Isotope Probing of the UDP-Apiose/UDP-Xylose Synthase Reaction: Evidence of a Mechanism via a Coupled Oxidation and Aldol Cleavage

Article abstract of DOI:10.1002/anie.201609288

The C-branched sugar d-apiose (Api) is essential for plant cell-wall development. An enzyme-catalyzed decarboxylation/pyranoside ring-contraction reaction leads from UDP-α-d-glucuronic acid (UDP-GlcA) to the Api precursor UDP-α-d-apiose (UDP-Api). We examined the mechanism of UDP-Api/UDP-α-d-xylose synthase (UAXS) with site-selectively²H-labeled and deoxygenated substrates. The analogue UDP-2-deoxy-GlcA, which prevents C-2/C-3 aldol cleavage as the plausible initiating step of pyranoside-to-furanoside conversion, did not give the corresponding Api product. Kinetic isotope effects (KIEs) support an UAXS mechanism in which substrate oxidation by enzyme-NAD⁺and retro-aldol sugar ring-opening occur coupled in a single rate-limiting step leading to decarboxylation. Rearrangement and ring-contracting aldol addition in an open-chain intermediate then give the UDP-Api aldehyde, which is intercepted via reduction by enzyme-NADH.

Full text of DOI:10.1002/anie.201609288

Angewandte
Communications
Chemie
International Edition: DOI: 10.1002/anie.201609288
German Edition: DOI: 10.1002/ange.201609288
Enzyme Catalysis
Isotope Probing of the UDP-Apiose/UDP-Xylose Synthase Reaction:
Evidence of a Mechanism via a Coupled Oxidation and Aldol Cleavage
Thomas Eixelsberger, Doroteja Horvat, Alexander Gutmann, Hansjçrg Weber, and
Bernd Nidetzky*
Abstract: The C-branched sugar d-apiose (Api) is essential for
plant cell-wall development. An enzyme-catalyzed decarbox-
ylation/pyranoside ring-contraction reaction leads from UDP-
a-d-glucuronic acid (UDP-GlcA) to the Api precursor UDP-
a-d-apiose (UDP-Api). We examined the mechanism of UDP-
Api/UDP-a-d-xylose synthase (UAXS) with site-selectively
2H-labeled and deoxygenated substrates. The analogue UDP-
2-deoxy-GlcA, which prevents C-2/C-3 aldol cleavage as the
plausible initiating step of pyranoside-to-furanoside conver-
sion, did not give the corresponding Api product. Kinetic
isotope effects (KIEs) support an UAXS mechanism in which
substrate oxidation by enzyme-NAD+ and retro-aldol sugar
ring-opening occur coupled in a single rate-limiting step
leading to decarboxylation. Rearrangement and ring-contract-
ing aldol addition in an open-chain intermediate then give the
UDP-Api aldehyde, which is intercepted via reduction by
enzyme-NADH.
skeleton rearrangement (4!5!6) then occurs probably via
a retro-aldol/aldol reaction,[6] and reduction of UDP-a-d-
apiose 3’-aldehyde (6) by enzyme-NADH gives 1.[5–7] The
alternative reaction product, UDP-a-d-xylose (7), is derived
from 4, also by NADH-dependent reduction. UDP-a-d-
xylose synthase (UXS) is structurally and mechanistically
related to UAXS, but lacks the ability to catalyze the
pyranoside-into-furanoside conversion.[8] A plausible point
of divergence in the proposed pathways of UAXS and UXS is
therefore intermediate 4.
Its widespread acceptance in the literature notwithstand-
ing,[5–7] the mechanism of Scheme 1 raises disquiet in that it
requires UAXS to recognize 4 equally for aldol ring cleavage
and for reduction by NADH. How the enzyme distinguishes
between these possibilities is not clear. Moreover, there is
only indirect evidence in support of the retro-aldol/aldol
route of conversion of 4 into 6. A 2-deoxy-2-fluoro analogue
of 3, rendering impossible the C-2/C-3 aldol cleavage in
a corresponding 2-fluoro derivative of 4, was completely
unreactive with UAXS.[6a] A chemically stable phosphonate
analogue of 1 (Scheme 1, 1a) was converted by UAXS into
the corresponding xylosyl compound. A xylose cyclic phos-
phonate (7b) instead of the expected product 7a (Scheme 1)
was formed. This was interpreted to involve an enzymatically
deprotonated C-2 hydroxy group, which would also be
involved in the “native” retro-aldol conversion of 4.[6b] The
current study was performed to re-investigate the catalytic
steps for conversion of substrate 3 into 1 and 7. Evidence
supporting an updated mechanism, involving retro-aldol ring
opening prior to the decarboxylation, is presented.
