Human carnitine biosynthesis proceeds via (2S,3S)-3-hydroxy-Nε-trimethyllysine

Article abstract of DOI:10.1039/c6cc08381a

N^ε-Trimethyllysine hydroxylase (TMLH) catalyses the first step in mammalian biosynthesis of carnitine, which plays a crucial role in fatty acid metabolism. The stereochemistry of the 3-hydroxy-N^ε-trimethyllysine product of TMLH has not been defined. We report enzymatic and asymmetric synthetic studies, which define the product of TMLH catalysis as (2S,3S)-3-hydroxy-N^ε-trimethyllysine.

Full text of DOI:10.1039/c6cc08381a

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Human carnitine biosynthesis proceeds via
(2S,3S)-3-hydroxy-Ne-trimethyllysine†
Cite this:DOI: 10.1039/c6cc08381a
Robert K. Lesniak,a Suzana Markolovic,a Kaspars Tarsb and
´
Christopher J. Schofield*a
Received 18th October 2016,
Accepted 29th November 2016
DOI: 10.1039/c6cc08381a
www.rsc.org/chemcomm
Ne-Trimethyllysine hydroxylase (TMLH) catalyses the first step in
mammalian biosynthesis of carnitine, which plays a crucial role in
fatty acid metabolism. The stereochemistry of the 3-hydroxy-Ne-
trimethyllysine product of TMLH has not been defined. We report
enzymatic and asymmetric synthetic studies, which define the
product of TMLH catalysis as (2S,3S)-3-hydroxy-Ne-trimethyllysine.
Carnitine plays key roles in mammalian metabolism by enabling
the transport of fatty acids into mitochondria as O-acyl carnitine
esters and in maintaining acetyl group homeostasis.1–3 There is
considerable biomedical interest in carnitine and its biosynthesis.
Carnitine is biosynthesised from (2S)-Ne-trimethyllysine (TML,
(1)),4 which is derived from naturally-occurring TML residues in
proteins following proteolysis.5–7 Two 2-oxoglutarate (2OG)-
dependent oxygenases, Ne-trimethyllysine hydroxylase (TMLH)
and g-butyrobetaine hydroxylase (BBOX), catalyse the first and
final steps of carnitine biosynthesis, respectively (Fig. 1).8,9
BBOX is one of the proposed targets of Meldonium (Mildronate,
THP, Met-88),10,11 a drug that is used for treatment of cardio-
vascular disease12 and by athletes due to perceived performance-
Fig. 1 Carnitine biosynthesis. The first and final steps of L-carnitine
biosynthesis in mammals are catalysed by the 2-oxoglutarate-dependent
oxygenases, Ne-trimethyllysine hydroxylase (TMLH) and g-butyrobetaine
hydroxylase (BBOX).
enhancing properties.13,14 Carnitine is proposed to promote We employed the Dixon methodology,22 which involves Ag(I)-
atherosclerosis by acting as a precursor for trimethylamine catalysed aldol-type reactions in the presence of a cinchona
oxide.15 There are also reported links between TMLHE gene alkaloid-derived pre-catalyst (7).22,23 We envisaged this
mutations and autism in males.16–18 Whilst BBOX has been approach could enable the requisite introduction of differently
extensively characterised, including by detailed kinetic and protected Na- and Ne-amino groups in a precursor of (13). Thus,
biophysical studies,19,20 relatively little is reported on TMLH,21 dibenzyl aldehyde (6), prepared in two steps from (5), was reacted
notably including on the stereochemistry of the product of its with tert-butyl isocyanoacetate in the presence of Ag2O and the
catalysis.
pre-catalyst (7) to yield trans-oxazoline (8) ( J2–3 = 7.0 Hz)22,24 in
To define the stereochemistry of the TMLH-catalysed 3-hydroxy- good yield (78%; Scheme 1).
