19F NMR studies on γ-butyrobetaine hydroxylase provide mechanistic insights and suggest a dual inhibition mode

Article abstract of DOI:10.1039/c9cc06466d

The final step in the biosynthesis of l-carnitine in humans is catalysed by the 2-oxoglutarate and ferrous iron dependent oxygenase, γ-butyrobetaine hydroxylase (BBOX). ¹H and ¹⁹F NMR studies inform on the BBOX mechanism including by

Full text of DOI:10.1039/c9cc06466d

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Timothy D. W. Claridge, Christopher J. Schofield et al.
19F NMR studies on γ-butyrobetaine hydroxylase provide
mechanistic insights and suggest a dual inhibition mode

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19F NMR studies on c-butyrobetaine hydroxylase
provide mechanistic insights and suggest a dual
inhibition mode†
Cite this: Chem. Commun., 2019,
55, 14717
Received 20th August 2019,
Accepted 23rd September 2019
Robert K. Lesniak, Anna M. Rydzik, ‡ Jos J. A. G. Kamps, ‡ Amjad Kahn,
´
Timothy D. W. Claridge * and Christopher J. Schofield
*
DOI: 10.1039/c9cc06466d
rsc.li/chemcomm
The final step in the biosynthesis of L-carnitine in humans is
catalysed by the 2-oxoglutarate and ferrous iron dependent oxygenase,
c-butyrobetaine hydroxylase (BBOX). 1H and 19F NMR studies inform on
the BBOX mechanism including by providing evidence for cooperativity
between monomers in substrate/some inhibitor binding. The value of
the 19F NMR methods is demonstrated by their use in the design of new
BBOX inhibitors.
The final step in L-carnitine (1) biosynthesis in humans is catalysed
by the 2-oxoglutarate (2OG, 2) and ferrous iron dependent
oxygenase, g-butyrobetaine hydroxylase (BBOX) (Fig. 1A).1,2
Carnitine plays a crucial role in lipid metabolism by enabling
long-chain fatty acid transport into mitochondria for b-oxidation.3,4
Approximately a quarter of total carnitine in humans is produced
endogenously, with the remainder from alimentation, e.g. red
meat.5,6 Excess carnitine is proposed as a cardiovascular disease
risk, due to its gut metabolism to N-trimethylamine oxide.7 Carnitine
is proposed to indirectly regulate carbohydrate metabolism via
modulation of the acetyl-CoA/CoA ratio,8,9 which affects pyruvate
dehydrogenase activity.10,11 Targeting carnitine biosynthesis to
therapeutically regulate cellular energy metabolism to treat cardio-
vascular diseases is thus of interest.12,13
Fig. 1 (A) Dimeric g-butyrobetaine hydroxylase (BBOX) is a 2-oxoglutarate
oxygenase. (B) BBOX inhibitors: Mildronate (4); isoquinoline (5). (C) Overlaid
g-butyrobetaine (GBB, 3; green sticks) and N-oxalylglycine (NOG; salmon
sticks) binding residues of hBBOX (blue/orange cartoon (dimer view)) blue
lines (PDB: 3O2G)1 and a psBBOX model23 (yellow lines). psBBOX Tyr201
(Y194 hBBOX,1 pink sticks) is involved in GBB quaternary ammonium ion
recognition;24 it is located in a ‘flexible-loop’ region. (D) Titrations of (4)
manifest only B60% GBB displacement using a 1H NMR reporter assay;25
by contrast (5) apparently displaces B90% GBB.
Mildronate (4) (Meldonium, THP, Met-88) (Fig. 1B) is a
clinically used cardioprotective agent,14 which has received attention
given its proposed performance enhancing abilities leading to
(ab)use in the sporting community.15,16 Definitive evidence for is interest in carnitine fermentation. Carnitine is biosynthe-
its effects, and the biological modes of action of Mildronate, are sized by microbes, including Pseudomonas spp (e.g. sp. AK1)
lacking; it is proposed to cause a change in metabolism from producing a human BBOX homologue.21,22 Like human BBOX
mitochondrial fatty acid b-oxidation towards peroxisomal meta- (hBBOX, sequence similarity: B30%) Pseudomonas sp. AK1
bolism and glycolysis, via reduction of carnitine levels, due to BBOX (psBBOX) is a 2OG and Fe(II) using oxygenase.21
inhibition of BBOX and of carnitine uptake.9
Crystallography reveals BBOX to be dimeric; each monomer
Carnitine is used as a fat-loss aid17,18 and to treat cardio- containing a 2OG oxygenase characteristic double-stranded
vascular conditions and carnitine deficiency.19,20 Hence, there b-helix fold and typical Fe(II) and (co)substrate binding elements
(Fig. 1C).1 Recombinant psBBOX is produced efficiently in
The Department of Chemistry, University of Oxford, 12 Mansfield Road, Oxford,
Escherichia coli (80 mg LÀ1);23 by contrast, recombinant hBBOX
is more difficult to produce in bacteria. Given its high yield,
OX1 3TA, UK. E-mail: tim.claridge@chem.ox.ac.uk,
christopher.schofield@chem.ox.ac.uk
recombinant psBBOX is a useful model enzyme for studying
BBOX and related enzymes, like trimethyllysine hydroxylase.23
† Electronic supplementary information (ESI) available. See DOI: 10.1039/c9cc06466d
‡ These authors contributed equally.