U
ridine 5’-diphosphate (UDP)-a-d-apiose (1) is the pre-
cursor of the C-branched pentose d-apiose [3-C-(hydroxy-
methyl)-d-glycero-tetrose; 2].[1] Compound 2 is present in the
cell-wall polysaccharides rhamnogalacturonan II and apioga-
lacturonan, as well as in various secondary metabolites in
plants.[1–4] Sugar nucleotide 1 is derived from UDP-a-d-
glucuronic acid (3) in a decarboxylation/pyranoside ring-
contraction reaction catalyzed by UDP-a-d-apiose/UDP-a-d-
xylose synthase (UAXS).[5] The proposed mechanism of this
chemically intriguing biotransformation (Scheme 1) involves
nicotinamide adenine dinucleotide (NAD+)-assisted oxida-
tion at C-4 of substrate 3 and subsequent decarboxylation to
give UDP-b-l-threo-pentopyranosid-4-ulose (4).[5–7] Carbon
Purified UAXS from Arabidopsis thaliana recombinantly
expressed in Escherichia coli was used (Figure S1 in the
Supporting Information). Reactions were performed at
pH 8.5 because the enzyme activity was highest (Table S1,
Supporting Information) and the ratio of product 1 to 7
maximized under these conditions. No intermediary products
(e.g., 4)[6b,7] were released. Under the conditions used, a-d-
apiofuranosyl-1,2-cyclic phosphate (8; Scheme 1) was sponta-
neously formed from 1.[5,7] Product 1 was therefore always
detected as 8. Site-selectively 13C- or 2H-labeled analogues of
substrate 3 were synthesized from the corresponding isotopi-
cally labeled d-glucoses (Scheme S1, Figure S2–S11).[9a] Unla-
beled 3 was prepared identically and used as a reference. A 2-
deoxy analogue of 3 was synthesized from 1,5-anhydro-2-
deoxy-d-arabino-hex-1-enitol via 2-deoxy-d-glucose-1-phos-
phate, exploiting the reaction of cellobiose phosphorylase
(Scheme S2, Figure S12–S18).[9b] Each compound was isolated
[*] Dr. T. Eixelsberger, D. Horvat, Dr. A. Gutmann, Prof. Dr. B. Nidetzky
Institute of Biotechnology and Biochemical Engineering
Graz University of Technology, NAWI Graz
Petersgasse 12, 8010 Graz (Austria)
E-mail: bernd.nidetzky@tugraz.at
Prof. Dr. H. Weber
Institute of Organic Chemistry
Graz University of Technology, NAWI Graz
Stremayrgasse 9, 8010 Graz (Austria)
Prof. Dr. B. Nidetzky
Austrian Centre of Industrial Biotechnology (acib)
Petersgasse 14, 8010 Graz (Austria)
Supporting information (the coding gene used (Figure S29–S30); the
preparation of the enzymes used; the substrate synthesis and
characterization; the methods of analysis; and determination of the
KIEs) and the ORCID identification number(s) for the author(s) of
this article can be found under http://dx.doi.org/10.1002/anie.
201609288.
1
and its identity confirmed by H and 13C NMR spectroscopy.
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demonstrated enzymatic conversion to
give 2-deoxy-7 as the product (Figure 1B,
Figure S26–S28). Noticing that UAXS
might be inhibited by UDP/UMP
released from 2-deoxy-3 due to decom-
position, we re-examined UAXS under
addition of alkaline phosphatase to
hydrolyze the nucleosides present. Syn-
thesis of a tiny amount of 2-deoxy-7 was
shown under these conditions (Fig-
ure 1B).