Ne-trimethyllysine (3HO-TML) product (2), we investigated the
Importantly, high diastereoselectivity favouring the trans-
asymmetric synthesis of (2S,3R)-3HO-TML (13) (Scheme 1). diastereomers (2S,3R/2R,3S : 2S,3S/2R,3R; d.r. 4 95 : 1) of (8)
was observed (2S,3R : 2R,3S; 3 : 1 inferred from analyses on (9)).
a Department of Chemistry, University of Oxford, Chemistry Research Laboratory,
12 Mansfield Road, Oxford OX1 3TA, UK.
Oxazoline (8) was unstable at room temperature (and over
prolonged periods at À20 1C), decomposing to give formamide
(9). We found that conversion of (8) to (9) is promoted by
aqueous citric acid, as reported for other oxazolines,22 or by
aqueous acetic acid in THF in near quantitative yield (Scheme 1).
The stereochemistry of the major stereoisomer of formamide (9)
E-mail: christopher.schofield@chem.ox.ac.uk
b Biomedical Research and Study Centre, Ratsupites 1, LV1067 Riga, Latvia
† Electronic supplementary information (ESI) available: Synthesis procedures,
assay conditions, NMR assignments and spectra, and MS analyses. See DOI:
10.1039/c6cc08381a
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Scheme 1 Stereoselective synthesis of (2S,3R)-3-hydroxy-Ne-trimethyllysine
(13) and (2S,3R)-3-hydroxylysine (11) via oxazoline (8). The oxazoline ring
hydrolysis and reduction steps can be carried out separately or via a one-pot
reaction, as displayed.
Fig. 2 TMLH catalysis produces (2S,3S)-3-hydroxy-Ne-trimethyllysine
(14). (A) 1H NMR assignment of the product resulting from TMLH-
catalysed C-3 hydroxylation of (2S)-Ne-trimethyllysine (1). Superimposition
of 1H NMR spectra of the reaction mixture before (blue) and after (red)
addition of TMLH shows 3HO-TML formation. Signals arising between
d = 3.5–3.75 ppm (including glycerol) are omitted for clarity. (B) Overlaid
extracted ion chromatograms (m/z = 375.2, corresponding to the mass of
derivatised 3HO-TML) for (i) TML incubated with (red) or without (black; at
baseline) TMLH and (ii) TMLH-treated TML (red) and TMLH-treated TML
spiked with synthetic (2S,3R)-3HO-TML ((13), black). (C) The stereochemistry
of TMLH- and BBOX-catalysed hydroxylation is the same relative to the
quaternary ammonium and carboxylate groups.
was assigned as (2S,3R) by 1H NMR analysis of the corresponding
Mosher’s esters (Fig. S1, ESI†).25,26
(2S,3R)-3-Hydroxylysine (3HO-Lys) (11) was efficiently
obtained from (8) using H2/Pd/C in aqueous citric acid followed
by the removal of formamide and tert-butyl ester protecting
groups via acid hydrolysis to give (11) (Scheme 1). The reduction
and hydrolysis steps to give (10) from (8) via (9) were initially
carried out separately; however, use of MeOH/5% citric acid as a
solvent during hydrogenation enabled one-pot conversion of (8)
to (10) in high yield (96%). Comparison of the optical rotation of
3HO-Lys (11) with that of enantiopure (2S,3R)-3HO-TML24,27 retention of the (2S)-stereochemistry in the TML substrate;
implied an e.r. for (11) as B3 : 1, consistent with the stereo- Fig. 2C and Fig. S3C, ESI†). This assignment was validated by
selectivity observed during oxazoline (8) formation.
amino acid analysis, using derivatisation with 6-aminoquinolyl-
3HO-TML (13) was then synthesised from (8) via initial N-hydroxysuccinimidyl carbamate. The enzyme-catalysed product
hydrogenation in acidic solvent to give the Ne-amine (10). and synthetic standard have identical masses (observed m/z =
Following treatment with methyl iodide to give (12), acid 375.2114), but were clearly separated by ultra performance liquid
promoted hydrolysis yielded (13) with an e.r. of 3 : 1 in favour chromatography (UPLC). These results reveal exclusive (495%)
of (2S,3R)-3HO-TML (Scheme 1, (13)), as determined by formation of the (2S,3S)-stereoisomer (14) as the TMLH-catalysed
Mosher’s analysis of the corresponding amide (Fig. S2, ESI†).
product (Fig. 2B and Fig. S4, ESI†).
