Mechanistic characterization of UDP-glucuronic acid 4-epimerase

Article abstract of DOI:10.1111/febs.15478

UDP-glucuronic acid (UDP-GlcA) is a central precursor in sugar nucleotide biosynthesis and common substrate for C4-epimerases and decarboxylases releasing UDP-galacturonic acid (UDP-GalA) and UDP-pentose products, respectively. Despite the different reactions catalyzed, the enzymes are believed to share mechanistic analogy rooted in their joint membership to the short-chain dehydrogenase/reductase (SDR) protein superfamily: Oxidation at the substrate C4 by enzyme-bound NAD⁺ initiates the catalytic pathway. Here, we present mechanistic characterization of the C4-epimerization of UDP-GlcA, which in comparison with the corresponding decarboxylation has been largely unexplored. The UDP-GlcA 4-epimerase from Bacillus cereus functions as a homodimer and contains one NAD⁺/subunit (k_cat?=?0.25?±?0.01?s^?1). The epimerization of UDP-GlcA proceeds via hydride transfer from and to the substrate’s C4 while retaining the enzyme-bound cofactor in its oxidized form (≥?97%) at steady state and without trace of decarboxylation. The k_cat for UDP-GlcA conversion shows a kinetic isotope effect of 2.0 (±0.1) derived from substrate deuteration at C4. The proposed enzymatic mechanism involves a transient UDP-4-keto-hexose-uronic acid intermediate whose formation is rate-limiting overall, and is governed by a conformational step before hydride abstraction from UDP-GlcA. Precise positioning of the substrate in a kinetically slow binding step may be important for the epimerase to establish stereo-electronic constraints in which decarboxylation of the labile β-keto acid species is prevented effectively. Mutagenesis and pH studies implicate the conserved Tyr149 as the catalytic base for substrate oxidation and show its involvement in the substrate positioning step. Collectively, this study suggests that based on overall mechanistic analogy, stereo-electronic control may be a distinguishing feature of catalysis by SDR-type epimerases and decarboxylases active on UDP-GlcA.

Full text of DOI:10.1111/febs.15478

PROF. BERND NIDETZKY (Orcid ID : 0000-0002-5030-2643)
Received Date : 06-Mar-2020
Revised Date : 22-Jun-2020
Accepted Date : 30-Jun-2020
Mechanistic characterization of UDP-glucuronic acid 4-epimerase
Running title: Mechanism of UDP-glucuronic acid 4-epimerase
Annika J. E. Borg[a], Alexander Dennig[a,b], Hansjörg Weber[c] and Bernd Nidetzky[a,b]*
[a] Institute of Biotechnology and Biochemical Engineering, Graz University of Technology,
NAWI Graz, Petersgasse 12, 8010 Graz, Austria
[b] Austrian Centre of Industrial Biotechnology, Petersgasse 14, 8010 Graz, Austria
[c] Institute of Organic Chemistry, Graz University of Technology, NAWI Graz, Stremayrgasse 9,
8010 Graz, Austria
* Corresponding author (B.N., bernd.nidetzky@tugraz.at)
Keywords: SDR, short-chain dehydrogenase/reductase, epimerase, decarboxylase, UDP-
glucuronic acid, UDP-galacturonic acid, NADH, kinetic isotope effect
Conflict of interest: The authors declare no conflict of interest.
Abbreviations: KIE, kinetic isotope effect; SDR, short-chain dehydrogenase/reductase; UGAepi;
UDP-glucuronic acid 4-epimerase
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Abstract
UDP-glucuronic acid (UDP-GlcA) is a central precursor in sugar nucleotide biosynthesis and
common substrate for C4-epimerases and decarboxylases releasing UDP-galacturonic acid (UDP-
GalA) and UDP-pentose products, respectively. Despite the different reactions catalyzed, the
enzymes are believed to share mechanistic analogy rooted in their joint membership to the short-
chain dehydrogenase/reductase (SDR) protein superfamily: oxidation at the substrate C4 by
enzyme-bound NAD+ initiates the catalytic pathway. Here, we present mechanistic
characterization of the C4-epimerization of UDP-GlcA, which in comparison to the corresponding
decarboxylation has been largely unexplored. The UDP-GlcA 4-epimerase from Bacillus cereus
functions as a homodimer and contains one NAD+/subunit (kcat = 0.25 ± 0.01 s-1). The
epimerization of UDP-GlcA proceeds via hydride transfer from and to the substrate’s C4 while
retaining the enzyme-bound cofactor in its oxidized form (≥ 97%) at steady state and without trace
of decarboxylation. The kcat for UDP-GlcA conversion shows a kinetic isotope effect of 2.0 (± 0.1)
derived from substrate deuteration at C4. The proposed enzymatic mechanism involves a transient
UDP-4-keto-hexose-uronic acid intermediate whose formation is rate-limiting overall, and is
governed by a conformational step before hydride abstraction from UDP-GlcA. Precise
positioning of the substrate in a kinetically slow binding step may be important for the epimerase
to establish stereo-electronic constraints in which decarboxylation of the labile -keto acid species
is prevented effectively. Mutagenesis and pH studies implicate the conserved Tyr149 as the
catalytic base for substrate oxidation and show its involvement in the substrate positioning step.
Collectively, this study suggests that based on overall mechanistic analogy, stereo-electronic
control may be a distinguishing feature of catalysis by SDR type epimerases and decarboxylases
active on UDP-GlcA.
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Introduction
Uridine 5’-diphosphate α-D-glucuronic acid (UDP-GlcA) is a central intermediate at the
crossroads of diverse metabolisms, including the biosynthesis of essential monosaccharides (e.g.,
D-xylose) [1] and polysaccharides (e.g., pectin, alginate) [2], the biosynthesis of secondary
metabolites [3], and detoxification [4,5]. In plants and microorganisms, UDP-GlcA is the
immediate precursor of three important monosaccharides (D-galacturonic acid, D-xylose, D-apiose)
required in cell wall polysaccharide biosynthesis [6,7]. Besides their importance in cell biology,
the sugar nucleotide syntheses from UDP-GlcA are of considerable interest for mechanistic
enzyme research. Three enzymes (UDP-GlcA epimerase, UGAepi, EC 5.1.3.6; UDP-xylose
synthase, UXS, EC 4.1.1.35; UDP-apiose/xylose synthase, UAXS, EC 4.1.1.-) share a common
substrate in order to form distinct products from it [8-10]. The enzymes are evolutionary related by
common membership to the short-chain dehydrogenase/reductase (SDR) protein superfamily [11].
All belong to the extended subclass of SDRs that are distinct from the canonical SDR type in
having NAD+ coenzyme tightly bound to their structure [11-13]. Common feature of their
mechanism appears to be a transient UDP-4-keto-hexose-uronic acid intermediate which is formed
by proton abstraction from C4-OH together with hydride transfer to enzyme-NAD+ [13-19]. At
this stage, the catalytic paths of the individual enzymes diverge (Figure 1) [11]. Remarkably, only
UGAepi avoids decarboxylation of UDP-GlcA. Further, the enzyme is able to catalyze re-addition
of the hydride at the equatorial position of the C4 to achieve the D-glucose to D-galactose switch
in stereochemistry [20]. Little is known about determinants of the finely tuned reactivity in each of
these enzymes. While the reactions of UDP-xylose synthase (UXS) and UDP-apiose/xylose
synthase (UAXS) have been characterized mechanistically [13,17,21], the one of UDP-GlcA
epimerase (UGAepi) is yet unexplored for direct evidence in support of the central claims of
enzymatic mechanism.
A transient UDP-4-keto-hexose-uronic acid species in the UGAepi reaction is currently supported
solely by inference to the UXS and UDP-galactose 4-epimerase (GALE) reactions [13,16,22].
