Trapping of a chromophoric intermediate in the Pdx1-catalyzed biosynthesis of pyridoxal 5′-phosphate

Article abstract of DOI:10.1002/anie.200704390

(Chemical Equation Presented) I₃₂₀ has it: The serendipitous trapping, by aromatization, of an enzyme-bound chromophoric intermediate (I ₃₂₀; see picture, blue box) involved in vitamin B₆ formation using a common biological reducing agent provides new information regarding the structure of the chromophoric species.

Full text of DOI:10.1002/anie.200704390

Communications
DOI: 10.1002/anie.200704390
Trapping Reactions
Trapping of a Chromophoric Intermediate in the Pdx1-Catalyzed
Biosynthesis of Pyridoxal 5’-Phosphate**
Jeremiah W. Hanes, Ivan Keresztes, and Tadhg P. Begley*
Pyridoxal 5’-phosphate (PLP, 1) is the biologically active form
of vitamin B6, and is essential for performing the chemistry of
primary metabolism in all varieties of life. The most common
pathway for the biosynthesis of PLP involves only two
enzymes, Pdx1 (SNZ) and Pdx2 (SNO). This biosynthetic
activity has recently been reconstituted in vitro and was
shown to use ribose-5-phosphate (R5P, 2), glutamine 3, and
glyceraldehyde-3-phosphate (G3P, 4) as substrates
(Scheme 1).[1,2]
Our previous attempts to remove I320 from Pdx1 using
either heat or acid or base denaturation failed to yield a
discrete product, presumably because the released product
decomposed under the harsh conditions used to remove it
from the enzyme. However, we noticed that in the presence of
tris(2-carboxyethyl)phosphine (TCEP), a newcompound was
observed by HPLC analysis of the small molecule pool. The
procedure that resulted in this new species was as follows:
Pdx1 was preincubated with R5P and then NH4Cl was added
to trigger I320 formation (Figure 1A). Under these
conditions, Pdx1–I320 is stable for several hours.
After purification of Pdx1–I320 from unbound small
molecules by gel filtration, the complex was
incubated overnight at 158C in 7m urea and 4 mm
TCEP. The protein was then removed by ultra-
filtration and the filtrate was analyzed by HPLC. A
newspecies eluting at 13.2 min was observed
Scheme 1. Major biosynthetic route for pyridoxal-5’-phosphate 1. Fragments of
ribose-5-phosphate (2), glutamine 3, and glyceraldehyde-3-phosphate (4) marked
in bold are incorporated into 1. Pi=inorganic phosphate.
(Figure 1B). The formation of this species (X230
)
was absolutely dependent on TCEP, reconstitution
of I320, and also required protein denaturation. The
absorption spectrum of X230 is shown in (Fig-
ure 1C) and shows the loss of the 320 nm absorb-
ance maximum of I320.
The new compound was collected following HPLC
purification and investigated by NMR spectroscopy and
mass analysis. The 1H NMR spectrum is shown in Figure 2A.
From this spectrum and COSY analysis, the structure of X230
The mechanistic details of this complex reaction have
captured considerable recent attention owing to the interest-
ing organic chemistry involved as well as the potential for the
development of antimicrobial agents.[3–10] The glutaminase
subunit (Pdx2) generates ammonia and delivers it through a
channel to the active site of the PLP synthase subunit
(Pdx1).[11–14] Biochemical studies on the Pdx1-catalyzed
reaction revealed that an intermediate (I320), formed from
R5P and glutamine (or ammonia) accumulates at the active
site of Pdx1.[15,16] I320 forms stoichiometrically with the
enzyme, is covalently attached to an active site lysine, and
has
a
high extinction coefficient (ca. 16200mÀ1 cmÀ1@
320 nm).[15] The structure of this intermediate has not yet
been firmly established. Herein we report an unanticipated
trapping reaction of I320 and the structure of the trapped
product.
[*] Dr. J. W. Hanes, Dr. I. Keresztes, Prof. T. P. Begley
Department of Chemistry and Chemical Biology
Cornell University
Ithaca, NY 14853 (USA)
Fax: ( +1)607-255-7133
E-mail: tpb2@cornell.edu
Figure 1. Trapping and isolation of I320. A) Experimental approach for
isolating I320. After preparation, I320 was separated from excess NH4Cl
and R5P by gel filtration and was then present in sodium phosphate
buffer (50 mm, pH 7.6) with NaCl (100 mm) and TCEP (4 mm).