We then investigated the stereochemistry of the TMLH-
The overall results define the stereochemical outcome of the
catalysed product (2) using recombinant TMLH28 and synthetic TMLH-catalysed hydroxylation of TML as (2S,3S)-3-hydroxy-Ne-
(2S,3R)-3HO-TML (13) as a standard for comparison by NMR and trimethyllysine (14). Interestingly, BBOX catalyses hydroxylation
amino acid analysis. (2S)-Ne-Trimethyllysine (1) was converted to of g-butyrobetaine (3) to give carnitine (4) with the (3R)-stereo-
3-hydroxy-Ne-trimethyllysine (2), as shown by 1D and 2D NMR chemistry (Fig. 1). Thus, the stereochemical outcomes of TMLH
analyses (Fig. 2A and Fig. S3A–F, ESI†). Addition of the synthetic and BBOX catalysis are the same relative to the trimethylammonium
(2S,3R)-3HO-TML standard (13) to the TMLH reaction mixture led and carboxylic acid groups (Fig. 2C), reflecting the likely common
to the appearance of non-redundant peaks, implying the TMLH- evolutionary origins of TMLH and BBOX, as revealed by structural
catalysed product to be the (2S,3S)-stereoisomer (14) (assuming analyses.19,29,30
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The results also highlight the continued important role of
Communication
W. H. Tang, F. D. Bushman, A. J. Lusis and S. L. Hazen, Nat. Med.,
2013, 19, 576–585.
16 R. O. Rosti, A. A. Sadek, K. K. Vaux and J. G. Gleeson, Dev. Med. Child
Neurol., 2014, 56, 12–18.
synthesis, including via efficient asymmetric catalysis, for bio-
molecular structural assignments. Modern proteomic and
other mass spectrometry (MS) methodologies are identifying
many new potential post-translational modifications (e.g. JMJD4-
catalysed formation of C-4 hydroxylysine),31,32 the regio- and
stereochemistries of which need to be confirmed, e.g. by NMR,
high resolution MS analyses and, at least in our view, wherever
possible by comparison with synthetic standards.
We thank the Wellcome Trust, Biotechnology and Biological
Sciences Research Council, the British Heart Foundation (R. K. L.),
and the Clarendon Fund (S. M.) for funding. We thank Dr Ju¨rgen
Brem, Dr Michael A. McDonough, and Dr Sarah E. Wilkins for
helpful advice.
17 C. Nava, F. Lamari, D. Heron, C. Mignot, A. Rastetter, B. Keren,
D. Cohen, A. Faudet, D. Bouteiller, M. Gilleron, A. Jacquette,
S. Whalen, A. Afenjar, D. Perisse, C. Laurent, C. Dupuits, C. Gautier,
M. Gerard, G. Huguet, S. Caillet, B. Leheup, M. Leboyer, C. Gillberg,
R. Delorme, T. Bourgeron, A. Brice and C. Depienne, Transl. Psychiatry,
2012, 2, e179.
18 P. B. Celestino-Soper, C. A. Shaw, S. J. Sanders, J. Li, M. T. Murtha,
A. G. Ercan-Sencicek, L. Davis, S. Thomson, T. Gambin,
A. C. Chinault, Z. Ou, J. R. German, A. Milosavljevic, J. S. Sutcliffe,
E. H. Cook, Jr., P. Stankiewicz, M. W. State and A. L. Beaudet, Hum.
Mol. Genet., 2011, 20, 4360–4370.
19 M. A. McDonough, C. Loenarz, R. Chowdhury, I. J. Clifton and
C. J. Schofield, Curr. Opin. Struct. Biol., 2010, 20, 659–672.
20 A. M. Rydzik, I. K. Leung, G. T. Kochan, N. D. Loik, L. Henry,
M. A. McDonough, T. D. Claridge and C. J. Schofield, Org. Biomol.
Chem., 2014, 12, 6354–6358.
21 A. H. K. Al Temimi, B. J. G. E. Pieters, Y. Reddy Vijayendar, P. B. White
Notes and references
´
and J. Mecinovic, Chem. Commun., 2016, 52, 12849–12852.
1 R. R. Ramsay, R. D. Gandour and F. R. van der Leij, Biochim. Biophys. Acta, 22 F. Sladojevich, A. Trabocchi, A. Guarna and D. J. Dixon, J. Am. Chem.
2001, 1546, 21–43.
Soc., 2011, 133, 1710–1713.
