Analysis of UDP-D-apiose/UDP-D-xylose synthase-catalyzed conversion of UDP-D-apiose phosphonate to UDP-D-xylose phosphonate: Implications for a retroaldol-aldol mechanism

Article abstract of DOI:10.1021/ja305322x

UDP-d-apiose/UDP-d-xylose synthase (AXS) catalyzes the conversion of UDP-d-glucuronic acid to UDP-d-apiose and UDP-d-xylose. An acetyl-protected phosphonate analogue of UDP-d-apiose was synthesized and used in an in situ HPLC assay to demonstrate for the first time the ability of AXS to interconvert the two reaction products. Density functional theory calculations provided insight into the energetics of this process and the apparent inability of AXS to catalyze the conversion of UDP-d-xylose to UDP-d-apiose. The data suggest that this observation is unlikely to be due to an unfavorable equilibrium but rather results from substrate inhibition by the most stable chair conformation of UDP-d-xylose. The detection of xylose cyclic phosphonate as the turnover product reveals significant new details about the AXS-catalyzed reaction and supports the proposed retroaldol-aldol mechanism of catalysis.

Full text of DOI:10.1021/ja305322x

Communication
pubs.acs.org/JACS
Analysis of UDP‑D‑Apiose/UDP‑D‑Xylose Synthase-Catalyzed
Conversion of UDP‑D‑Apiose Phosphonate to UDP‑D‑Xylose
Phosphonate: Implications for a Retroaldol−Aldol Mechanism
Sei-hyun Choi, Steven O. Mansoorabadi, Yung-nan Liu, Tun-Cheng Chien, and Hung-wen Liu*
Division of Medicinal Chemistry, College of Pharmacy, and Department of Chemistry and Biochemistry, University of Texas at
Austin, Austin, Texas 78712, United States
S
* Supporting Information
Scheme 1. Proposed Mechanisms for the AXS-Catalyzed
Biosynthesis of UDP-D-apiose (2) and UDP-D-xylose (7)
from UDP-GlcA (3)
ABSTRACT: UDP-D-apiose/UDP-D-xylose synthase
(AXS) catalyzes the conversion of UDP-D-glucuronic
acid to UDP-D-apiose and UDP-D-xylose. An acetyl-
protected phosphonate analogue of UDP-D-apiose was
synthesized and used in an in situ HPLC assay to
demonstrate for the first time the ability of AXS to
interconvert the two reaction products. Density functional
theory calculations provided insight into the energetics of
this process and the apparent inability of AXS to catalyze
the conversion of UDP-D-xylose to UDP-D-apiose. The
data suggest that this observation is unlikely to be due to
an unfavorable equilibrium but rather results from
substrate inhibition by the most stable chair conformation
of UDP-D-xylose. The detection of xylose cyclic
phosphonate as the turnover product reveals significant
new details about the AXS-catalyzed reaction and supports
the proposed retroaldol−aldol mechanism of catalysis.
D-Apiose (1) is a rare branched-chain furanose found in the cell
wall of plants.1 D-Apiose residues play an important role in
plant growth and development by cross-linking cell-wall
polysaccharides through the formation of a borate tetraester.2
UDP-D-apiose (2), the in vivo D-apiose donor, is formed from
uridine-5′-diphosphate-D-glucuronic acid (UDP-GlcA, 3) in an
unusual ring-contraction reaction catalyzed by UDP-D-apiose/
UDP-D-xylose synthase (AXS). The conversion of 3 to 2 by
AXS is NAD+-dependent and generates UDP-D-xylose (7) as a
side product.3 The proposed reaction sequence for the
formation of 2 (Scheme 1) involves oxidative decarboxylation
of 3 to afford a 4-ketoxylose intermediate (UDP-4-KX, 4).
Subsequent ring contraction (4 → 6) followed by reduction at
C3′ by NADH yields 2.
