Organic & Biomolecular Chemistry
Communication
2
4,25
26
substrates.
Both GBBF and GBBNF were found to be the natural product Anthopleurine
((2R,3S)-dihydroxy-
2
5
27,28
psBBOX substrates. For both BBOXs, GBBNF was a better γ-butyrobetaine, Fig. S24†),
which is produced as an alarm
substrate than GBBF, consistent with crystallographic analyses hormone in sea anemone Anthopleura elegantissima. This
which revealed little room for C-3 substitutions (Fig. 1C). The result raises the question of whether a BBOX related enzyme
coupling ratio between 2OG and GBB analogue oxidation was could be responsible for hydroxylation step in the biosynthesis
good for GBBNF, but uncoupling was observed when GBBF of anthopleurine in sea anemone.
was a substrate (Fig. S12–14†). With both BBOXs GBBNF dis-
Finally, we tested a set of trimethylammonium containing
played lower KM values than GBBF (Table 1, Fig. S15 and 16†). compounds, some of which were close BBOX substrate
psBBOX was not inhibited by GBBF nor GBBNF, at least up to analogues, e.g. acetylcholine (Fig. S25†). However, none of
0
.5 mM concentration of substrate. hBBOX was inhibited by these were found to be hydroxylated by either hBBOX or
low concentrations of GBBF, but much less inhibition was psBBOX.
observed in case of GBBNF (Fig. S15 and 16†). Because GBB
substrate inhibition was not observed at low concentrations
with psBBOX, the lack of substrate inhibition of psBBOX by
Conclusions
GBBF or GBBNF is unsurprising.
The observation that GBBNF is a better substrate for
psBBOX than hBBOX, prompted us to examine substrate ana-
logues with different chain lengths. Structural analogues of
In conclusion, the results clearly demonstrate that while
hBBOX and psBBOX share key properties, there are also
clear differences between them, including their differential
dependencies on ascorbate. As predicted based on the
hBBOX crystal structure, there are differences in their sub-
strate analogue selectivities, with psBBOX being more toler-
ant of modifications with increased steric demand in the
aromatic cage responsible for the trimethylammonium
binding. The substrate analogue results imply that engineer-
ing of the BBOX activity, might be productive, including
with respect to the production of vicinal diols and amino-
alcohols, such as present in some natural products, such as
Anthopleurine.
1
4
GBB are reported to be substrates for hBBOX. 3-Trimethyl-
aminopropionate (GBB-3) and 5-trimethylaminovalerate (GBB-5)
were found to be hydroxylated by hBBOX at C-2 and C-3,
respectively. Similar experiments with psBBOX revealed that
only GBB-5 was a substrate (within limits of detection), being
hydroxylated at C-3, as for hBBOX (assignments were made by
1
1
13
H and H– C HSQC NMR spectra, Fig. S17†). In the case of
hBBOX, GBB-3 was a reasonably good and GBB-5 a fair sub-
strate, with GBB-3 being hydroxylated at 58% and GBB-5 at
1
2% of the initial GBB hydroxylation rate. GBB-5 was found to
be a poor psBBOX substrate (1–2% of initial hydroxylation rate
compared to GBB) (Table 2). GBB-3 was not hydroxylated even
at a high concentration of psBBOX (13 µM). Both GBB-5 and
GBB-3 stimulated uncoupled 2OG turnover by psBBOX Acknowledgements
(
Fig. S18 and S19†), showing that GBB-3 and GBB-5 bind to
We thank the Wellcome Trust, Biotechnology and Biological
Sciences Research Council, Cancer Research UK, European
Union and Dulverton Trust (A.M.R.) financial support.
the active site, however, predominantly in an unproductive
manner. Neither hBBOX nor psBBOX oxidised 6-trimethyl-
aminohexanoate (GBB-6) or 2-trimethylaminoacetate (GBB-2).
BBOX is closely related to trimethyllysine hydroxylase
(
TMLH), which is the 2OG oxygenase that catalyses the first
4
step of carnitine biosynthesis in animals. It was therefore of
interest to investigate if the BBOXs can catalyse amino acid
hydroxylation. Neither BBOX was able to catalyse hydroxylation
of trimethyllysine. However, psBBOX but not hBBOX, was
found to catalyse hydroxylation of both (R)- and (S)-analogues
of 2-amino GBB (GBB-NH(R) and GBB-NH(S), respectively) to
give products arising from C-3 hydroxylation as assigned by
NMR (Fig. S20,† Table 2). The lack of activity with hBBOX with
GBB-NH(R) and GBB-NH(S) is in agreement with the structural
studies indicating that the hBBOX GBB binding site is too
small to accommodate C-2 substituted GBB analogues. We
then tested (2S)-hydroxy GBB (GBB-OH) as a substrate for
BBOXs. GBB-OH was found to be a substrate for psBBOX only,
giving the 2,3-dihydroxy GBB (Fig. S22†). GBB-OH was a better
substrate for psBBOX than the 2-amino derivatives, i.e.
GBB-NH(R) and GBB-NH(S) (the initial hydroxylation rate was
Notes and references
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2 M. A. McDonough, C. Loenarz, R. Chowdhury, I. J. Clifton
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3 R. P. Hausinger, Crit. Rev. Biochem. Mol. Biol., 2004, 39,
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4 F. M. Vaz and R. J. Wanders, Biochem. J., 2002, 361,
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5 K. Strijbis, F. M. Vaz and B. Distel, IUBMB Life, 2010, 62,
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6 S. Englard, J. S. Blanchard and C. F. Midelfort, Biochemistry,
1985, 24, 1110–1116.
7 C. J. Rebouche and H. Seim, Annu. Rev. Nutr., 1998, 18,
39–61.
8
times higher than for amino derivatives, Table 2, Fig. S23†).
Interestingly, the scaffold of 2,3-dihydroxy GBB is the same as
8 H. P. Kleber, FEMS Microbiol. Lett., 1997, 147, 1–9.
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