the first step in the reaction, the transfer of the amino group of
SAM to PLP, generates the corresponding oxo-acid 3 as shown
in Scheme 1.
This study was funded by BBSRC. RSB & MB thank
Syngenta Ltd. for support through CASE awards.
Notes and references
1 M. A. Eisenberg, Adv. Enzymol., 1973, 38, 317; M. A. Eisenberg,
Biosynthesis of Biotin and Lipoic Acid, vol. 1, American Society for
Microbiology, Washington DC, 1987.
2 A. R. Rendina, W. S. Taylor, K. Gibson, G. Lorimer, D. Raynor,
B. Locket, K. Kranis, B. Wexler, D. Marcovici-Mizrahi,
A. Nudelman, A. Nudelman, E. Marsilii, C. Hongji, Z. Wawrzak,
J. Calabrese, W. Huang, J. Jia, G. Schneider, Y. Linquist and
G. Yang, J. Pestic. Sci., 1999, 55, 236; O. Ploux, O. Breyne,
S. Carillon and A. Marquet, Eur. J. Biochem., 1999, 259, 63.
3 G. L. Stoner and M. A. Eisenberg, J. Biol. Chem., 1975, 259, 4029;
G. L. Stoner and M. A. Eisenberg, J. Biol. Chem., 1975, 259, 4037.
4 J. L. Martin and F. M. McMillan, Curr. Opin. Struct. Biol., 2002, 12,
783.
5 P. A. Frey and O. T. Magnusson, Chem. Rev., 2003, 103, 2129.
6 H. Kack, J. Sandmark, K. Gibson, G. Schneider and Y. Linqvist,
J. Mol. Biol., 1999, 291, 857.
7 A. C. Elliot, J. Sandmark, G. Schneider and J. F. Kirsch,
Biochemistry, 2002, 41, 12582.
Scheme 2 Reagents and conditions: (i) LDA, THF, (ii) ethyl 6-
iodohexanoate, HMPA, (iii) TiCl4, Et3N, (Me3Si)2NH, (iv) NaCNBH3,
DCM, (v) NBS, MeCN–H2O (4–1), (vi) aq. HCl (6 M).
form the quinonoid is rapid but the subsequent hydrolysis to
the bound PMP form is very slow and this appears to be the
rate determining step in the overall catalytic reaction.
Under the conditions used in the enzyme experiments we
found that SAM degrades slowly affording 5Ј-methylthio-
adenosine (t1/2 ∼ 20 h at 37 ЊC and pH 7.5).11 Under steady state
conditions12 (both substrates at ca 5 × KM) disappearance of
SAM was accompanied by the appearance of an HPLC peak
corresponding to a new product. On standing this compound
decomposed to give 5Ј-methylthioadenosine much more rapidly
than SAM suggesting that the oxo-acid 3 was indeed being
formed and subsequently disproportionated. Since we were
unable to find HPLC conditions13 which gave satisfactory
separation of this product from SAM, a series of incubations
was carried out under non-optimum catalytic conditions with
( )-1 (6 µM, 5 × KM) in excess but where the SAM concen-
tration (200–300 µM, 1.3–2.0 × KM) was limited so that it was
almost completely depleted from the incubation mixture within
fifteen minutes, thus minimising the contamination of the
product by SAM. HPLC-MS of the reaction mixtures showed
that the unstable product had a Mϩ peak at 400 amu. However
this is very close to the molecular weight of the SAM cation
itself (399 amu). So, to determine unambiguously whether the
compound formed in the enzymatic reaction was indeed the
putative oxo-acid, we elected to reduce the product in situ to
afford the more stable hydroxy-acid 4. Accordingly, authentic 4
was synthesised directly from SAM by partial diazotisation
essentially as described by Zappia.14,15
8 J. Sandmark, S. Mann, A. Marquet and G. Schneider, J. Biol. Chem.,
2002, 277, 43352.
9 S. Kuramitsu, K. Hiromi, H. Hayashi, Y. Morino and H. Kagami-
yama, Biochemistry, 1990, 29, 5469.
10 C. M. Leir, J. Org. Chem., 1972, 37, 887.
11 Commercially available SAM is a ∼3 : 2 mixture of the S and
R forms and can contain up to 10% 5Ј-methylthioadenosine. SAM
used in these experiments was purified by HPLC and contained less
than 1% 5Ј-methylthioadenosine. The unnatural R isomer is not a
substrate (see ref. 7).
12 Steady state incubations were typically carried out at 37 ЊC in 20
mM potassium phosphate buffer (pH 7.5) containing 20 µM DAPA
synthase (equilibrated with 100 µM PLP), 1 mM SAM and 6 µM
( )-1.
13 HPLC separations were typically carried out on a Phenomenex 5 µ
C8 (150 × 4.6 mm) column eluted isocratically with 5% MeOH–H2O
for 5 minutes followed by a gradient to 60%. MeOH–H2O, over 25
min. All eluents contained 0.1% TFA. For a flow rate of 0.5 cm3
minϪ1 the retention times of SAM, 3, 4 and 5Јthioadenosine were
3.9, 4.0, 4.9 and 21.4 min respectively.
14 V. Zappia, C. R. Zydek-Cwick and F. Schlenk, J. Biol. Chem., 1969,
244, 4499.
15 S-Adenosyl-(2-hydroxy-4-methylthio)butanoic acid 4 was prepared
as its TFA salt by treatment of the sulphate salt of SAM with excess
NaNO2 in citrate–phosphate buffer (1 M, pH 2.5) at 40 ЊC for 1 h.
The product was purified by sequential ion exchange chromato-
graphy (AG15 × 8 resin, Hϩ form) and HPLC. The 13C NMR (125
The enzyme was incubated under the above conditions with
the addition of [14C-methyl]SAM (2 nmoles, 0.024 M Bq), the
mixture was spin-filtered to remove the protein, and treated
with NaBH4 (5 mM, 5 min, on ice) to convert 3 to 4.
Under conditions where virtually all of the SAM was turned
over 88% of the radioactivity co-purified with 4 and 8% with
5Ј-methylthioadenosine. This provides compelling evidence that
2
MHz, H2O–H2O) of 4 showed a methine resonance at 68.4 ppm.
(CHOH) and no CHNH2 signal (at 51.6 ppm in the spectrum of
SAM). Fully concordant MS, NMR and UV data were obtained for
the product. The stereochemistry of the 2-hydroxyl group was not
determined.
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 3 4 9 8 – 3 4 9 9
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