Studies on phytanoyl-CoA 2-hydroxylase and synthesis of phytanoyl-coenzyme A

Article abstract of DOI:10.1016/S0960-894X(01)00494-2

Phytanoyl-CoA 2-hydroxylase (PAHX), an iron(II) and 2-oxoglutarate-dependent oxygenase, catalyses an essential step in the mammalian metabolism of β-methylated fatty acids. Phytanoyl-CoA was synthesised and used to develop in vitro assays for PAHX. The product of the reaction was confirmed as 2-hydroxyphytanoyl-CoA by NMR and mass spectrometric analyses. In accord with in vivo analyses, hydroxylation of both 3R and 3S epimers of the substrate was catalysed by PAHX. Both pro- and mature- forms of PAHX were fully active.

Full text of DOI:10.1016/S0960-894X(01)00494-2

Bioorganic & Medicinal Chemistry Letters 11 (2001) 2545–2548
Studies on Phytanoyl-CoA 2-Hydroxylase and Synthesis of
Phytanoyl-Coenzyme A
Nadia J. Kershawa,y, Mridul Mukherjia,y Colin H. MacKinnon,a
Timothy D. W. Claridge,a Barbara Odell,a Anthony S. Wierzbicki,b
Matthew D. Lloyda,* and Christopher J. Schofielda,*
aThe Oxford Centre for Molecular Sciences and The Dyson Perrins Laboratory, South Parks Road, Oxford OX1 3QY, UK
bDepartment of Chemical Pathology, King’s, Guy’s and St. Thomas’ Medical School, St Thomas’ Hospital Campus,
Lambeth Palace Road, London SE1 7EH, UK
Received 25 April 2001; accepted 11 July 2001
Abstract—Phytanoyl-CoA 2-hydroxylase (PAHX), an iron(II) and 2-oxoglutarate-dependent oxygenase, catalyses an essential step
in the mammalian metabolism of b-methylated fatty acids. Phytanoyl-CoA was synthesised and used to develop in vitro assays for
PAHX. The product of the reaction was confirmed as 2-hydroxyphytanoyl-CoA by NMR and mass spectrometric analyses. In
accord with in vivo analyses, hydroxylation of both 3R and 3S epimers of the substrate was catalysed by PAHX. Both pro- and
mature- forms of PAHX were fully active. # 2001 Elsevier Science Ltd. All rights reserved.
Phytanic acid (1) in the human diet is derived via
microbial cleavage of chlorophyll followed by reduc-
tion/oxidation of the resultant phytol side chain. The
presence of the 3-methyl group in 1 prevents its normal
degradation by the fatty acid b-oxidation pathway.
Phytanic acid (1) is normally present in small amounts
in human tissues, but accumulates in patients with
Refsum’s disease. Some of the symptoms of Refsum’s
disease can be ameliorated by treatment but others
including blindness, anosmia and deafness are irrever-
sible.1
with the peroxisomal membrane;4 (ii) phytanoyl-CoA
(3) is then oxidised to give 2-hydroxyphytanoyl-CoA (4)
in a step catalysed by phytanoyl-CoA 2-hydroxylase
(PAHX);3,5 (iii) the TPP-dependent enzyme 2-hydro-
xyphytanoyl-CoA lyase catalyses the unusual conver-
sion of (4) to pristanal (5) and formyl-CoA;6 (iv) finally,
pristanal (5) is oxidised, in an NAD+-dependent reac-
tion, to give pristanic acid (2).7
Comparison of the sequence and predicted structures
for PAHX suggests it is a member of the super-family of
2-oxoglutarate-dependent oxygenases.8À10 Conserved
motifs present in PAHX include the iron binding
HXD motif encompassing residues His-175 and
Asp-177 in the human PAHX sequence. Recently,
Mihalik et al. reported the cloning and expression
in Escherichia coli of the gene (pahx) for PAHX.3
PAHX, with its peroxisomal targeting sequence
attached, was produced in E. coli as a fusion pro-
tein (using the pMALc expression vector). This
enzyme was shown to catalyse the conversion of 3 to
4 in the presence of ferrous ions and 2-oxoglutarate by
an HPLC assay using radioactive phytanoyl-CoA (3)3
prepared by enzyme-catalysed condensation of phytanic
acid (1) and coenzyme A. Here, we report the chemical
synthesis of phytanoyl-CoA (3) and its use to study the
reaction of recombinant wild-type pro- and mature-
PAHX.
It is possible that some metabolism of phytanic acid (1)
occurs by an o-oxidation pathway. However, the major
pathway for its oxidation is in liver peroxisomes via a
preliminary a-oxidation pathway in which one carbon
atom is removed to give pristanic acid (2), which is then
metabolised via the normal b-oxidation pathway
(Scheme 1).2,3 The sequence of events in the preliminary
pathway has recently been elucidated as follows: (i)
phytanic acid (1) is converted to phytanoyl-CoA (3)
probably by a non-specific ligase that may be associated
*Corresponding authors. Tel.: +44-1865-275625; fax: +44-1865-
275674; e-mail: matthew.lloyd@chem.ox.ac.uk; christopher.schofield@
chem.ox.ac.uk
yNadia J. Kershaw and Mridul Mukherji contributed equally to this
work.
0960-894X/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(01)00494-2