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NMR based reporter BBOX assays, using either Zn(II) or
Mn(II) (making use of paramagnetic relaxation enhancement
(PRE)) to observe ligand binding are reported.25 These enable
monitoring of co/substrate 2OG/g-butyrobetaine (GBB; 3)
psBBOX binding and inform on binding modes of inhibitors,
including whether they displace GBB and/or 2OG. We now
report 19F NMR studies on ligand binding to BBOX; the work
was initiated following 1H NMR observations concerning the binding
of Mildronate to psBBOX. The results inform on the BBOX
mechanism by revealing cooperativity between monomers during
substrate binding. The value of the 19F NMR methods is demon-
strated by their use in identification of new BBOX inhibitors.
During 1H ligand observed studies on the binding of Mildronate
to psBBOX, we observed that attempted displacement of GBB by
Mildronate from the psBBOX-Zn(II)-2OG–GBB complex (and vice versa)
does not proceed to more than B60%, i.e. to give an apparent
B1 : 1, Mildronate: GBB complex (Fig. 1D). Mildronate is a close
structural analogue of GBB, that under catalytic conditions is a
competitive hBBOX substrate undergoing fragmentation via
Stevens type rearrangement to give several products.26 By contrast
with the Mildronate results, on titration of the 2OG-competing and
metal-chelating BBOX inhibitor (5) (IC50 = 0.9 mM)25 near complete
Fig. 2 (A) Labelling of the Y201C psBBOX (blue/orange surface) using
3-bromo-1,1,1-trifluoroacetone (BTFA) to give psBBOX*. (B) 19F NMR
spectra of apo-psBBOX*. (C) 19F NMR spectra obtained from titrations of
psBBOX*-Zn(II) with GBB/2OG. (See ESI† for details).
GBB displacement was observed (Fig. 1D). Since psBBOX is observed with psBBOX* by 19F NMR at À85.34 ppm relative to TFA
dimeric21,22 (Fig. 1C), these observations led to the proposal that (Fig. 2B), indicating that the flexible loop of dimeric psBBOX* exists
binding of a second molecule of GBB (or Mildronate) to the as a single distinct symmetrical conformer in solution and/or that
psBBOX-Zn(II)-2OG–GBB complex strengthens binding of the first flexible loop movement is fast on the NMR timescale, such that a
GBB molecule, i.e. there is cooperativity in substrate binding time averaged shift is observed.
between the monomers of the dimer.
To monitor psBBOX* ligand binding without turnover, we
We proposed further insights into the apparent cooperative used Differential Scanning Fluorimetry thermal shift studies
ligand binding could be achieved using protein-observed fluorine with wt-psBBOX to identify an Fe(II) surrogate: Ni(II) and Zn(II)
(PrOF) NMR spectroscopy.27 19F NMR was chosen over traditional were identified as candidates (Fig. S6, ESI†). Initial 19F NMR
methods using 15N/13C labelling due to the near 100% natural experiments with psBBOX* showed Zn(II) enabled visualisation
abundance of 19F,28 the high 19F signal sensitivity (83% relative to 1H),27 of co/substrate binding events (Fig. 2C); the results with Ni(II)
and, importantly for BBOX studies, the ease with which one can were more complex (Fig. S7, ESI†). Titration of GBB with
produce 19F labelled proteins and interpret spectra of large psBBOX*-Zn(II) manifested a second signal at À82.19 ppm
macromolecules.
which increased in intensity with increasing GBB concentration
Given a lack of crystallographic data for psBBOX, choice of (Fig. 2C). Thus, Zn(II) was used in subsequent 19F NMR psBBOX*
the position for 19F labelling was based on hBBOX crystallo- ligand binding studies.