No evidence of 2-deoxy-1 was found,
as expected. However, while UXS exhib-
ited substantial activity with 2-deoxy-3
(Figure 1B; Figure S26), the UAXS
activity was almost completely destroyed
(ꢁ 0.1% of activity with 3) on substitut-
ing the 2-OH in the substrate by a hydro-
gen atom. These results are important
mechanistically showing that 2-deoxy-3
was fully competent to undergo oxidative
decarboxylation in the UXS-type reac-
Scheme 1. The proposed mechanism of UAXS; E=enzyme.[5–7]
Purity was furthermore determined by capillary electropho-
resis and HPLC.
tion, plausibly via 2-deoxy-4. Assuming intermediate 4 to be
the point of divergence in the UAXS and UXS reaction paths,
the huge difference in activity of the two enzymes in forming
2-deoxy-7 was somewhat counterintuitive. Note that only
a small change in the reaction conditions (e.g. pH, ion type
and concentration) is sufficient to shift the distribution of
UAXS reaction products to favor 7, while at the same time the
overall activity is just moderately affected (data not shown;
see also Ref. [5f]). We therefore hypothesized that the
pyranoside ring-opening characteristic of UAXS might not
be as clearly decoupled from the oxidation–decarboxylation
common to both enzymatic conversions as the mechanistic
proposal of Scheme 1 assumes. Intermediate 4 might be
formed through a different path in UAXS than in UXS, and
we designed a KIE study to address the problem. It will
become clear below that the minute conversion of 2-deoxy-3
by UAXS is probably a relic of UXS-type activity in this
enzyme, which considering the relatedness of UAXS and
UXS at the level of amino acid sequence[10] is not a complete
surprise.
In situ NMR analysis of enzymatic reactions of [2-13C]-3
and [3-13C]-3 demonstrated rearrangement of the carbon
skeleton on formation of 1 as expected from Scheme 1.
Despite being confirmatory mainly,[5–7] the evidence was
nonetheless crucial for it enabled the precise assignment of all
the 13C signals from substrate and products (Figure S19–S24).
Reaction of unlabeled 3 in D2O resulted in a single deuterium
from solvent to be incorporated at C-4 of 1 and C-5 of 7 as
result of the decarboxylation, consistent with literature.[5d,e]
Reactions of [3-2H]-3 and [4-2H]-3 both gave [3’-2H]-1, as
expected from Scheme 1,[5e] however with clearly distinct
C2H1H groups at position 3’ (Figure 1A, panels a,b; Fig-
ure S25). Because reduction of 6 by the hydrogen or
deuterium abstracted from C-4 is stereospecific,[5d,e] the
absolute configuration at C-3’ in 1 will be opposite in the
two conversions. Irrespective of whether reaction of unla-
beled 3 was examined in D2O or reaction of [3-2H]-3 in water,
1
no H/2H exchange was observed at position 3’ (Figure 1A,
panels c,d; Figure S25) The result was important mechanisti-
cally for it validated the determination of kinetic isotope
effects (KIEs) through the deuteration at C-3 of 3. Studying
the UAXS from parsley or duckweed, however, with a rela-
tively complicated and indirect procedure of product analysis,
We determined the effect of [4-2H] in 3 on the rate of
substrate consumption (VS). Direct comparison of VS with
protio and deuterio substrates at saturating concentration
gave a large KIE of 2.72 ꢂ 0.20 (N = 5). The KIE measured by
intermolecular competition was also high and its value (2.47 ꢂ
0.43) was similar to the KIE on kcat (Table S2). Contrary to the
directly determined KIE, which influences the catalytic
constant (kcat), the KIE from the competition experiment
3
other authors reported uptake of 0.5 mol H molꢀ1 in 1 at
C-3’.[5b,e] We rule out a similar exchange with protons from
solvent in the enzymatic reaction under the conditions we
used.
2-Deoxy-3 was examined as a substrate of UAXS for it
prevents the C-2/C-3 retro-aldol cleavage to initiate pyrano-
side-into-furanoside ring conversion. Compared to 2-deoxy-2-
fluoro-3 used earlier with the same rationale,[6a] 2-deoxy-3
features only weak electronic perturbation at the position 2,
thus rendering it a mechanistic probe of the enzyme in its own
right. Because preliminary experiments suggested UAXS to
be inactive towards 2-deoxy-3, we also tested UXS and
necessarily influences the second-order rate constant (kcat
/
KmS).[11] Scheme 2 is used for interpretation of the data.