GALE, active on UDP-glucose (UDP-Glc), is mechanistically the best-characterized member of
the extended SDR family. GALE promotes a 180°-degree rotation of the formed 4-ketopyranose
intermediate around the phosphate backbone, therefore offering the opposite face of the sugar for
reduction by NADH [16,23]. A similar rotation has been assumed, although not shown, to be
involved in UGAepi reactions as well [22]. The central role of a highly conserved Tyr as the
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catalytic base for initiating the oxidation at C4 of the sugar has been proposed for UGAepi, based
on the similarity to the GALE reaction [22,24,25]. UGAepi has been described from plants and
bacteria in studies spanning several decades, with reported difficulties in recombinant production
[10,20,22,26-28]. The enzymes were often found to be quite unstable in isolated form, thus
complicating their detailed characterization.
Here, we report the identification of a new UGAepi from Bacillus cereus HuA2-4 (BcUGAepi).
The enzyme was conveniently produced in Escherichia coli (30 mg L-1) and proved quite robust
during isolation, storage and use. Thus, it was a practical candidate for the proposed mechanistic
study. We performed here a comprehensive biochemical and steady-state kinetic analysis of the
enzyme. We developed efficient enzymatic syntheses of UDP-GalA and UDP-4-[2H]-GlcA as
substrate for the reverse reaction of the epimerase and as mechanistic probe, respectively. Using
UDP-4-[2H]-GlcA and in situ real-time monitoring of the reaction by proton NMR, we
demonstrated that the overall net epimerization involves two stereospecific steps of hydride
transfer, from and to the sugar C4, and proceeds without loss of deuterium label from the substrate
in the product. We further used site-directed mutagenesis, kinetic isotope effects and pH studies to
obtain deepened insight into the enzyme mechanism. We provide evidence supporting catalytic
reaction via a transient UDP-4-keto-hexose-uronic acid intermediate. Reduction of the
intermediate is considerably faster than its formation. A kinetic isomerization of the enzyme-
substrate complex before the hydride abstraction from UDP-GlcA is rate-limiting. The
isomerization step arguably reflects a slow protein conformational change in which the reactive
groups on substrate and NAD+ are aligned with the catalytic groups on the enzyme. Based on
structural comparisons and mechanistic considerations, we put forth the idea that stereo-electronic
control is crucial in the epimerase reaction to protect the labile -keto acid species from
undergoing decarboxylation. Our study thus shows basic mechanistic analogy between SDR type
epimerases (UGAepi) and decarboxylases (UXS, AXS) active on UDP-GlcA and proposes
essential features of their mechanistic distinction.
Results
Identification and biochemical characterization of a new UGAepi
BLAST search against the known microbial UGAepis revealed an uncharacterized protein
(UniProt: J8BY31_BACCE) in Bacillus cereus HuA2-4 that was annotated as UDP-glucose 4-
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epimerase. Since the protein was similar to already known UGAepi enzymes, we wished to
examine its reactivity. Protein harboring C-terminal Strep-tag (referred to as BcUGAepi
throughout) was conveniently obtained from standard E. coli culture in a yield of 30 mg purified
protein/L (Figure S1). We showed with gel filtration that the isolated protein was a homodimer of
37-kDa subunits (Figure S2, S3). Absorbance spectrum indicated the presence of a protein-bound
nicotinamide cofactor, as expected (Figure S4). Using sensitive HPLC assay for detection (Figure
S5), we identified the cofactor as NAD, not NADP. We determined that the NAD was mainly
oxidized (NAD+). NADH was also present, the content varying slightly (4.3 ± 1.1% ; N = 3)
dependent on the enzyme batch used (Figure S5). The protein was stable during storage (-20°C; ≥
52 weeks) and under all assay conditions (pH 7.6; 23 °C) used later.
We then offered sugar nucleotides (1 mM; UDP-Glc, UDP-GlcA, UDP-Xyl) as substrates of the
putative epimerase (2 µM) and monitored their conversion with HPLC. UDP-Glc and UDP-Xyl
were not reactive above the detection limit (5 µM), irrespective of the addition of NAD+ (up to 1
mM) to the reaction. UDP-GlcA was a substrate and it was converted into a single product
detectable by HPLC (Figure 2a). Using authentic standard of UDP-GalA synthesized according to
herein developed procedure (see later), we confirmed the identity of the product and thus
demonstrated the enzymatic reactivity as epimerization at C4 of UDP-GlcA.
We show in Figure 2b the results of time-course study of conversion of UDP-GlcA. The reaction
proceeded to equilibrium whose position (Keq = [UDP-GalA]/[UDP-GlcA]) we determined as 2.0
± 0.1 (N = 10). We also showed that NAD+ (0 – 1000 µM) had no effect on the reaction rate nor
on the equilibrium position (Figure S6), indicating that the cofactor is tightly bound to the enzyme
during the recombinant expression. A specific activity for UDP-GlcA epimerization of 0.6 U/mg
was calculated from the data. The enzyme activity was unaffected by pH in the range 7.0 – 9.0. It
increased with temperature up to 37°C (Figure S7). However, to avoid convoluted effects of
temperature on enzyme and sugar nucleotide stability, we chose 23 °C for further experiments.
Besides demonstrating the enzymatic reactivity, our results also illuminate the substrate specificity
of the epimerase, showing that a carboxylate group at C5 of the UDP-sugar is required for
enzymatic function.
We further used proton NMR to monitor the epimerization directly from the enzymatic reaction
mixture in D2O solvent (pD 7.6). The conversion of UDP-GlcA into UDP-GalA was conveniently
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recorded from the change in the corresponding anomeric proton signals, as shown in Figure 2c.
The equilibrium ratio of UDP-GalA and UDP-GlcA was 2, consistent with results obtained in
H2O. The NMR data confirmed the results from HPLC analysis that UDP-GalA was the sole
product of the enzymatic reaction. Release of an intermediate, or of a potential side product
resulting from adventitious decarboxylation, was ruled out at level of approximately the enzyme
molarity used (⁓5 µM; HPLC).
Enzymatic synthesis of UDP-GalA and UDP-4-[2H]-GlcA
To characterize UGAepi kinetically, we required UDP-GalA as enzyme substrate for the reverse
direction of reaction. We considered previous protocol that had obtained UDP-GalA from
commercial -galacturonic acid 1-phosphate, using enzymatic reaction with UTP [29]. Since -
galacturonic acid 1-phosphate is no longer offered, we attempted its synthesis by anomeric
phosphorylation of GalA but were unsuccessful for lack of a suited kinase. In our hands, the GalA
kinase [30] suffered from poor stability in its isolated form and showed insufficient activity with
GalA. We considered isolation of UDP-GalA from an epimerase reaction mixture at equilibrium,
however, preparative HPLC separation of UDP-GalA from UDP-GlcA proved impractical due to
the low yield (1 mg) obtained. We improved on the isolation efficiency by exploiting the enzyme
ArnA for selective conversion of UDP-GlcA into UDP-4-keto-xylose (Scheme S1) [31,32]. The
ArnA does not utilize UDP-GalA within limits of detection. The UDP-GalA is conveniently
separated from UDP-4-keto-xylose, thus enabling its isolation in high purity (≥ 98%) and in
amounts of 10 - 50 mg (Figure S8, S9). The identity of UDP-GalA was confirmed by HPLC and
NMR (Figure S10, S11).