Two plausible mechanisms for the ring-contraction step have
been proposed.4 In the first mechanism (Scheme 1, pathway
A), the C2−C3 bond of 4 is cleaved in a retroaldol reaction,
yielding an enediol intermediate (5) that cyclizes in an aldol
reaction to give 6. In the second mechanism (Scheme 1,
pathway B), 6 is formed from 4 directly through a 1,2-shift. In
contrast to the stepwise retroaldol−aldol route, the C−C bond
migration in this concerted mechanism would bypass the
formation of a discrete intermediate such as 5. In both
mechanisms, the formation of 7 could result from a premature
reduction of the decarboxylated 4-keto intermediate 4 by
NADH prior to the rearrangement step.
One curious observation from previous studies is that AXS is
unable to convert 7 to 2,5 suggesting that there is no
mechanistic link between these two products and that each is
formed irreversibly.5c However, testing this hypothesis is
challenging because of the instability of 2 in aqueous solution,6
which makes direct detection of its formation in vitro difficult.
One potential strategy to circumvent this issue and gain insight
into the mechanism of the AXS-catalyzed ring contraction may
be to assay the reaction in the energetically favorable reverse
direction (2 → 7) using a stable UDP-D-apiose analogue as the
substrate. Accordingly, we designed and synthesized 8, a
phosphonate analogue of 2 (Figure 1), and examined its
interaction with AXS. Compound 8 was chosen because
phosphonates are well-established as stable analogues of
Figure 1. Phosphonate analogues of UDP-D-apiose (8) and UDP-
xylose (9).
Received: June 7, 2012
Published: July 25, 2012
© 2012 American Chemical Society
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Communication
phosphates.7 In addition, density functional theory (DFT)
calculations provided insight into the energetics of the AXS-
catalyzed reaction. The results are described herein.
Because of the tertiary carbon center at C3, the key step in
the synthesis of D-apiose analogues is the introduction of the
hydroxymethyl moiety. Several synthetic strategies were
attempted to introduce the C3-methyl branch in the synthesis
of D-apiose analogue 8 (Scheme 2). Starting from isopropyli-
osmium tetroxide would approach from the less sterically
hindered Si face during the hydroxylation step (Figure 2B).
The introduction of the hydroxymethyl group with the
desired configuration was then attempted using ylide chemistry
(Scheme 2B). As with OsO4, a sulfur ylide would be expected
to attack from the Si face. The nuclear Overhauser effect
spectroscopy (NOESY) NMR spectrum of the hydrolyzed
product 17 revealed that it contained the desired configuration
(Figure S1B). Consecutive protection of the hydroxyls with
benzyl groups, deprotection of the TBDPS group, bromination
with CBr4, and phosphonylation afforded protected apiose
phosphonate analogue 20. Hydrolysis of the methyl phospho-
nate to the corresponding phosphonic acid (21) and coupling
with uridine-5′-monophosphate (UMP) morpholidate afforded
22, a benzyl-protected UMP phosphonate analogue of D-
apiose.9 However, after deprotection of the benzyl groups,
cyclic phosphonate 23 was obtained.
Scheme 2. Attempts To Synthesize 8
With the hope that this intramolecular cyclization problem
could be overcome under mildly basic conditions, a tri-O-
acetyl-protected UMP phosphonate analogue of D-apiose, 25,
was synthesized (Scheme 2C). However, even under mild
conditions (0.2 M NH3 in MeOH, 0 °C), 23 and UMP were
the only products obtained. A possible explanation for these
results is that the 2-alkoxide ion formed during the reaction is
suitably aligned to attack the vicinal phosphorus center, forming
the thermodynamically stable five-membered cyclic phospho-
nate with concomitant release of UMP, which is a good leaving
group.