Whereas kcat involves all the unimolecular steps of the
reaction, kcat/KmS includes only those steps up to the first
irreversible step, which in UAXS is the decarboxylation. The
large KIE on kcat/KmS implies that hydride abstraction from
substrate is partly rate limiting for the steps covered by kcat
/
KmS. The UAXS reaction involves three isotope-sensitive
steps (Scheme 1 and Scheme 2) and each could contribute to
2
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Figure 1. A) Formation of product 1 (detected as 8) by UAXS. a),b) 1H NMR signal of the 3’-H in 8 obtained from [3-2H]-3 (a) and [4-2H]-3 (b).
Both reactions were carried out in H2O (pH 8.5). c),d) 1H NMR signal of 3’-H in 8 obtained from unlabeled 3 in D2O (c) and H2O (d). In panels
(a)–(d), the product was analyzed directly from the reaction mixture. Enzyme: 20 mm; substrate 3: 2 mm; pH(D)=8.5. B) Formation of 2-deoxy-7
1
by UAXS and UXS. In blue: the H NMR spectrum of a reaction mixture of 2-deoxy-3 (d=5.67 ppm, 5.68 ppm) converted partially into 2-deoxy-7
1
(d=5.59 ppm, 5.60 ppm) by UXS. The black stack plot shows an in situ H NMR experiment of the conversion of 2-deoxy-3 (d=5.67 ppm,
5.68 ppm) by UAXS. It shows that 2-deoxy-7 (d=5.59 ppm, 5.60 ppm) is formed in small amounts. UXS: 20 mm; UAXS: 100 mm; substrate 2-
deoxy-3: 2 mm. Reactions were performed in D2O (pD=8.5) for 2 h (UXS) and over 12 h with spectra recording in 2 h intervals (UAXS).
Next, we determined in intermolecular competition
experiments the effect from [3-2H] in 3 on kcat/KmS. We
considered that a KIE would be a secondary one, arising from
hybridization change at C-3 during sugar ring opening and
closure. And additionally, because substrate oxidation was
a rate-limiting step, a KIE different from unity would be
Scheme 2. A minimal kinetic mechanism of UAXS is shown. “I”
represents an enzyme-bound intermediate suggested to be the acyclic
possible only in the case that the reaction step(s) affecting C-3
happened within the rate-determining cascade of oxidation
and decarboxylation. Starting from a substrate composed
initially of roughly equal amounts of 3 and [3-2H]-3, we
determined by NMR spectroscopy at three different levels of
form 5. A primary deuterium KIE could arise in steps 1, 2a and 2b.
The sequence of reaction steps included in kcat/Km is shown in the box.
the KIE on kcat. We measured under rapid-mixing conditions
by absorbance at 340 nm the reduction of enzyme-bound
NAD+. NADH did not accumulate in detectable amounts,
which it would if the two hydride transfers from NADH were
limiting for kcat. This result together with the evidence that
kcat/KmS and kcat were affected by a similar KIE, identified
hydride abstraction from substrate to NAD+ as major rate-
determining step of the UAXS reaction. Although hydride
transfers from enzyme-NADH were not slow steps of the
overall reaction, the ratio of the products 1 and 7 was
nonetheless affected by [4-2H] in 3. The ratio changed from
a value of 1.84 with undeuterated 3 to a lower value of 1.48
with [4-2H]-3 (Table S3). Therefore, this suggested a larger
deuterium KIE on reduction of 6 than on reduction of 4.
Moreover, the result demonstrated the ability of UAXS to
reversibly interconvert intermediates 4 and 6, in good agree-
ment with the results of Liu and co-workers,[6b] and to rapidly
equilibrate their enzyme-bound forms.
1
conversion the H/2H isotope ratio at C-3 in the remaining
substrate. A KIE of 1.20 ꢂ 0.03 was obtained (Table S4),
indicating that [3-2H]-3 reacted significantly more slowly than
3. The observed effect was in the upper region of the KIEs
previously reported from enzyme-catalyzed aldol/retro-aldol
reactions.[12] It would be consistent with a fully developed
secondary KIE resulting from complete sp3!sp2 hybridiza-
tion change between the ground state and the rate-limiting
transition state of a retro-aldol carbon–carbon bond cleavage
for enzymatic pyranosyl ring opening. It may be noted that
secondary deuterium KIEs of similar magnitude were
observed in other enzymes catalyzing aldol reactions.[12]
1
Additionally we measured the H/2H isotope ratio at C-3’ in
1 and C-3 in 7 at approximately 50% conversion and found its
value of approximately 1.2 to be the same in both products
and to reflect exactly the corresponding isotope ratio (i.e. 3-
2H/3-1H) in the residual substrate (Table S5). These results
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indicate that reaction steps after the decarboxylation involv-
Acknowledgements
ing hybridization change at C-3, namely the reversible aldol
addition to give 4 and the reduction of 6, did not have
a significant secondary KIE. Had there been a KIE in one of
these steps, the isotope ratio in 1 and 7 would not have been
the same, and would also have been different from that in the
3 converted.