The UDP-4-[2H]-GlcA was prepared as mechanistic probe to analyze hydrogen transfer steps of
the overall C4 epimerization of UDP-GlcA. The synthetic route was effectively that reported
earlier [33,34], except that regeneration of NAD+ for two-fold oxidation of UDP-glucose was done
using the pyruvate/L-lactate dehydrogenase system [35]. This seemingly small change in
procedure was however crucial to enhance the efficiency of the key oxidative step catalyzed by
UDP-glucose dehydrogenase (Scheme S2). The efficient NAD+ regeneration system also allowed
higher substrate concentration (up to 30 mM) while retaining full conversion to the desired
product. UDP-4-[2H]-GlcA was obtained in high purity (≥ 99.5%) and in amounts of 40 - 50 mg
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(Figure S12, S13). The yield from 4-[2H]-glucose was 50%. The identity of UDP-4-[2H]-GlcA
was confirmed by HPLC and NMR (Figure S14, S15). The [2H] content at C4 was 99% or greater.
Kinetic characterization of UGAepi
Initial rates of C4 epimerization were recorded using UDP-GlcA and UDP-GalA as the substrate.
Time courses of substrate consumption were linear in the early phase of the reaction (Figure 2b,
S16) and there was a closed balance between substrate utilized and product formed at all
conditions used. Kinetic parameters (kcat, Km) were obtained from nonlinear fits of regular
hyperbolic dependencies of the initial rate upon the substrate concentration, as shown in Figure
S17. The Km was 0.36 ± 0.02 mM for UDP-GlcA and 0.89 ± 0.13 mM for UDP-GalA. The kcat was
0.25 ± 0.01 s-1 for UDP-GlcA and 0.32 ± 0.02 for s-1 for UDP-GalA. The catalytic efficiencies
(kcat/Km) for forward (0.71 mM-1 s-1) and reverse (0.35 mM-1 s-1) epimerization are used to
calculate the Keq from the Haldane relationship for the reaction [36]. The kinetically determined
Keq of 2.0 is in excellent agreement with the equilibrium constant measured directly. Kinetic
parameters for the forward epimerization agree between this epimerase and other such enzymes of
microbial and plant origin [20,22,26,27]. However, this is the first full kinetic study of a UDP-
GlcA 4-epimerase at steady state.
pH studies of wild-type enzyme and Y149F variant
The canonical active site of SDR enzymes involves an invariant tyrosine whose canonical role in
catalysis is that of a general base to facilitate alcohol oxidation by NAD+ [11]. Sequence
comparison with the well-studied UDP-Gal 4-epimerase revealed Tyr149 as the candidate residue
in UGAepi (Figure S18). We used replacement with phenylalanine to disrupt the catalytic
functionality of the original tyrosine. The purified Y149F variant (Figure S19) contained protein-
bound NAD+ as the wild-type enzyme. As anticipated from the proposed role of Tyr149 in
catalysis, the Y149F variant was highly impaired in epimerase activity that was about 1.2  103-
fold lower than the wild-type activity (Figure S20). Nonetheless, the Y149F variant converted
UDP-GlcA into UDP-GalA without trace formation of side products (e.g., due to decarboxylation)
or intermediates. Note: we rigorously excluded contamination by the wildtype enzyme during
purification of the Y149F variant (see Materials and methods). In addition, the triplet codon
exchange (TAC  TTC) used for mutagenesis made translational errors during incorporation of
Phe149 into the Y149F variant extremely unlikely [37-40]. Generally, removal of base catalytic
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assistance to the reaction does not, by necessity, lead to a complete loss of activity in the variant
enzyme. Other factors effective in catalysis (e.g. positioning) might suffice to reduce the energetic
barrier such that reaction can still occur at a detectable rate [41]. To further address the question of
acid-base catalysis in UGAepi, we carried out pH studies with wild-type enzyme and Y149F
variant. Results are shown in Figure 3.
The pH dependence of the wild-type enzyme was sigmoidal with low and high levels of activity
(kcat) at low and high pH, respectively. Model for a mechanism with a single ionizable group that
must be unprotonated for enzyme activity was used to fit the data [42]. An apparent pKa of 5.90 (±
0.05) was calculated. The Y149F variant was inactive at pH 6.5 or lower. Activity was found at
pH 7 and the logarithmic kcat increased linearly with pH, showing a slope of 0.85 (±0.07). These
results suggested that the Y149F variant might exploit alternative forms of base catalysis, perhaps
from H2O/OH- or from another enzyme residue, as proposed for an analogous tyrosine-to-
phenylalanine variant of GALE [24,43]. They provide strong evidence in support of a catalytic
base function of Tyr149 in the wild-type UGAepi.
Isotope-labeling studies and kinetic isotope effects
To explore the epimerization mechanism used by the enzyme, we analyzed with in situ NMR the
conversion of UDP-4-[2H]-GlcA catalyzed by wild-type UGAepi. We show in Figure S21 that
deuterium label at the C4 of substrate was retained completely at the C4 of UDP-GalA product.
The overall epimerization is thus shown to consist of two stereospecific steps of hydrogen transfer:
abstraction (to NAD+) of the axial C4 hydrogen/deuterium in the D-gluco configuration and
transfer back to C4 (from NADH) in an equatorial position to establish the D-galacto
configuration. The reaction proceeds through a transient UDP-4-keto-hexose-uronic acid
intermediate that is not released from the enzyme.
Having determined that epimerization involves cleavage and formation of C4-H bond, we
analyzed the effect of deuterium labeling at C4 on the enzymatic reaction rates of wild-type
UGAepi and Y149F variant. We showed that the apparent kcat for the wild-type reaction with
UDP-4-[2H]-GlcA was 2.0 (± 0.1)-fold (N = 5) slower than the kcat for the corresponding reaction
with unlabeled substrate (prepared identically as the isotopically labeled one). This demonstrates
D
that the reaction is subject to a normal primary kinetic isotope effect (KIE). The KIE (= kcatH/kcat
)
was independent of pH in the range 6.0 – 9.5. At pH 5.0, the KIE was 3.5 (± 0.3). The
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corresponding KIE for reaction of the Y149F variant was dependent on pH and increased from a
value of 1.17 (± 0.01) at pH 7.0 to higher values of 2.39 (± 0.09) at pH 8.0 and 3.53 (± 0.02) at pH
9.0.
The relative rates of the enzymatic half-reactions leading to and from the UDP-4-keto-hexose-
uronic acid intermediate determine the reduced-state portion of the nicotinamide cofactor at steady
state. Absorbance and fluorescence are useful to detect the enzyme-bound NADH, but the data are
challenging to quantify. We therefore measured directly with a rapid-quenching HPLC assay the
relative NADH content in the wild-type enzyme while reacting with UDP-GlcA or UDP-4-[2H]-
GlcA. The NADH content normalized on the total amount of cofactor present was 0.014 and 0.016
in reactions with unlabeled and labeled substrate, respectively (Figure S22, S23). It was not
different within limits of error from the relative NADH content in the enzyme as isolated (0.031)
(Figure S5).
Discussion
Functional annotation of UDP-GlcA epimerases
Phylogenetic analysis places UDP-GlcA epimerases into a distinct subgroup within the sugar
nucleotide epimerase family of SDR enzymes (Figure S24). The UDP-pyranose 4-epimerases
(GALE, UDP-GlcNAc 4-epimerase) [44-46] and CDP-D-paratose 2-epimerases [47,48] constitute
further subgroups of that family. The UDP-GlcA epimerases are separated from UDP-GlcA
decarboxylases (UXS, UAXS, ArnA) that represent a different SDR family. They are also clearly
separated from two-site sugar nucleotide epimerases such as GDP-D-mannose 3,5-epimerase
(Figure S24) [49] as well as from sugar nucleotide dehydratases (not shown). The UDP-GlcA
epimerases are further subdivided according to phylogenetic origin and specificity. Besides the
main 4-epimerase found in plant and bacterial subgroups, a second bacterial subgroup additionally
contains 5-epimerase that converts UDP-GlcA into UDP-L-IdoA [50]. However, a recent
computational analysis suggested that these 5-epimerases might in fact all be UDP-GlcA 4-
epimerases [51]. Studies of UGAepi substrate specificity show that the C5 carboxylate group of
UDP-GlcA is essential for substrate binding recognition and/or catalysis. UDP-Glc, UDP-Gal,
UDP-GlcNAc and UDP-Xyl are often not accepted as substrates by UGAepis [20,26,27] or give
strongly decreased activity [22].