graphy.1,29 psBBOX Tyr201 (Tyr194, hBBOX) is located on a
The chemical shift change (DdF) of 3.15 ppm, relative to the
‘flexible-loop’ which plays a role in catalysis via recognition of psBBOX* signal at À85.34 ppm, observed on GBB addition to
the GBB quaternary ammonium group (Fig. 1C).24 To study psBBOX*-Zn(II) indicates a significant change in local environ-
psBBOX using 19F NMR via use of the thiol-selective reagent ment for the 19F nucleus, consistent with the labelling position
3-bromo-1,1,1-trifluoroacetone (BTFA), site-specific cysteine being close to the GBB trimethylammonium binding site (Fig. S8,
substitution of psBBOX at Tyr201 was carried out (Fig. 2A).30 ESI†). By contrast to GBB, titration of 2OG with psBBOX*-Zn(II)
Treatment of wt-psBBOX with BTFA manifested no labelled complex did not yield a second signal, but manifested broadening
product by MS (Fig. S1 and S2, ESI†); by contrast Y201C was and a slight shift of the 19F resonance, in a 2OG concentration
efficiently labelled. BTFA was apparently selective for Cys201, dependent manner (Fig. S8, ESI†). This type of observation is
independent of incubation time and equivalents of BTFA used, typically observed in PrOF NMR with weak binding ligands that
despite the presence of other cysteines in psBBOX. These results exhibit equilibrium binding kinetics in an intermediate exchange
support the proposed solvent exposed nature of Tyr/Cys201 (at regime. These results are consistent with the unusually high Km
least in uncomplexed psBBOX) and are consistent with the of wt-psBBOX for 2OG (532 mM).23
proposed dynamic nature of the ‘flexible-loop’ region (Fig. 2).1
Addition of 2OG to the psBBOX*-Zn(II)-GBB complex manifested
BTFA labelled psBBOX-Y201C (psBBOX*) was catalytically a shift of the signal at À82.19 ppm to À82.40 ppm (Fig. 2C), likely
active by 1H NMR (Vmax = 2.6 mM sÀ1, kcat = 1.4 sÀ1, Km = 362 mM, representing a state in which all co/substrates are bound.
Fig. S3–S5, ESI†), though less so than wt-psBBOX (Vmax = 1.3 mM sÀ1
,
Attenuation of the putative psBBOX* signal at À85.34 ppm was
kcat = 5.2 sÀ1, Km = 696 mM, Fig. S5, ESI†). A sharp singlet was also observed, suggesting 2OG addition promotes GBB binding.
14718 | Chem. Commun., 2019, 55, 14717--14720
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The combined effects of 2OG on the psBBOX*-Zn(II)-GBB support catalysed GBB turnover by Mildronate by 1H NMR (Fig. S10,
the results obtained with the 1H NMR reporter assay, i.e. GBB has a ESI†). Addition of Mildronate to psBBOX*-Zn(II)–GBB-2OG, did
relatively high binding affinity for psBBOX in the presence of 2OG not manifest detectable changes using 19F NMR (Fig. 3D).
(KD = 5 mM).25 They are also consistent with the proposed These results agree with the reported relatively weak affinity
cooperativity on binding of a second GBB molecule being due of Mildronate for hBBOX (IC50 = 34–60 mM).29,31
to enhancement of binding of the first, via promotion of closure
of the flexible loop (Fig. 3A). A comparable binding affinity was (Fig. S11, ESI†) and substrate/product analogues (Fig. S12,
observed for GBB with psBBOX* (KD = 8.9 mM, Fig. S9, ESI†).
ESI†), further reveal utility of the 19F NMR method for monitoring
Studies with different types of reported BBOX inhibitors25,32
Titration of Mildronate with psBBOX*-Zn(II) manifested only subtle differences in ligand binding modes for even closely
low levels of a comparable second signal, so contrasting with related compounds, e.g. for 2OG and its close analogue N-oxalyl-
GBB titrations; high Mildronate concentrations were required glycine (NOG) (Fig. S13 and S14, ESI†) and for compounds in the
(B70 : 1, Mildronate : psBBOX* Zn(II)) to observe binding (Fig. 3C). same series (Fig. S11, ESI†).
