results in insoluble protein during expression (27), consistent with the proposed structural role for this residue.
Medicinal and Biological Implications-Knowledge of the 2OG binding site of PAHX may help to develop new treatments for RD and in the design of selective inhibitors for human 2OG oxygenases. Mukherji et al. (60) have shown that it is possible to partially rescue the activity of a purified 2OG binding pocket mutant using complementary 2OG analogues. An R275Q mutant showed a 500-fold increase in activity using 2-oxobutyrate in place of 2OG as the co-substrate. Structural knowledge of the 2OG binding pocket of PAHX may enable the design of more efficient substituted 2-oxoacids, which are useful for "chemical co-substrate rescue" of mutant PAHX activity.
Human 2OG oxygenases involved in the hypoxic response, including FIH and the hypoxia-inducible factor (HIF) prolyl-hydroxylases, are targets for inhibition with the medicinal objective of up-regulating HIF target genes (64). Inhibitors of 2OG oxygenases based upon analogues of 2OG (e.g. N-oxalylamino acids) have been shown to be active against the HIF hydroxylases (65), including FIH. N-oxalylglycine is also an inhibitor of PAHX 4 and probably inhibits many human 2OG oxygenases and other 2OG-utilizing enzymes. Recently, it has been reported that N-oxalylamino acids with the D-stereochemistry and hydrophobic side chains are selective for FIH over a HIF prolyl-hydroxylase (human prolyl-hydroxylase domain isoform 2) (66). Given the differences in the 2OG binding pocket between FIH and PAHX, it might be possible to use the PAHX structure to design FIH inhibitors that do not significantly inhibit PAHX and possibly its nearest human relative, PHYHD1.
The observation of homozygous patients possessing inactive mutant PAHX that can still metabolize phytanoyl-CoA, albeit at a very reduced rate (67), have led to proposals for an alternative pathway for phytanoyl-CoA metabolism via -oxidation (68), or the existence of other phytanoyl-CoA-metabolizing enzymes. The putative human 2OG oxygenase, PHYHD1 (phytanoyl-CoA dioxygenase domain-containing 1) (52), is clearly related to PAHX, and both have additional homologues in a wide range of metazoans and bacteria. Although the Fe(II)-and 2OGbinding residues (including His 175 , Asp 177 , His 264 , Arg 275 , Lys 120 , and Ser 266 ), together with other active site residues (e.g. His 281 ), are conserved among the homologues, the three disordered regions in PAHX proposed to be involved in substrate binding are very different. This suggests that phytanoyl-CoA is not a common substrate. However, the evolutionary relationship of PAHX to its homologues might indicate a new subfamily of coenzyme A-binding 2OG-dependent oxygenases.