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Although basic functional attributes are assignable from the sequence-based categorization,
specific correlations between molecular structure and enzymatic reactivity remain elusive in the
absence of detailed mechanistic-kinetic analysis. Except for the Tyr/Ser/Lys catalytic triad of
residues that are universally conserved, the residues involved in sugar binding are highly variable.
There is effectively very little conservation in UGAepi of the relevant sugar-binding residues of
UDP-pyranose 4-epimerases [45,52], as presented with an example of GALE in Figure S25. The
epimerase and the decarboxylase reaction both face the challenge of having to combine specificity
in substrate recognition with flexibility in the positioning of the respective 4-keto-intermediate
(Figure 1). Each mechanism additionally requires that the different chemical steps of the
enzymatic reaction are precisely coordinated with at least one physical step of
reorientation/repositioning of the 4-keto-intermediate. Particular task of UGAepi is to handle the
highly reactive UDP-4-keto-hexose-uronic acid species for epimerization which in the
corresponding decarboxylases is immediately taken further to the UDP-4-keto-pentulose
intermediate via decarboxylation [13,14,17]. Only little can be inferred from the well-
characterized epimerases (GALE) and UDP-GlcA decarboxylases (UXS, AXS) as to the particular
catalytic strategy employed by UGAepi, thus necessitating the current study.
Kinetic properties of UGAepi
Despite their overall structural similarity and mechanistic analogy, UGAepi and GALE show
interesting differences in the observable kinetic behavior. The proposed reaction pathway for
UGAepi is shown in Figure 4.
This pathway may be largely identical with that of GALE [46] but differs from it in respect to the
kinetic significance of the reductive half-reaction, leading from the 4-keto-intermediate to the D-
galacto configured product. The GALE reaction with UDP-glucose was shown to involve 19%
enzyme-NADH of total enzyme, hence enzyme-bound 4-keto-intermediate, at the steady state
[53,54]. This indicates that both half-reactions of the GALE mechanism proceed at rates of the
same magnitude order. Absence of enzyme-NADH in the UGAepi reaction with UDP-GlcA
shows that effectively no 4-keto-intermediate is accumulated on the enzyme at the steady state.
Two kinetic scenarios are consistent with enzyme-NADH below detection in the catalytic reaction:
(1) The overall sequence of steps leading to enzyme-NADH is rate-limiting. (2) Release of the
UDP-GalA product from enzyme-NAD+ is rate-limiting. The KIE of 2.0 is in accordance with the
first scenario while it refutes the second for which no isotope effect (KIE = 1) is expected.
UGAepi reaction with hydride transfer to NAD+ as the rate-limiting step is expected to show a
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sizeable KIE on kcat (≥ 3.5), substantially larger than the one observed [55-57]. Plausible
explanation is that the enzymatic mechanism involves a slow physical step, most likely a
conformational rearrangement of the enzyme/NAD+/UDP-GlcA complex (Figure 4), that precedes
the hydride transfer step; and this physical step suppresses the intrinsic KIE on the hydride transfer
step to the one effectively measurable in kcat. The proposed precatalytic step is further supported
by evidence from pH studies of wild-type enzyme and Y149F variant. At low pH, where the kcat of
wild-type UGAepi decreases, the KIE increases to a value of 3.5 (pH 5.0). This result suggests that
the hydride transfer step of the enzymatic mechanism requires a group of enzyme/NAD+/UDP-
GlcA to be unprotonated and therefore, is dependent upon the pH. It further suggests that an
additional pH-independent step (the proposed conformational rearrangement) partly limits the kcat
at the pH optimum. Effect of substitution of Tyr149 by Phe on the pH dependencies of the kcat and
its associated KIE suggests that Tyr149 is part of the ionizable molecular group (apparent pKa =
5.9) critical for the hydride transfer; and the tyrosine is also involved in the precatalytic physical
step whose probable role is to align precisely the reactive groups in enzyme/NAD+/UDP-GlcA
complex (Figure 4). By analogy with the reaction of GALE, the conformational rearrangement can
also be important to increase the redox potential of the enzyme-bound NAD+ through favorable
change in electrostatic environment [58,59]. We note furthermore that pH studies of GALE have
also pointed to an unusually low pKa (= 6.9) for the catalytic tyrosine [43].
UGAepi: stereo-electronic control for catalytic epimerization under prevention of
decarboxylation
How then does UGAepi prevent decarboxylation of the labile -keto-acid moiety of the transient
4-keto-intermediate? The combined evidence from kinetic analysis and structural comparison
(discussed below) suggests an important role for stereo-electronic control in enzyme catalysis to
the epimerization of UDP-GlcA (for general case, see ref. 60). Using structure-based, multiple
sequence alignment (Figure 5a), we show that residues involved in binding of the substrate’s
carboxylate group differ between UGAepis and UDP-GlcA decarboxylases (UXS, UAXS, ArnA).
Crystal structure of MoeE5 [28] provides the essential structural basis for UGAepi and a
homology model of BcUGAepi shows excellent agreement with the experimental template (Figure
5b). In UGAepi, the substrate carboxylate is embedded in a hydrogen bond network of four
coordinating residues, namely Thr126, Ser127, Ser128 and Thr178 (main chain). In the
decarboxylases (UAXS, Figure 5c; UXS, Figure S26a; ArnA, Figure S26b), the carboxylate group
appears to be more loosely bound. The overall structure of the binding pocket is changed to
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provide fewer interactions (compare Figure 5c to Figure 5b); and a binding residue (Ser128) of the
epimerase is replaced by an acidic residue (Glu; Figure 5a), known from earlier studies of UXS
and UAXS to play an important role in the decarboxylase catalytic mechanism. In the epimerase,
the threonine from the SDR catalytic triad (Thr126 in BcUGAepi) is in hydrogen bond distance to
both the C5 carboxylate and the reactive 4-hydroxy group of the substrate. By way of comparison
(Figure S27), the 6-OH of UDP-Glc in E. coli GALE has just two coordinating residues (Asn179
and Tyr299) and is oriented away from Ser124 (the positional/functional homologue of Thr126 in
BcUGAepi). The unique substrate binding mode in UGAepi gives rise to the suggestion that the
carboxylate of UDP-GlcA not only serves as a recognition site for binding, but it can also have an
immediate involvement in substrate positioning for the catalytic event.