Notably, addition of 2OG to the psBBOX*-Zn(II)–Mildronate complex
Titration of a potent psBBOX inhibitor (6) (IC50 = 0.2 mM25
)
clearly gave a second signal at À82.15 ppm, with a similar DdF to with psBBOX*-Zn(II) manifested signals at À85.18 ppm and
that observed on addition of 2OG to the psBBOX*-Zn(II)-GBB À85.44 ppm (Fig. 3E and F). By 1H NMR (6) was observed to
complex (DdF 3.19 ppm, Fig. 2C and 3C, KD = 12.4 mM, also be a potent psBBOX* inhibitor (IC50 = 1.6 mM, Fig. S15,
Fig. S10, ESI†). We were unable to detect inhibition of psBBOX* ESI†). Titration of (6) with psBBOX*-Zn(II)-GBB manifested
concentration dependent decrease of the putative psBBOX*-
Zn(II)-GBB signal at À82.19 ppm, concomitant with increases
in the assigned psBBOX*-Zn(II)-(6) signals at À85.18 and
À85.44 ppm. A similar result was obtained with another potent
hBBOX inhibitor, AR692B (7) (IC50 hBBOX = 0.2 mM),32 with
concentration dependent formation of signals at À85.19 and
À85.43 ppm and attenuation of the psBBOX*-Zn(II) signal at
À85.34 ppm being observed (Fig. 3G). The two signals obtained
with (6) and (7) may reflect different binding modes which the
ligand can adopt in the same dimer, as observed by crystallo-
graphy with (7) with hBBOX (Fig. 3H).32 Note that, at least by
1H NMR, (7) appears to be a relatively poor inhibitor of psBBOX*
(IC50 = 245 mM, Fig. S15, ESI†) compared to wt-psBBOX, suggest-
ing psBBOX* is an imperfect model for hBBOX.
The combined NMR studies on ligand binding led to the
proposal that a new BBOX inhibitor scaffold could be identified
by directly incorporating analogues of identified 2OG and GBB
binding motifs, comprising appropriately positioned metal chelating
and quaternary ammonium moieties, giving a ‘co-substrate/
substrate’ mimic. With this in mind, RL190B (8) was synthesised
(ESI†); it is a relatively potent psBBOX inhibitor (IC50 = 5 mM
(fluoride release assay)31). Inhibition of psBBOX* by (8) was
1
confirmed by H NMR (IC50 = 15 mM Fig. S15, ESI†). Pleasingly,
(8) was observed to displace both GBB and 2OG from the psBBOX
active site using a 1H NMR reporter assay (Fig. 4A); 19F NMR
titrations of (8) with psBBOX*-Zn(II) showed significant broadening
and almost complete attenuation of the original psBBOX* signal
Fig. 3 19F Labelled psBBOX* can be used to monitor ligand binding.
(A) We propose binding of GBB to a second monomer strengthens binding
of GBB to the first monomer, via a conformational change. Site of BTFA
labelling: pink stars. psBBOX* monomers: blue or orange surfaces. Circles:
C- and N-terminus Zn(II) binding sites. (B) 19F NMR of titrations of GBB with
psBBOX*. (C) Evidence 2OG influences Mildronate (4) binding to psBBOX*.
(D) Mildronate does not change the 19F NMR spectra observed with
psBBOX*-Zn(II)-GBB–2OG under the tested conditions. (E) hBBOX inhibitors
(6) and (7). (F) Addition of (6) attenuates the GBB binding signal. (G) 19F NMR
spectra of titrations of AR692B (7) with psBBOX* may reflect two crystallo-
graphically observed binding modes. (H) The two conformations of (7) with
hBBOX (PDB: 4C8R). Overlays of the structures of GBB (green sticks), and
NOG (green sticks) at the hBBOX active site (yellow sticks), and (7) (blue sticks)
bound to hBBOX (white sticks) are shown.
(Fig. 4C). By contrast with other ligand titrations using psBBOX*
such significant line broadening was not observed (e.g. Fig. S11–S13,
ESI†). Broadening of signals in PrOF NMR is often typical of weaker
inhibitors; the significant (and unusual amongst studied com-
pounds) signal attenuation observed with (8) may be a result of
its binding in both 2OG and GBB cavities. This may cause
changes of conformational mobility/protein destabilisation, poten-
tially yielding a number of indistinct conformational states.
The combined results highlight the power of PrOF to reveal
insights into cooperative binding, especially when combined
with ligand observed NMR. Although such information can be
obtained by other methods, including classic kinetics and other
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We thank the Biotechnology and Biological Sciences Research
Council (BBSRC, BB/E527620/1), Cancer Research UK (C8717/
A18245), the Wellcome Trust (091857/7/10/7/099141/Z/12/Z) for
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Research Council for a studentship to JK via the Centre for Doctoral
Training in Synthesis for Biology and Medicine (EP/L015838/1), and
a Clarendon Scholarship.
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Conflicts of interest
There are no conflicts to declare.
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