In addition to the phosphonate analogue of D-apiose, we also
attempted to synthesize 9, a UMP phosphonate analogue of D-
xylose, for use as a product standard (Scheme 3). The reducing
Scheme 3. Synthesis of Xylose Cyclic Phosphonate 10
dene-protected xylofuranose (11), sequential protection of the
free hydroxyl groups gave protected xylose 12. Deprotection of
the isopropylidene group, reduction of the anomeric carbon,8
and oxidation of the 3-hydroxyl moiety afforded 3-keto-4-
deoxysugar 14. For the introduction of the hydroxymethyl
moiety at C3, a Wittig reaction followed by a dihydroxylation
protocol was attempted. However, careful NMR assignment
[Figure S1A in the Supporting Information (SI)] and X-ray
crystallographic analysis (Figure 2A) revealed that the stereo-
chemistry of the product at C3 was opposite that of the desired
product. This result can be rationalized by considering that
end of benzyl-protected D-xylose 27 was converted into linear
alkene 28 by a Wittig reaction and subsequently transformed
into iodomethyl compound 29 by a successive oxymercura-
tion−iodination reaction.10 Only the α-isomer of 29 was
isolated, possibly because of an anomeric effect. With a method
similar to that applied for the synthesis of 22, we obtained 32,
the benzyl-protected UMP phosphonate analogue of D-xylose.
Hydrogenation to remove the benzyl groups also afforded
cyclic phosphonate 10 and UMP as the only products.
Although phosphonates are known to be more stable than
the corresponding phosphates in terms of their anomeric bond
strength, these results suggest that the P−OUMP bond of the
phosphonate analogues can still be readily cleaved when the 2-
Figure 2. (A) X-ray structure of 16. (B) Explanation of the observed
stereochemistry of the methylenation of 14 and dihydroxylation of 15.
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OH group is well-aligned for nucleophilic attack. One potential
way to overcome the intrinsic instability of 8 would be to
generate it in situ during the assay of AXS activity. A coupled
assay using an acetyl esterase and AXS was therefore designed
in which 8 would be generated from acetyl-protected substrate
25. Orange peel acetyl esterase (AE, E.C. 3.1.1.6) was chosen
because it is known to deacetylate various peracetylated NDP
sugars in good yield.11 25 was incubated with AE for 24 h, and
mass spectrometry (MS) analysis showed that it was
deacetylated to give the desired product, 8 (see the SI).
Having established that 8 could be synthesized enzymatically
from 25 using AE, an in situ HPLC assay of AXS activity was
attempted using dual UV−vis and Corona-charged aerosol
detection.12 The latter is able to visualize nonvolatile
compounds regardless of whether they have a chromophore.
The Corona HPLC traces of the AXS reaction mixtures (Figure
3a,b) contain a broad peak between 11 and 13 min.
Figure 4. HPLC traces of the isolated 11−12.5 min peak using DEAE
resin ion-exchange chromatography. For detailed experimental
procedures, see the SI.
indicating that AXS can catalyze the conversion of UDP-D-
apiose analogue 8 to the corresponding UDP-D-xylose analogue
9, which degrades into 10.
The observation that AXS can catalyze the reverse reaction,
the conversion of 8 to 9, contrasts with the previous result that
7 could not be converted to 2 by AXS.5 To gain insight into
these different reactivities, the energetics of the reaction was
examined by DFT calculations using Gaussian 98.13 The
calculations (performed at the B3LYP/6-311+G(d,p) level
using a scaling factor of 0.9877 and the polarizable continuum
model to mimic solvent) showed that in aqueous solution, the
chair conformation of D-xylose with the hydroxyl groups in the
equatorial position (e.g., 7) is only 0.4 kcal/mol more stable
than D-apiose (e.g., 2). Such a small difference in free energy is
unable to account for the apparent inability of AXS to convert 7
to 2. Surprisingly, however, it was found that the alternative
chair conformation of D-xylose in which the hydroxyl groups
are in the axial position (e.g., 33) is 1.6 kcal/mol more stable
than D-apiose.
The less stable D-xylose conformer, 7, would be expected to
bind to AXS in a manner analogous to the substrate, 3, with the
4-H properly oriented for transfer to NAD+ (Scheme 4). In
contrast, the more stable D-xylose conformer, 33, overlays quite
well with the product, 2 (Scheme 4 inset). In this binding
mode, the catalytic base required for the oxidation of the
hydroxymethyl moiety of 2 would interact with the C3
hydroxyl instead of the C4 hydroxyl of 33. Thus, 33 would
not be positioned for efficient hydride transfer to NAD+, and its
binding to AXS would result in the formation of a dead-end
complex. The combined observations give a possible
explanation for the apparent inability of AXS to catalyze the
conversion of 7 to 2: the former can adopt two conformations
in solution, the more stable of which inhibits the enzyme. This
inhibition may have prevented the detection of 2 under the
conditions and time scale used in the previous studies.5
The detection of xylose cyclic phosphonate 10 instead of 9 as
the product of the incubation of 8 with AXS is another
significant finding. Since compound 10 must be derived from 9
via nucleophilic attack at the vicinal phosphorus center by the
2-alkoxide anion, the above observation strongly suggests that
Figure 3. HPLC traces for the in situ AXS assay with dual detection
(black line, Corona detector; red line, UV detector): (a) reaction
mixture at 0 h; (b) reaction mixture at 47 h; (c) apiose cyclic
phosphonate 23; (d) xylose cyclic phosphonate 10; (e) NAD+; (f)
Tris.