Analyzing the epimerase-bound conformation of UDP-GlcA in more detail, we notice that the
4
pyranose moiety adopts an undistorted C1 ring pucker and has the carboxylate group in a
perfectly equatorial position, as shown in Figure 6a. Based on stereo-electronic considerations [61-
64], decarboxylation of the 4-keto intermediate would be optimal with the carboxylate group
oriented axially, for the dihedral angle between the C=O bond of the ketone and the C5-C6 bond
could thus approach the ideal 90°. It follows that, by maintaining the dihedral angle at close to
0° due to the equatorial carboxylate, the epimerase might constrain its catalytic reaction so as to
minimize decarboxylation. To achieve this, the epimerase has to limit sugar ring distortion in its
active site, which is noteworthy in light of chemical studies showing that distortion from chair
conformation influences the reactivity of 2-oxocyclohexane carboxylic acid isomers for
decarboxylation in solution [61,62]. The appealing idea of differential stereo-electronic control in
epimerase and decarboxylase reactions receives additional support from mechanistic studies of
UXS and UAXS. In UXS, as revealed by molecular dynamics simulations, the enzyme-bound
UDP-GlcA features a binding energy-driven sugar ring distortion (4C1  BO,3) that forces the
carboxylate group into an almost fully axial position (Figure 6b) [13]. Thus, the UXS Michaelis
complex provides optimal stereo-electronic conditions for immediate decarboxylation subsequent
to the oxidation at C4. The UAXS reaction exploits substrate oxidation at C4 to promote retro-
aldol ring opening between C2 and C3 and the decarboxylation happens only after the ring
opening has occurred [17]. Stereo-electronic conditions in the active site of UAXS meet
requirements for the particular timing of the catalytic steps in the enzymatic conversion. The
Michaelis complex involves a largely undistorted UDP-GlcA substrate that has the carboxylate in
a closely equatorial position (Figure 6c). As suggested from molecular dynamics simulation, the
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ring-opened conformation of the 4-keto-intermediate will only change later during the reaction to
move the carboxylate into a quasi-axial position suitable for decarboxylation [17]. The special
importance of stereo-electronic control for 4-keto-intermediate protection in the UGAepi reaction
is emphasized by the catalytic behavior of active-site variants of UXS and UAXS which despite
showing only trace activity lack the ability to uncouple substrate oxidation at the C4 from
decarboxylation at C5 [13,14,17].
Central task of catalysis by SDR-type epimerases is to position their keto-intermediates flexibly
for non-stereospecific reduction. Studies of GALE show that the requirement can be realized
through a relatively weak binding of the intermediate’s sugar moiety, compared to binding of its
UDP moiety [25]. Therefore, mechanistic conundrum posed for UGAepi is to establish proper
balance between stereo-electronic control, due to a relatively more constrained substrate binding
compared to GALE (compare Figure 5b and Figure S27), and free rotation of the 4-keto hexose
intermediate. It can be significant, therefore, that in terms of their kcat the known UGAepis (this
work, 0.25 s-1; B. cereus isozyme, 3.2 s-1, [20]; Arabidopsis thaliana, 24 s-1, [27]) are much slower
enzymes than GALE (760 s-1 [24]). Added binding interactions with the UDP-sugar substrate in
,
UGAepi compared to GALE may be responsible for the slowing down of the catalytic reaction.
Figure 4 shows the particular distribution of fluxes across the reaction pathway of BcUGAepi. The
conformational restraint imposed by the enzyme on the rate of 4-keto-intermediate formation
(which also limits the kcat) might reflect the structural requirements of the active site to implement
the proposed stereo-electronic control. Rotation of the 4-keto-hexose-uronic acid intermediate
bound to enzyme-NADH is part of the sequence of microscopic steps involved in the reduction of
the 4-keto-intermediate to UDP-GalA (kred in Figure 4). Although considerably faster than the
enzyme’s kcat, the rotation step in BcUGAepi could still be appreciably slower than the analogous
rotation step is in GALE. To keep a tight “grip” on the equatorial C5 carboxylate group during a
comparably slow rotation may be important for BcUGAepi to retain stereo-electronic control
during conversion of the 4-keto intermediate into UDP-GalA.
In summary, this study provides biochemical foundation for enzymatic C4 epimerization in UDP-
GlcA. The proposed catalytic mechanism involves a transient UDP-4-keto-hexose-uronic acid
intermediate whose formation and conversion via hydride transfer to and from enzyme-bound
nicotinamide coenzyme is facilitated by Tyr149 acting as the general base/acid, respectively.
While largely analogous to the GALE mechanism in its base features, the UGAepi mechanism is
special in requiring the handling of a highly decarboxylation-prone 4-keto-intermediate. Our study
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suggests stereo-electronic control as the catalytic strategy developed by UDP-GlcA 4-epimerase to
achieve this task. Precisely orchestrated structural dynamics will be essential in their
implementation. Importantly, stereo-electronic control provides an essential mechanistic
distinction between UDP-GlcA 4-epimerase and SDR type decarboxylases using UDP-GlcA as
the substrate.
Materials and methods
Materials
The synthetic genes of BcUGAepi and ArnA were ordered in pET17b-expression vector
(pET17b_BcUGAepi; pET17b_ArnA) from GenScript (USA). Uridine monophosphate (UMP,
98% purity) was from Carbosynth (Compton, UK). Uridine diphosphate (UDP), UDP-D-
glucuronic acid (UDP-GlcA; >98% purity) and sodium pyruvate were from Sigma-Aldrich
(Vienna, Austria). NAD+ (>98% purity) was from Roth (Karlsruhe, Germany). Deuterium oxide
(99.96% 2H) was from Euriso-Top (Saint-Aubin Cedex, France). All other reagents and chemicals
were of highest available purity. For plasmid DNA isolation, the GeneJET Plasmid Miniprep Kit
(Thermo Scientific; Waltham, MA, USA) was used. DpnI and Q5® High-Fidelity DNA
polymerase were from New England Biolabs (Frankfurt am Main, Germany). Hexokinase and D-
lactate dehydrogenase were from Megazyme (Vienna, Austria). All other enzymes were prepared
in-house. Oligonucleotide primers were from Sigma-Aldrich (Vienna, Austria). E. coli NEB5α
competent cells were from New England Biolabs (Frankfurt, Germany). E. coli Lemo21(DE3)
cells were prepared in-house.
Enzymes
The site-directed substitution Tyr149 by Phe was introduced by a modified QuikChange protocol
(see SI for details). Both BcUGAepi wild-type and Y149F variants were produced by expression
in E. coli Lemo21 (DE3) cells that harbored the pET17b-expression vector containing the gene of
interest. The proteins were purified utilizing the C-terminal Strep-tag, and the size and purity of
the proteins were confirmed by SDS-PAGE. For purification of the Y149F variant, new Strep-tag
columns were used to avoid any possible contamination by the wild-type enzyme. Full details of
the expression and purification conditions used are given in the SI. ArnA was obtained by
expression in E. coli BL21 (DE3) harboring the pET17b_ArnA vector using the same expression
and purification procedure as for BcUGAepi.
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Human UDP-glucose 6-dehydrogenase (hUGDH) was expressed in E. coli Rosetta 2 (DE3) cells
harboring the vector pBEN_SGC_hUGDH [65]. Inorganic pyrophosphatase (iPPase) was
produced by expression in E. coli BL21 (DE3) cells carrying pET-STREP3_iPPase vector. UDP-
glucose pyrophosphorylase (UGPase) was expressed in E. coli BL21 (DE3) Gold cells harboring
pET30_UGPase plasmid. hUGDH and iPPase were purified by affinity chromatography using
Strep-tag following the same procedure as for BcUGAepi, UGPase was isolated by His-tag
purification (see the SI for details). The activity of iPPase was determined as described in
literature [66], the activity measurement of UGPase is described in the SI.
Substrate synthesis
UDP-α-D-galacturonic acid
The reaction mixture (20 ml) contained 4.6 mM UDP-GlcA (60 mg, 0.09 mmol), 1 mM NAD+
(13.3 mg, 0.02 mmol) and 2.7 µM BcUGAepi (0.1 mg/ml, 2 mg) in buffer (50 mM Na2HPO4, 100
mM NaCl, pH 7.6). Incubation was at 30 °C until UDP-GalA and UDP-GlcA were present at the
equilibrium ratio of 2:1, respectively, confirmed by HPLC. BcUGAepi was removed by
ultrafiltration (Vivaspin Turbo centrifugal filter tube, 30 kDa cut-off). The remaining UDP-GlcA
was converted to UDP-α-D-4-keto-xylose (Scheme S1) by adding the following components into
the reaction mixture: 10 mM sodium pyruvate (22 mg, 0.2 mmol), 20 U/ml LDH (400 U) and 4.7
µM ArnA (0.2 mg/ml, 2.8 mg). The reaction mixture was incubated at 30 °C for 16 h until all
UDP-GlcA was consumed. ArnA and LDH were removed from the mixture by ultrafiltration (30
kDa cut-off).