Financial support was from the Austrian Science Fund (DK
Molecular Enzymology W901). Dr. Motomitsu Kitaoka
(National Food Research Institute, Ibaraki, Japan) provided
the gene of the uridylyltransferase used in substrate synthesis.
Taken together, the primary and secondary KIEs also
suggest a relative timing of substrate oxidation and ring
opening. A distinctly slow retro-aldol reaction occurring after
the oxidation at C-4 would likely make the hydride transfer to
NAD+ come to equilibrium. This scenario is inconsistent with
the large primary KIEs observed. Therefore, a concerted
transformation is supported in which hydride abstraction and
ring opening take place coupled one to another in a single
rate-determining reaction step (Scheme 3). Following decar-
Conflict of interest
The authors declare no conflict of interest.
Keywords: aldol reactions · carbohydrates · enzyme catalysis ·
reaction mechanism · ring contraction
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Scheme 3. Updated mechanistic proposal for UAXS. “EB” indicates an
enzyme base in the active site.
boxylation, ring closure would thus yield intermediates 4 and
6, probably in rapid equilibrium, which are then reduced to
products 7 and 1, respectively.[13] This way of product
formation seems attractive as it avoids the dual use of
intermediate 4 as substrate for enzymatic aldol cleavage and
for NADH-dependent reduction. Selective stabilization of
the acyclic intermediate after the decarboxylation could be
a catalytic strategy of UAXS to facilitate rapid interconver-
sion of the more stable cyclic forms 4 (in particular) and 6. In
this mechanistic scenario, formation of 2-deoxy-7 from 2-
deoxy-3 in the absence of ring opening is considered a minor
side activity of UAXS.
In summary, KIE and substrate analogue studies suggest
an updated UAXS mechanism (Scheme 3). Aldol cleavage for
pyranosyl ring opening is proposed to occur early in the
reaction, concerted with the oxidation at C-4. The reactions of
UAXS and UXS would not, therefore, proceed on the same
path up to intermediate 4.[5–7] Formation of the two UAXS
products 1 and 7 involves reduction by enzyme-NADH at
distinct “exit points” of the aldol/retro-aldol rearrangement
cycle in rapid equilibrium. The UAXS mechanism, although
highly specialized, possesses fundamental significance, as
other C-branched carbohydrates, such as l-streptose (3-C-
formyl-5-deoxy-l-lyxose),[14]
l-dihydrostreptose
(3-C-
hydroxymethyl-5-deoxy-l-lyxose),[14] d-hamamelose (2-C-
hydroxymethyl-d-ribose),[1] and aceric acid (3-C-carboxy-5-
deoxy-d-xylose)[1] might be formed via mechanistically sim-
ilar transformations.
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4
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Manuscript received: September 22, 2016
Revised: November 9, 2016
Final Article published: && &&, &&&&
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Communications
Enzyme Catalysis
T. Eixelsberger, D. Horvat, A. Gutmann,
H. Weber, B. Nidetzky*
&&& — &&&
Isotope Probing of the UDP-Apiose/UDP-
Xylose Synthase Reaction: Evidence of
a Mechanism via a Coupled Oxidation
and Aldol Cleavage
Coupled up: The reaction performed by
UDP-apiose/UDP-xylose synthase pro-
ceeds by a mechanism in which substrate
oxidation with NAD+ and sugar ring-
opening by aldol cleavage are coupled
with each other in the rate-determining
step and occur before the irreversible
decarboxylation. The C-branch of d-
apiose is then installed by a ring-con-
tracting aldol reaction and the exocyclic
hydroxymethyl group is formed by
NADH-dependent reduction.
6
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