2546
N. J. Kershaw et al. / Bioorg. Med. Chem. Lett. 11 (2001) 2545–2548
Chemistry
with an N-terminal PTS-2 signal sequence) was pro-
duced as an N-terminal MBP-fusion,3 whilst mature
PAHX was prepared without a fusion ‘tag’. Both forms
of the enzyme were purified to >95% purity (by SDS-
PAGE analysis).20
Phytanic acid (1) was synthesised from an E/Z mixture
of phytol (6) according to a modification of the pub-
lished procedure.11 Hydrogenation of the double bond
to (3RS)-phytanol (7) was achieved using Rh/alumina in
methanol. This procedure reduced the proportion of
hydrogenolysis of the allylic alcohol obtained using
PtO2 in ethanol.11 In contrast to the reported use of
Jones reagent, oxidation of (3RS)-phytanol (7) to phy-
tanic acid (1) was achieved by initial reaction with
TPAP/NMO12,13 to give a crude mixture of aldehyde
and acid products (approx. 2:1), which was immediately
treated with potassium permanganate in aqueous ace-
tone to effect oxidation of residual aldehyde.
Initial assays measured 2-oxoglutarate oxidation21 in
order to test with highly purified PAHX if phytanic
acid (1) was a substrate and to confirm the conver-
sion of 3. These demonstrated that 2-oxoglutarate
conversion was enhanced in the presence of phyta-
noyl-CoA (3), but not phytanic acid (1), indicating that
only the former is a substrate. Phytanoyl-CoA (3) was
then incubated with both pro- and mature-PAHX.22 We
used a modification of the method of Mihalik et al. to
assay for conversion of epimeric phytanoyl-CoA (3) to
the hydroxylated product.22 Optimised conditions
included ATP23 as well as tris(carboxyethyl)phosphine
in place of dithiothreitol.24 Reverse-phase HPLC ana-
lysis of the reaction mixtures revealed the presence of a
new peak, absent from negative controls, with a reten-
tion volume of 16 mL compared to that of 18 mL for the
starting material. The intensity of the new peak
increased with increasing enzyme concentration and
incubation times.
Preliminary attempts at the coupling of phytanic acid
(1) with the lithium salt of CoASH using the N-hydro-
xysuccinimide ester of phytanic acid prepared using
DCCI/NHS14 led to poor yields. This may have reflec-
ted difficulties in purifying the product via reported
procedures for fatty acid CoA derivatives15À17 probably
due to the amphiphilic nature of the product and, pos-
sibly, steric hindrance in the coupling reaction. Subse-
quently, carbonyl diimidazole was successfully used to
directly couple phytanic acid (1) and CoASH.18 The
crude product was solubilised with the aid of b-cyclo-
dextrin and purified by reverse-phase HPLC (Scheme
2).19
Multiple incubations were used to produce enough
1
material for characterisation by H NMR and electro-
spray ionisation mass spectrometry (ESI MS).25 Pro-
duct was again isolated by reverse-phase HPLC,
Negative ion ESI MS analysis was consistent with for-
mation of 2-hydroxyphytanoyl-CoA (4): (m/z: 1076
[MÀH]À). Analyses by 1-D and 2-D 1H NMR (500MHz)
confirmed the product as 2-hydroxyphytanoyl-CoA (4).
Selected NMR data: Starting material, phytanoyl-CoA
Incubation of Phytanoyl-CoA with PAHX
The pahx gene was cloned from a human liver cell
cDNA library and expressed in E. coli. Pro-PAHX (i.e.,
Scheme 1. Metabolism of phytanic acid (1).
Scheme 2. Synthesis of phytanoyl-CoA (3). Reagents: (i) rhodium on alumina, MeOH (60%); (ii) tetrapropylammonium perruthenate (TPAP) and
N-methylmorpholine N-oxide (NMO) in CH3CN then KMnO4 in acetone/H2O (80%); (iii) carbonyl diimidazole, THF, then coenzyme A (CoASH)
in THF/H2O (2:1) (50% after HPLC purification).