UDP-α-D-4-2H-glucuronic acid
The synthesis strategy is indicated in Scheme S2. The reaction mixture (37 ml) contained 15 mM
4-2H-α-D-glucose (100 mg, 0.55 mmol), 50 mM uridine 5’-triphosphate (UTP, 716.5 mg, 1.3
mmol), 5 mM MgCl2 (17.6 mg, 0.18 mmol), 0.13% (w/v) bovine serum albumin (BSA, 48.1 mg)
and 10 µM α-D-glucose 1,6-bisphosphate dissolved in 50 mM Tris/HCl buffer (pH 7.5).
Hexokinase (13.8 U/ml, 510.6 U), 6.8 U/ml phosphoglucomutase (251.6 U), 0.2 mg/ml UGPase
(7.4 mg) and 0.077 mg/ml iPPase (2.8 mg) were added and the reaction mixture was incubated at
30 °C for 20 h. After confirming the completeness of the reaction on HPLC, the final oxidation
step towards UDP-4-2H-GlcA was initiated by the addition of 2 mM NAD+ (49 mg, 0.074 mmol),
40 mM sodium pyruvate (163 mg, 1.48 mmol), 20 U/ml LDH (740 U) and 1.23 mg/ml hUGDH
(45.5 mg), and the reaction was incubated at 37 °C for 24 h. The enzymes were removed with
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Vivaspin Turbo centrifugal filter tubes (10 kDa cut-off, Sartorius) prior to column purification of
the nucleotide sugar.
Product isolation
Both UDP-α-D-galacturonic acid and UDP-α-D-4-2H-glucuronic acid were isolated by two column
chromatographic purification steps. First the desired product was separated from the other
components using ÄKTA FPLC system (GE Healthcare) connected to a 125 ml TOYOPEARL
SuperQ-650M anion exchange column (GE Healthcare) and a 10 ml sample loop. 20 mM sodium
acetate solution (pH 4.3) was used as binding buffer. Compounds bound to the column were eluted
with a step-wise gradient of 1 M sodium acetate buffer at pH 4.3, see SI for details. Fractions
containing the desired product were detected via UV absorption (ʎ = 254 nm), identified by HPLC
and combined prior to concentrating under reduced pressure on a Laborota 4000 rotary evaporator
(Heidolph, Schwabach, Germany) at 40 °C to a final volume of approximately 5 to10 ml. NaOAc
was removed from the concentrated product preparation using an ÄKTA FPLC connected to
Superdex G-10 size-exclusion column (GE Healthcare) and 5 ml sample loop. The product was
detected based on its UV absorption at 254 nm and eluted with deionized water. The product-
containing fractions were combined and concentrated under reduced pressure at 40 °C to a final
volume of approximately 20 ml. Residual H2O was removed by lyophilization (Christ Alpha 1-4
lyophilizer, B. Braun Biotech International, Melsungen, Germany), and the pure nucleotide sugar
obtained as white powder. The purity and identity of UDP-α-D-galacturonic acid and UDP-α-D-4-
2H-glucuronic acid were determined on HPLC and 1H-NMR.
Determination of enzyme-bound coenzyme
The dimeric oligomeric state of BcUGAepi wild-type was determined by gel filtration
chromatography (see SI for details).
Enzyme-bound NAD+ / NADH
For studying the occupancy of BcUGAepi by NADH, the enzyme (50 µl, 18 mg/ml) was
denatured by incubating with 50 µl of methanol for 3 h at room temperature. The enzyme
precipitation was removed by centrifugation (16100 g, 80 min, 4 °C) and the supernatant analyzed
on HPLC. The amount of released NADH was calculated based on a calibration curve (Figure
S22), where defined concentrations of NADH were prepared in double distilled water and directly
used for measurements. UV-absorbance at 262 nm was used for detection of NAD+ and NADH.
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The occupancy of NADH in BcUGAepi was calculated in % from the concentration of released
NADH (in µM) divided by the total protein concentration (in µM) used in the experiment.
Rapid-quench procedure to analyze enzyme-bound NADH from reaction
Reaction mixture containing 1 mM UDP-GlcA or UDP-4-2H-GlcA and 1 µM (0.035 mg/ml) of
purified BcUGAepi was prepared in 10 ml of sodium phosphate buffer (50 mM Na2HPO4, 100
mM NaCl, pH 7.6). The reaction was run for 2 min at 23 °C and afterwards 10 ml of ice-cold
phosphate buffer (50 mM Na2HPO4, 100 mM NaCl, pH 1.3) was added to decrease the pH of the
reaction mixture to 4.4 and terminate the enzymatic activity, while retaining the enzyme soluble.
The reaction mixture was immediately transferred into an ice-cold Vivaspin Turbo centrifugal
filter tube (30 kDa cut-off) and centrifuged at 0 °C (2880 g) until 19 ml of the mixture had flowed
through the filter. The flow through was analyzed on HPLC and the enzyme was washed twice
with 5 ml of ice-cold phosphate buffer (50 mM Na2HPO4, 100 mM NaCl, pH 4.4). The flow
through from each washing step was analyzed on HPLC to confirm that the UDP-GlcA/UDP-
GalA ratio remained unchanged over the washing procedure. The enzyme was concentrated up to
the final volume of 50 µl and denatured with 50 µl of MeOH as described in the section “Enzyme-
bound NAD+ / NADH”. The NADH released from the active site of BcUGAepi was detected on
HPLC. As a control for the stability of NADH, the same procedure was performed for NADH
standard solution to confirm that the coenzyme was not degrading during the denaturation
experiment.
BcUGAepi activity assays
Standard BcUGAepi reaction mixtures (250 µl final volume) contained 1 mM UDP-GlcA,
100 µM NAD+ and 2 µM (0.07 mg/ml) purified recombinant BcUGAepi (135 µM / 5 mg/ml for
variant Y149F) in sodium phosphate buffer (50 mM Na2HPO4, 100 mM NaCl, pH 7.6). The
reactions were incubated at 23 °C, quenched with methanol (50% (v/v) final concentration) at
desired time points and the precipitated enzyme removed by centrifugation (16100 g, 4 °C, 30
min) prior to HPLC analysis. The initial rates were determined from the linear part of the time-
course by dividing the slope of the linear regression (mM/min) by the enzyme concentration
(mg/ml) giving the initial rate in µmol/(min mg protein). The apparent kcat values (s-1) were
calculated from the initial rate with the molecular mass of the functional enzyme monomer (wild-
type BcUGAepi: 37003 g/mol; Y149 variant: 36987 g/mol). For the temperature optimum study,
the reactions were performed under standard conditions but in varying temperatures. For NAD+
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concentration study the reactions were carried out under standard conditions except that lower
enzyme concentration (1 µM, 0.037 mg/ml) was used.
For determination of the kinetic parameters, BcUGAepi (2 µM, 0.07 mg/ml) was mixed with
NAD+ (50 µM) in 50 mM Na2HPO4 buffer (containing 100 mM NaCl, pH 7.6). Varying
concentrations of substrate (UDP-GlcA/UDP-GalA) were added into the 100 µl reaction mixture
at room temperature and the reactions were quenched after 10 min incubation time with 25% (v/v)
acetonitrile and heated up to 95 °C for 5 min. Each reaction was carried out in duplicate. After
centrifugation (16100 g, 4 °C, 30 min) the samples were analyzed on HPLC and the initial rates
were plotted against the substrate concentration used. The kinetic parameters were calculated with
SigmaPlot 10.0 based on fitting to the Michaelis-Menten kinetic model. The kcat is based on the
molarity of the enzyme subunit.