23 A. Franchino, P. Jakubec and D. J. Dixon, Org. Biomol. Chem., 2016,
14, 93–96.
24 P. F. Hughes, S. H. Smith and J. T. Olson, J. Org. Chem., 1994, 59,
5799–5802.
25 H. S. M. J. A. Dale, J. Am. Chem. Soc., 1973, 95, 512–519.
2 J. D. McGarry, Biochem. Soc. Trans., 1995, 23, 321–324.
3 C. Hoppel, Am. J. Kidney Dis., 2003, 41, S4–S12.
4 D. S. Sachan and C. L. Hoppel, Biochem. J., 1980, 188, 529–534.
5 J. LaBadie, W. A. Dunn and N. N. Aronson, Jr., Biochem. J., 1976, 160,
85–95.
6 W. A. Dunn, G. Rettura, E. Seifter and S. Englard, J. Biol. Chem., 26 T. R. Hoye, C. S. Jeffrey and F. Shao, Nat. Protoc., 2007, 2, 2451–2458.
1984, 259, 10764–10770.
7 L. Servillo, A. Giovane, D. Cautela, D. Castaldo and M. L. Balestrieri,
PLoS One, 2014, 9, e84589.
8 R. P. Hausinger, Crit. Rev. Biochem. Mol. Biol., 2004, 39, 21–68.
9 C. Loenarz and C. J. Schofield, Nat. Chem. Biol., 2008, 4, 152–156.
27 C. E. Masse, A. J. Morgan and J. S. Panek, Org. Lett., 2000, 2,
2571–2573.
28 A. Kazaks, M. Makrecka-Kuka, J. Kuka, T. Voronkova, I. Akopjana,
S. Grinberga, O. Pugovics and K. Tars, Protein Expression Purif., 2014,
104, 1–6.
10 E. Liepinsh, R. Vilskersts, D. Loca, O. Kirjanova, O. Pugovichs, I. Kalvinsh 29 I. K. Leung, T. J. Krojer, G. T. Kochan, L. Henry, F. von Delft,
and M. Dambrova, J. Cardiovasc. Pharmacol., 2006, 48, 314–319.
11 E. Liepinsh, I. Konrade, E. Skapare, O. Pugovics, S. Grinberga,
T. D. Claridge, U. Oppermann, M. A. McDonough and C. J. Schofield,
Chem. Biol., 2010, 17, 1316–1324.
J. Kuka, I. Kalvinsh and M. Dambrova, J. Pharm. Pharmacol., 2011, 30 K. Tars, J. Rumnieks, A. Zeltins, A. Kazaks, S. Kotelovica, A. Leonciks,
63, 1195–1201.
J. Sharipo, A. Viksna, J. Kuka, E. Liepinsh and M. Dambrova,
Biochem. Biophys. Res. Commun., 2010, 398, 634–639.
12 V. Dzerve and M. I. S. Group, Medicina, 2011, 47, 544–551.
13 C. Gorgens, S. Guddat, J. Dib, H. Geyer, W. Schanzer and M. Thevis, 31 T. Feng, A. Yamamoto, S. E. Wilkins, E. Sokolova, L. A. Yates,
Drug Test. Anal., 2015, 7, 973–979.
14 H. K. Greenblatt and D. J. Greenblatt, Clin. Pharmacol. Drug Dev.,
2016, 5, 167–169.
M. Munzel, P. Singh, R. J. Hopkinson, R. Fischer, M. E. Cockman,
J. Shelley, D. C. Trudgian, J. Schodel, J. S. O. McCullagh, W. Ge,
B. M. Kessler, R. J. Gilbert, L. Y. Frolova, E. Alkalaeva, P. J. Ratcliffe,
C. J. Schofield and M. L. Coleman, Mol. Cell, 2014, 53, 645–654.
15 R. A. Koeth, Z. Wang, B. S. Levison, J. A. Buffa, E. Org, B. T. Sheehy,
E. B. Britt, X. Fu, Y. Wu, L. Li, J. D. Smith, J. A. DiDonato, J. Chen, 32 S. Markolovic, S. E. Wilkins and C. J. Schofield, J. Biol. Chem., 2015,
H. Li, G. D. Wu, J. D. Lewis, M. Warrier, J. M. Brown, R. M. Krauss,
290, 20712–20722.
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