N. J. Kershaw et al. / Bioorg. Med. Chem. Lett. 11 (2001) 2545–2548
2547
(3): d 2.58 and 2.39 (2Â1H, CH2C(O)SCoA); product,
2-hydroxyphytanoyl-CoA (4): d 4.12, 1H, CH(OH)-
C(O)SCoA (Fig. 1). Note that the H NMR signals for
the 3R/3S epimers of the starting material and those for
the two diastereoisomeric products were coincident.
Phytanic acid (1) in the mammalian diet is present as a
mixture of epimers and it seems PAHX has evolved to
accept both substrates with approximately equal effi-
ciency. The results for PAHX also suggest that the next
two reactions in the b-oxidation pathway are non-ste-
reoselective, that is the lyase will accept both (2S,3R and
2R,3S) stereoisomers of 2-hydroxyphytanoyl-CoA (4)
and the oxidoreductase will accept both 2R and 2S
pristanal to give 2R/2S pristanic acid (2). However, only
the 2S stereoisomer of pristanic acid (2) is processed via
the a-oxidation pathway,26 and thus the results are
consistent with the presence of a racemase. The recent
characterisation of an a-methylacyl-CoA racemase defi-
ciency28 supports this hypothesis.
1
Under appropriate conditions, that is with high enzy-
me:substrate ratios, >95% conversion of the 3-epimeric
phytanoyl-CoA (3) to products could be achieved,
demonstrating both epimers to be substrates for PAHX.
Previous in vivo studies using rat liver are consistent
with this observation,26 implying both 3R- and 3S-
methylhexadecanoic acid are equally well metabolised.
The earlier work also elegantly demonstrated that the
3R epimer is converted to the 2S,3R threo product and
the 3S epimer to the 2R,3S threo product. The observa-
tion that both epimers of the natural substrate are sub-
strates indicates that PAHX activity is due to a single
enzyme that is responsible for the metabolism of both
stereoisomers. Further, it seems that the individual epi-
mers are converted to the products with the same rela-
tive stereochemistry. With the obvious exception of
racemases/epimerases, most enzymes, including non-
haem oxygenases are highly stereoselective.27 It will be
interesting to study how PAHX binds its epimeric
susbtrate. It is difficult to envisage how a ‘rigid’ three
point attachment of CoA, b-methyl and hydrocarbon
side-chain can lead to the observed selectivity. One
possibility is that a two point attachment occurs, most
probably involving selective binding of the b-methyl
group, but only one of the CoA and hydrocarbon side-
chain moieties.
Acknowledgements
We thank Dr. R. T. Aplin for mass spectrometric ana-
lyses and Dr. V. Lee for helpful discussions and also the
BBSRC, MRC, EPSRC, Felix foundation (scholarship
to MM), E.U. Biotechnology project and the Wellcome
Trust for financial support of the work.
References and Notes
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Figure 1. Partial 1H NMR spectra (500 MHz, CD3CN–D2O, recorded
using a 3 mm microprobe) of phytanoyl-CoA (3) (lower trace) and 2-
hydroxyphytanoyl-CoA (4) (upper trace). Arrows indicate the reso-
nance changes discussed in the text. The broad lines observed result
from the poor solubility and probable aggregation of these materials
due to the presence of both hydrophobic and hydrophilic moieties.