B) HPLC analysis (detected at 240 nm) of the nonproteinaceous
fraction after filtration following an overnight incubation of Pdx1-I320 in
[**] This research was supported by grants from the National Institutes
of Health to T.P.B. (GM069618). We would like to thank Prof. Frank
C. Schroeder for helpful discussions regarding the NMR charac-
terization. Pdx1=pyridoxal phosphate synthase.
Supporting information for this article is available on the WWW
under http://www.angewandte.org or from the author.
7m urea in the presence of TCEP. C) UV-absorbance scan of X230
.
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Figure 3. 15N decoupling analysis of X230. I320 was constituted using
Figure 2. Structural characterization of X230 (5). A) 1H NMR spectrum
(600 MHz) of purified X230 in D2O. The protons corresponding to b
and c exchanged with solvent over time (b: several hours, c: several
days). The solvent signal at d=4.8 ppm was suppressed by presatura-
tion. B) ESI-MS analysis of X230 performed in positive mode. The peak
labeled at m/z=251.2 corresponds to the +1 ion of TCEP, which is
likely a fragment of the species responsible for the peak seen at m/
z=346.1. m/z=346.1 is consistent with the +1 ion of 5.
15NH4Cl, and then isolated according to Figure 1A. A) 1H NMR spec-
trum (600 MHz) of purified I320 in 20% D2O/80% H2O (20% D2O was
used because the signals corresponding to a, c, and d exchanged with
solvent over time). Processing of the data included solvent subtrac-
tion. B) Comparison of sections of the 1H NMR spectrum shown in
(A) to a spectrum obtained with broadband 15N decoupling applied.
Signals corresponding to a, b, c, and d were significantly simplified by
15N decoupling. Processing of the data included the application of an
unshifted sine–bell function prior to Fourier transformation to increase
resolution.
was tentatively assigned as 5. ESI-MS analysis was also in
good agreement with the proposed structure (Figure 2B).
Although this analysis was consistent with a nitrogen-
containing heterocycle, it did not directly demonstrate the
presence of the nitrogen atom.
in a covalent manner, and that X230 is a novel adduct of TCEP
and I320
.
From previous work, it was shown that 6 is a likely
[16]
structure for I320
.
This proposal is based on the following:
Establishing the presence of a nitrogen atom in I320 is of
critical importance for our analysis of the mechanism of PLP
formation. Previous data, obtained by high-resolution MS
analysis of an I320-labeled peptide from a tryptic digest,
1) I320 reacts with G3P to give PLP in the absence of ammonia,
suggesting that ammonia is covalently bound; 2) I320 must be a
highly conjugated system to account for its long-wavelength
absorption; 3) the observation of a primary deuterium kinetic
isotope effect on the formation of I320 using C5 pro-R
deuterium-labeled suggests that one of the CH2 protons of
R5P is absent from I320; 4) phosphate elimination is stoichio-
metric with the production of I320, demonstrating that I320 does
not contain phosphate; and 5) analysis of the formation of I320
from 9 by ESI-FTMS is consistent with structure 6. A
mechanistic proposal for the formation of 6 is shown in
Scheme 2.
suggested that the glutamine-derived nitrogen atom was not
[15]
covalently incorporated into I320
.
However, separate
experiments demonstrated that gel-purified I320 can be con-
verted into PLP by the addition of G3P in the absence of any
ammonia source.[16] This suggests that the ammonia is
covalently bound to I320
.
The isolation of X230 allowed us to perform a direct
experiment to probe for the presence of the nitrogen atom in
I
320. To do this, Pdx1–I320 formation was triggered using 15N-
In the absence of other substrates, R5P is ring-opened to
the aldehyde 7, and imine formation with the active-site lysine
would give 8 which then rearranges to ketone 9. This species is
poised for ammonia addition at C2 to give imine 10. This
1
enriched NH4Cl, and X230 was purified by HPLC. The H
NMR spectrum obtained in 20% D2O is shown in Figure 3A,
and is consistent with structure 5. This assignment is also
supported by a variety of additional 2D NMR spectra (see
Supporting Information). The large doublet of apparent
triplets at d = 9.8 ppm suggested that Ha was coupled to 15N.