Comparison with the HPLC traces of the individual reaction
components showed that NAD+ (Figure 3e) and Tris buffer
(Figure 3f) both elute in this region. Xylose cyclic phosphonate
10, the product of the attempted synthesis of 9, also elutes
within this region (Figure 3d). After a 47 h reaction period, a
new peak with a retention time of 10 min was observed,
consistent with the formation of a small amount of apiose cylic
phosphonate 23 (Figure 3b,c). In addition, a shoulder appeared
to form on the broad peak centered at ∼12 min.
To verify that this shoulder indicates the formation of 10,
and thus the conversion of 8 to 9 by AXS, the peak between 11
and 12.5 min was collected and analyzed by MS. The MS data
are indeed consistent with the peak containing both 10 ([M −
H]−, m/z 209) and Tris ([M + H]+, m/z 122) (see the SI). To
ensure that the species detected by MS corresponded to xylose
cyclic phosphonate 10 and not contaminating apiose cyclic
phosphonate 23, the isolated peak was fractionated using
diethylaminoethyl (DEAE) resin ion-exchange chromatogra-
phy, and each fraction was reanalyzed by HPLC. As shown in
Figure 4, Tris (11 min) eluted in the H2O wash fractions (f1
and f2), whereas 10 (12 min) eluted at high ammonium
bicarbonate concentrations (f14−f18). There was no evidence
for the presence of 23 (10 min) in any of the fractions. These
results clearly show that the broad peak indeed contains 10,
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Communication
Notes
Scheme 4. Energy Differences between the Two Conformers
of UDP-D-xylose
a
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank Dr. Mikio Fuji for his contributions to the initial
synthetic efforts. This work was supported by grants from the
National Institutes of Health (GM035906 and GM054346) and
the Welch Foundation (F-1511).
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of 2 to 7. Of the two mechanisms proposed for AXS, only the
cleavage of the C2−C3 bond of 4 in a retroaldol process
requires the ionization of the 2-OH group (4 → 5; Scheme 1,
pathway A). In contrast, the 1,2-shift mechanism (Scheme 1,
pathway B) requires deprotonation of the C3 hydroxyl group
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retroaldol−aldol route as the catalytic mechanism of AXS.
In summary, we synthesized 8, a protected phosphonate
analogue of UDP-D-apiose (2), for use in mechanistic studies of
AXS. To overcome the intrinsic instability of 8 during chemical
synthesis, a coupled assay in which this substrate was generated
in situ using acetyl esterase was developed. The data show that
AXS can accept phosphonate in addition to the natural
phosphate substrates. More importantly, our results reveal that
AXS converts 8 into the corresponding UDP-D-xylose analogue
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2 was obtained from DFT calculations, which suggested that
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chair conformations in aqueous solution, the more favorable of
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activity. This product inhibition of the AXS-catalyzed reaction
by UDP-D-xylose may play a physiological role in the regulation
of cell-wall biogenesis in plants. Moreover, the isolation of 10
instead of 9 as the turnover product of 8 with AXS provides
strong evidence in support of the retroaldol−aldol mechanism
for this intriguing enzyme.
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ASSOCIATED CONTENT
* Supporting Information
Materials and methods and spectroscopic and structural data.
This material is available free of charge via the Internet at
http://pubs.acs.org.
■
S
AUTHOR INFORMATION
Corresponding Author
h.w.liu@mail.utexas.edu
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