In situ 1H NMR
For in situ NMR experiments, the reaction was carried out in D2O buffer (50 mM
K2HPO4/KH2PO4) titrated to pD 7.6, where pD corresponds to the reading of pH meter + 0.4. The
reaction mixture contained 2.7 mM UDP-GlcA (or UDP-4-2H-GlcA), 91 µM NAD+ and 2.4 µM
(0.09 mg/ml) purified recombinant BcUGAepi. The acquisition was carried out on a Varian
INOVA 500-MHz NMR spectrometer (Agilent Technologies, Santa Clara, California, USA) every
1
30 s from the enzyme addition. The VNMRJ 2.2D software was used for the measurements. H-
NMR spectra (499.98 MHz) were recorded on a 5 mm indirect detection PFG-probe with pre-
saturation of the water signal by a shaped pulse. The spectra were analyzed using MestReNova
16.0 (Mestrelab Research, S.L.).
pH studies and kinetic isotope effects
All the experiments were performed with 1 mM UDP-GlcA. No significant increase in activity
was observed if higher substrate concentration was used.
pH studies
For pH optimum studies (at 23 °C) the reaction buffer (50 mM Na2HPO4, 100 mM NaCl) was
prepared in desired pH values (pH 3-9) and BcUGAepi wild-type was confirmed as stable over
this pH range. The pH was adjusted either with 5 mM NaOH or with 10% HCl. UDP-GlcA (1
mM), NAD+ (100 µM) and BcUGAepi (2 µM, 0.07 mg/ml) were mixed with the buffer and
samples were taken for HPLC analysis at defined time points from 0 to 150 min.
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The initial rates (s-1) were calculated at each pH value and plotted against the pH. The data for the
BcUGAepi wild-type was fitted to an equation (2) where logkcat increases with a slope of +1 below
pK and is level above pK. [42]
퐶
logkcat = log
(2)
[
]
+
[
]
1 + 퐻 /퐾
where C is the pH independent value of kcat, [H+] is the proton concentration and K is a proton
dissociation constant.
For Y149F variant the data was fitted using standard linear regression.
Kinetic isotope effect measurements
For kinetic isotope effect measurements BcUGAepi concentration of 0.037 mg/ml (1 µM) was
used and the reactions (30 µl final volume) were performed in quintuplicates for both UDP-GlcA
and 4-2H-UDP-GlcA (1 mM). The substrate concentration used was saturating. The KIE reaction
mixtures were quenched (incubation in 50% (v/v) methanol at 90 °C for 5 min) after 5 min and
analyzed on HPLC. Initial reaction rates (V) were calculated from the linear dependence of the
product formed and time used. The KIE was calculated from the ratio of the reaction rates
(effectively, kcat) for the unlabeled and deuterium-labeled substrate (KIE = Vunlabeled/Vlabeled).
Analytics
HPLC
The sugar nucleotides and NAD+/NADH were separated with Shimadzu Prominence HPLC-UV
system (Shimadzu, Korneuburg, Austria) on a Kinetex C18 column (5 µm, 100 Å, 50 x 4.6 mm)
using an isocratic method with 5% acetonitrile and 95% tetrabutylammonium bromide buffer (40
mM TBAB, 20 mM K2HPO4/KH2PO4, pH 5.9) as mobile phase. UDP-sugars, NAD+ and NADH
were detected by UV at 262 nm wavelength. The amount of UDP-GlcA/UDP-GalA formed was
determined based on the relative integrated peak areas and referred to a calibration curve of a
commercial standard of UDP-GlcA.
NMR
1
The purity and identity of the synthesized compounds was determined by H NMR. All the
acquisitions were carried out in D2O on a Varian INOVA 500-MHz NMR spectrometer (Agilent
Technologies, Santa Clara, California, USA).
Author contributions
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The experimental work included in this paper has been performed by A.J.E.B. A.J.E.B. and H.W.
performed the NMR analyses. A.D. contributed in designing the synthesis route for UDP-GalA.
B.N. designed and supervised the research. B.N. and A.J.E.B. wrote the paper.
Acknowledgements
Financial support from the Austrian Science Fund FWF (project number I 3247) is gratefully
acknowledged. The authors thank Dr. Mario Klimacek and Dr. Manuel Eibinger for assistance in
fitting the pH and kinetic data.
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Legends to Figures
Figure 1. Simplified reaction pathways for UDP-apiose/xylose synthase (UAXS), UDP-xylose
synthase (UXS) and UDP-glucuronic acid 4-epimerase (UGAepi). The routes leading to
decarboxylation are highlighted in red (UAXS) and blue (UXS) and position of CO2 is assigned
with a yellow circle. Orientation of the C4-hydroxyl group in the products is equatorial in case of
UDP-xylose (UAXS, UXS) and axial in UDP-GalA (UGAepi). UDP-GlcA = Uridine
diphosphate glucuronic acid, UDP-GalA = Uridine diphosphate galacturonic acid.
Figure 2. Analysis of the reaction of BcUGAepi a. Achiral HPLC analysis of the reaction mixture
of BcUGAepi with UDP-GlcA as substrate (sample at equilibrium state) is shown in blue. HPLC
chromatogram of a control reaction (without enzyme) is shown in black. b. Time course of
BcUGAepi reaction with UDP-GlcA as a substrate. The reaction mixture contained 1 mM UDP-
GlcA, 100 µM NAD+ and 2 µM (0.07 mg/ml) purified recombinant BcUGAepi in sodium
phosphate buffer (50 mM Na2HPO4, 100 mM NaCl, pH 7.6). c. Snapshot of in situ 1H-NMR of the
BcUGAepi reaction (in D2O) with UDP-GlcA as a substrate (0 to 16.8 min). Signals from the
anomeric proton of UDP-GlcA (5.57 ppm) and UDP-GalA (5.62 ppm) are shown. UDP-GlcA =
Uridine diphosphate glucuronic acid, UDP-GalA = Uridine diphosphate galacturonic acid.
Figure 3. Apparent kcat values for the BcUGAepi reaction with UDP-GlcA dependent on pH.
Symbols show the data and the solid line is the corresponding fit. Closed circles correspond to
wild-type and open circles to Y149F variant of BcUGAepi.
Figure 4. Proposed kinetic mechanism of UGAepi. The first step is the binding of uridine
diphosphate glucuronic acid (UDP-GlcA) into the free enzyme (E-NAD+) including a slow
conformational change (highlighted with gray background) in the enzyme to E*-NAD+. Hydride
transfer from C4-H to NAD+ yields E*-NADH and UDP-4-keto-hexose-uronic acid as a reaction
intermediate. This 4-keto intermediate rotates around the phosphate backbone of the substrate
positioning the opposite face of the 4-keto group towards NADH. Hydride transfer from NADH
yields the product uridine diphosphate galacturonic acid (UDP-GalA) which is released from
the enzyme regenerating E-NAD+. The flux through the reduction (kred) of the 4-keto-intermediate
to UDP-GalA is considerably higher than the net flux through the steps involved in the oxidation
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(kox), which the thickness of arrows should illustrate. One can estimate that, for just 3% or less of
total enzyme accumulated as enzyme-NADH at the steady state when UDP-GlcA is saturating, the
flux from enzyme-NADH to give enzyme-NAD+ (kred) must exceed by two magnitude orders (≥
30-fold) the net flux from enzyme-NAD+ to enzyme-NADH (kox). The portion of enzyme-NADH
is given by the relationship kox/(kred + kox).