2548
N. J. Kershaw et al. / Bioorg. Med. Chem. Lett. 11 (2001) 2545–2548
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19. To a stirred solution of carbonyl diimidazole (20 mg,
120 mmol) in THF (2 mL) was added phytanic acid (36 mg,
100 mmol) in THF (2 mL). After 2 h the solvent was removed
in vacuo and the residue dissolved in 2:1 THF–water (1 mL).
A solution of the lithium salt of coenzyme A (80 mg, 100 mmol)
in 2:1 THF–water (2.5 mL) was added and the reaction stirred
under argon for 18 h. THF was removed in vacuo and the
residue diluted with water (2 mL). The pH was adjusted to pH
3–4 by addition of Dowex-50 resin then filtered. The filtrate was
lyophilised and the white solid triturated with EtOAc (10 mL) to
remove any unreacted phytanic acid. The crude product was
collected by filtration, dissolved in 5 mM Tris–HCl, pH 8,
containing 5 mM ammonium bicarbonate in 5% (v/v) aceto-
nitrile, and solubilised with b-cyclodextrin, with sonication for
1H NMR (500 MHz), CD3CN–D2O: 0.75 (3H, s, 200-
CCH3), 0.85–0.95 (18H, m, 5ÂCH3, 200-CCH3), 1.55 (1H,
m, CH(CH3)2), 1.95 (1H, m, CH(CH2C(O)S), 2.41 (3H, m,
2000-CH2, 1 of CH2C(O)S), 2.58 (1H, m, 1 of CH2C(O)S),
2.98 (2H, m, 20000-CH2), 3.30 (2H, m, 10000-CH2), 3.40 (2H,
m, 1000-CH2), 3.54 (1H, m, 100-CH2), 3.85 (1H, m, 100-CH2),
4.02 (1H, s, 300-CH), 4.24 (2H, m, 50-CH2), 4.55 (1H, m, 40-
CH), 4.82 (2H, m, 20 and 30-CH), 6.12 (1H, d, 10-CH), 8.23
(1H, s, 8-CH), 8.52 (1H, s, 2-CH). A 2-D COSY spectrum
was consistent with the connectivity. m/z (Àve ESI-MS):
1060 [MÀH]À.
20. Mukherji, M.; Kershaw, N. J.; Chien, W.; Clifton, I. J.;
Schofield, C. J.; Wierzbicki, A. S.; Lloyd, M. D. Hum. Mol.
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5 min. This solution was purified by HPLC using
a
10Â250 mm Hypersil ODS reverse phase column. Samples
were eluted with a linear gradient from 10 mM ammonium
bicarbonate in 10% (v/v) acetonitrile to 50% (v/v) acetonitrile
at a flow rate of 4 mL/min over 10 min. This was followed by
an isocratic gradient for 10 min, at 4 mL/min, and a linear
gradient from 50% (v/v) acetonitrile to 10 mM ammonium
bicarbonate in 10% (v/v) acetonitrile over 2 min to return to
the initial conditions. Absorbance of samples was monitored
by ultraviolet absorbance of the adenine group of CoASH at
254 nm. The fraction eluting at 14 min was evaporated in
vacuo to remove acetonitrile and lyophilised to give phyta-
noyl-CoA (60 mg, 50%) as a white solid.
25. Incubations for product isolation were performed as
described20 using 50Â100 mL incubations containing 6.4 nmol/
min of PAHX for 60 min. HPLC analysis suggested a >95%
conversion to product. Product was isolated by reverse phase
HPLC (ODS Hypersil, 250Â4.6 mm). Samples were eluted
using a flow rate of 1 mL/min and a linear gradient of 30–15 mM
NH4OAc and 35–75% (v/v) acetonitrile over 10 min, reverting to
30 mM NH4OAc and 35% (v/v) acetonitrile over 2 min and re-
equilibrating under these conditions for a further 8 min. Product
1
eluted with a retention volume of 16 mL. A H NMR (COSY)
spectrum was consistent with the proposed connectivity.
26. Croes, K.; Casteels, M.; Dieuaide-Noubhani, M.; Man-
naerts, G. P.; Van Veldhoven, P. P. J. Lipid Res. 1999, 40, 601.
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367.
28. Ferdinandusse, S.; Denis, S.; Clayton, P. T.; Graham, A.;
Rees, J. E.; Allen, J. T.; McLean, B. N.; Brown, A. Y.; Vre-
ken, P.; Waterham, H. R.; Wanders, R. J. A. Nature Genet.
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