To confirm this, the proton spectrum was measured with and
without broadband 15N decoupling. From the pair of spectra
(Figure 3B), it is clear that the heterocycle contains an 15N
atom that is coupled to each of the protons responsible for
signals downfield of d = 3.5 ppm (a–d). As expected, the
signals present in the aliphatic region of the sprectrum (d =
2.5–2.8 ppm) were not affected by 15N decoupling (e–f). This
analysis demonstrates that ammonia is incorporated into I320
event triggers the next series of reactions leading to I320
.
Elimination of water gives 11, and then rearrangement to 12
occurs by deprotonation at C5. Elimination of the lysine from
C1 generates 13, and then the same lysine residue adds to C5
resulting in 14, which facilitates phosphate elimination to give
I320 6. During the normal catalytic cycle of Pdx1, the addition
of G3P to I320 results in PLP formation in a series of reactions
that are not yet well understood.
The detection of 5 as a trapped product derived from I320 is
consistent with our assignment of structure 6 to the chromo-
phoric intermediate. We propose (Scheme 3) that upon
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N2 for storage. At this point the protein
concentration
was
approximately
2.15 mm.
To prepare X230
,
Pdx1 (2.15 mm,
1.5 mL) was mixed with a final concen-
tration of d-ribose-5-phosphate (1.5 mm,
Sigma–Aldrich) and incubated at room
temperature for 20 min. Only 1.5 mm
ribose-5-phosphate was used because
approximately 60% of the active sites
contain the pentose-5P as a result of
copurification. NH4Cl was added as a
solid to a final concentration of 1m, and
the reaction mixture was incubated for
1 hour. At this time the Pdx1-I320 was
purified from the excess salt and small
molecules using a 10DG gel filtration
column (Bio-Rad) according to the prod-
uct instructions. The column was pree-
quilibrated with sodium phosphate
buffer (50 mm, pH 7.6) containing
300 mm NaCl and 4 mm TCEP. Solid
Scheme 2. Mechanistic proposal for the formation of I320. For compounds 2–14, carbon atoms are
defined as (right to left) C1–C5. See text for details.
urea was then added to the protein to a
final concentration of 7m and the mix-
ture was vortexed until the urea was dissolved. The sample was
then incubated for 12 h at 158C to allowthe reaction to take
place. Many products were observed if this incubation temper-
ature was raised to 378C. The protein was removed from X230
by ultrafiltration using a 10000 kDa-cutoff Amicon Ultra-4
centrifugal filter unit at 48C (Millipore). X230 was purified by
HPLC using a Supelcosil LC-18-T column (25 cm ” 10 mm,
5 mm) equilibrated with water containing 0.1% trifluoroacetic
acid (TFA), and eluted with a gradient of MeOH containing
0.1% TFA. The HPLC method is reported in the Supporting
Information.
The major species was collected over the course of several
injections, pooled, and then the solvent removed with a rotary
evaporator prior to lyophilization. Following lyophilization the
solid was directly dissolved in water or D2O for MS or NMR
spectroscopic analysis.
Scheme 3. Proposed route for the formation of 5. See text for details.
denaturation, TCEP (15) adds to 6 to give 16, which then
tautomerizes to 17 and cyclizes to 18 releasing it from the
lysine residue. Aromatization gives pyrrole 5.
Received: September 23, 2007
Published online: February 7, 2008
The unanticipated trapping of I320 by TCEP demonstrates
that ammonia is covalently bound to I320, and supports our
assignment of structure 6 to this intermediate.[15] However, a
previous MS analysis of a peptide fragment (trypsin digest)
containing I320 suggested that it was not a nitrogen-containing
compound. One explanation for this discrepancy is that 6
isomerizes upon exposure to solvent and the resulting imine
undergoes hydrolysis, leading to the loss of ammonia. The
current study also suggests that TCEP (and other phos-
phines), which is generally viewed as an inert buffer
component used to protect thiols from oxidation, may have
applications in the trapping of other enzyme-bound a,b-
unsaturated aldehydes, ketones and imines.
Keywords: biosynthesis · intermediates · phosphines ·
pyridoxal · vitamin B6
.
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Experimental Section
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