Figure 5. Structural comparison of UDP-glucuronic acid 4-epimerases (UGAepis) and SDR-
type decarboxylases active on UDP-GlcA. UXS = UDP-xylose synthase, UAXS = UDP-
apiose/xylose synthase, ArnA = UDP-glucuronic acid decarboxylase. a. Partial multiple
sequence alignment (aligned with Clustal Omega and visualized with ESPript). The epimerase
residues involved in binding of the carboxylate group of UDP-GlcA are highlighted in yellow and
the corresponding residues of BcUGAepi are labelled in blue at the top of the alignment. UniProt
entries: UGAepi_BcUGAepi (J8BY31), UGAepi_MoeE5 (A0A003), UGAepi_GlaKP_analog
(Q9RP53), UGAepi_Arabidopsis_1 (Q9M0B6), UGAepi_Arabidopsis_3 (O81312),
UGAepi_Arabidopsis_4
(O22141),
UGAepi_Arabidopsis_5
(Q9STI6),
UGAepi_Arabidopsis_6 (Q9LIS3), UGAepi_Zea_mays (Q304Y2), UGAepi_Cap1J (P96481),
UGAepi_UGlcAE (A7GQD3), UGAepi_Rhiz._mel. (O54067), UXS_Rattus_norveg.
(Q5PQX0),
UXS_Homo_sapiens (Q8NBZ7), UXS_Arabidopsis_1 (Q8VZC0), UXS_Arabidopsis_2
(Q9LZI2), UXS_Arabidopsis_3 (Q9FIE8), UXS_Arabidopsis_5 (Q9SN95),
UXS_Danio_rerio
(Q6GMI9),
UXS_Mus_musculus
(Q91XL3),
UXS_Arabidopsis_6 (Q9ZV36), UAXS (Q9ZUY6), ArnA_Shigella_flex. (Q83QT8),
ArnA_Salm._typhi (O52325), ArnA_E.coli_K12 (P77398), ArnA_E.coli_O157 (Q8XDZ3),
ArnA_Yersinia_pseud. (Q93PD8) and ArnA_Pseud._aerug. (Q9HY63). b. Active-site close up
of BcUGAepi modeled with YASARA from the experimental UDP-GlcA complex structure of
the epimerase MoeE5 [28]. c. Active-site close up of the UDP-GlcA complex structure of
Arabidopsis thaliana UAXS (C100A variant; PDB: 6H0P), showing the relevant residues from the
alignment. PyMOL was used to generate the structures.
Figure 6. PyMOL representation of the position of the C5 carboxylate group of UDP-GlcA
bound to UGAepi, UXS and UAXS. The yellow carbon atoms show BcUGAepi (modeled). a.
Overlay of the structures of MoeE5 (green; PDB: 6KV9) and BcUGAepi (modeled). The
carboxylate group is equatorial. b. Overlay of the structures of UXS (cyan; PDB: 2B69, sugar
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modeled into the active site [13]) and BcUGAepi. The axially oriented carboxylate can be seen in
the structure of UXS. c. Overlay of the structures of UAXS (pink; PDB: 6H0P) and BcUGAepi,
showing the nearly equatorial carboxylate group in UAXS.
Supporting Information
Figure S1. Results of SDS-PAGE of purified BcUGAepi (⁓37 kDa).
Figure S2. Calibration curve for HiLoad 16/60 Superdex 200 gel filtration column prepared
with gel filtration standard mixture #1511901.
Figure S3. Gel filtration chromatogram of BcUGAepi detected by UV absorbance at 280 nm.
Figure S4. Absorbance spectrum of wild-type BcUGAepi indicating the presence of a
protein-bound nicotinamide cofactor (260 nm, 340 nm).
Figure S5. HPLC chromatograms of NAD+, NADP+ and NADH standards (a-c) and
supernatant of denatured BcUGAepi showing the cofactor content (d).
Figure S6. Influence of NAD+ concentration (0-1000 µM) on the catalytic activity of
BcUGAepi with UDP-GlcA.
Figure S7. Catalytic activity of BcUGAepi in the reaction with UDP-GlcA at different
temperatures.
Figure S8. Anion exchange chromatogram recorded during the purification of UDP-α-D-
galacturonic acid (UDP-GalA).
Figure S9. Chromatogram recorded during the desalting step of UDP-α-D-galacturonic acid.
Figure S10. HPLC chromatogram of purified and desalted UDP-α-D-galacturonic acid. The
purity of >98% was obtained.
1
Figure S11. H NMR spectrum (500 MHz, D2O) of purified and desalted UDP-α-D-
galacturonic acid, δ 5.62 ppm (dd, 1H), 4.41 ppm (s, 1H), 4.23 ppm (m, 1H), 3.92 ppm (dd,
1H), 3.75 ppm (dd, 1H).
Figure S12. Chromatogram recorded during the purification of UDP-α-D-4-2H-glucuronic
acid.
Figure S13. Chromatogram recorded during the desalting of UDP-α-D-4-2H-glucuronic acid.
1
Figure S15. H NMR spectrum (500 MHz, D2O) of purified and desalted UDP-α-D-4-2H-
glucuronic acid, δ 5.58 ppm (dd, 1H), 4.17 ppm (d, 1H), 3.74 ppm (d, 1H), 3.55 ppm (dd, 1H).
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Figure S16. Time course of BcUGAepi reaction with UDP-GalA as a substrate.
Figure S17. Michaelis-Menten kinetics of the forward (a. UDP-GlcA → UDP-GalA) and
reverse (b. UDP-GalA → UDP-GlcA) reaction catalyzed by BcUGAepi.
Figure S18. Multiple sequence alignment (prepared with Clustal Omega) of UDP-galactose
4-epimerases (GALEs) and BcUGAepi.
Figure S19. Results of SDS-PAGE of purified BcUGAepi _Y149F (⁓37 kDa).
Figure S20. Time course of BcUGAepi_Y149F catalyzed reaction with UDP-GlcA as a
substrate.
Figure S21. In situ NMR experiments with BcUGAepi.
Figure S22. NADH calibration curve on HPLC.
Figure S23. HPLC chromatograms from denaturation experiments of BcUGAepi reacted
with UDP-GlcA and UDP-4-[2H]-GlcA.
Figure S24. Phylogenetic analysis of SDRs active on sugar nucleotides.
Figure S25. a. A part of a sequence alignment (aligned with Clustal Omega and visualized
with ESPript) of UGAepis and GALEs.
Figure S26. a. Interactions with the carboxylate moiety of UDP-GlcA (yellow carbons) in the
active site of UXS (cyan carbons; PDB: 2B69, sugar modeled into the active site).
Figure S27. Residues (Asn179 and Tyr299) on the active site of GALE (green carbons; PDB:
1XEL) responsible for binding the 6-OH group of UDP-Glc (yellow carbons
Scheme S1. NAD+ dependent oxidative decarboxylation of UDP-α-D-glucuronic acid yielding
UDP-4-keto-α-D-xylose (and CO2) catalyzed by E. coli enzyme ArnA.
Scheme S2. One-pot synthesis of UDP-α-D-4-2H-glucuronic acid with lactate dehydrogenase-
based regeneration system of NAD+.
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NAD+
HO
4
O
O
HO
+ CO2
UDP
or
HO
O
OH
UDP
HO
O
OH
UDP-xylose
UDP-apiose
UAXS
NAD+
NADH
UGAepi
UXS
UAXS
NAD+
H
OH
CO2
O
CO2
CO2
H
O
4
UGAepi
O
O
O
O
H
Tyr
HO
HO
HO
OH
OH
OH
OUDP
OUDP
OUDP
UDP-4-keto-
hexose-
uronic acid
UDP-GalA
UDP-GlcA
UXS
NAD+
4
O
HO
HO
+ CO2
OH
O
UDP
UDP-xylose