Regioselective glucuronidation of flavonols by six human UGT1A isoforms

Article abstract of DOI:10.1007/s11095-011-0418-5

Purpose: Glucuronidation is a major barrier to flavonoid bioavailability; understanding its regiospecificity and reaction kinetics would greatly enhance our ability to model and predict flavonoid disposition. We aimed to determine the regioselective glucuronidation of four model flavonols using six expressed human UGT1A isoforms (UGT1A1, 1A3, 1A7, 1A8, 1A9, 1A10). Methods: In vitro reaction kinetic profiles of six UGT1A-mediated metabolism of four flavonols (all with 7-OH group) were characterized; kinetic parameters (K_m, V_max and CL_int∈=∈V_max/K_m) were determined. Results: UGT1A1 and 1A3 regioselectively metabolized the 7-OH group, whereas UGT1A7, 1A8, 1A9 and 1A10 preferred to glucuronidate the 3-OH group. UGT1A1 and 1A9 were the most efficient conjugating enzymes with K _m values of 1 μM and relative catalytic efficiency ratios of 5.5. Glucuronidation by UGT1As displayed surprisingly strong substrate inhibition. In particular, K_si values (substrate inhibition constant) were less than 5.4 μM for UGT1A1-mediated metabolism. Conclusion: UGT1A isoforms displayed distinct positional preferences between 3-OH and 7-OH of flavonols. Differentiated kinetic properties between 3-O- and 7-O- glucuronidation suggested that (at least) two distinct binding modes within the catalytic domain were possible. The existence of multiple binding modes should provide better "expert" knowledge to model and predict UGT1A-mediated glucuronidation.

Full text of DOI:10.1007/s11095-011-0418-5

Pharm Res (2011) 28:1905–1918
DOI 10.1007/s11095-011-0418-5
RESEARCH PAPER
Regioselective Glucuronidation of Flavonols by Six Human
UGT1A Isoforms
Baojian Wu & Beibei Xu & Ming Hu
Received: 1 December 2010 /Accepted: 4 March 2011 /Published online: 7 April 2011
#
Springer Science+Business Media, LLC 2011
ABSTRACT
the catalytic domain were possible. The existence of multiple
binding modes should provide better “expert” knowledge to
model and predict UGT1A-mediated glucuronidation.
Purpose Glucuronidation is a major barrier to flavonoid
bioavailability; understanding its regiospecificity and reaction
kinetics would greatly enhance our ability to model and predict
flavonoid disposition. We aimed to determine the regioselec-
tive glucuronidation of four model flavonols using six expressed
human UGT1A isoforms (UGT1A1, 1A3, 1A7, 1A8, 1A9,
1A10).
.
.
.
KEY WORDS flavonols glucuronidation regioselectivity
substrate inhibition UGTs
.
ABBREVIATIONS
Methods In vitro reaction kinetic profiles of six UGT1A-
mediated metabolism of four flavonols (all with 7-OH group)
were characterized; kinetic parameters (Km, Vmax and
CLint=Vmax/Km) were determined.
Results UGT1A1 and 1A3 regioselectively metabolized the 7-
OH group, whereas UGT1A7, 1A8, 1A9 and 1A10 preferred
to glucuronidate the 3-OH group. UGT1A1 and 1A9 were the
most efficient conjugating enzymes with Km values of ≤1 μM
and relative catalytic efficiency ratios of ≥5.5. Glucuronidation
by UGT1As displayed surprisingly strong substrate inhibition. In
particular, Ksi values (substrate inhibition constant) were less
than 5.4 μM for UGT1A1-mediated metabolism.
DHF
MS
NMR
QHF
QSAR
SN2
dihydroxyflavone
mass spectroscopy
nuclear magnetic resonance
tetrahydroxyflavone
quantitative structure activity relationship
bimolecular nucleophilic substitution
trihydroxyflavone
THF
UDPGA uridine diphosphoglucuronic acid
UGTs
UPLC
UDP-glucuronosyltransferases
ultra performance liquid chromatography
Conclusion UGT1A isoforms displayed distinct positional
preferences between 3-OH and 7-OH of flavonols. Differen-
tiated kinetic properties between 3-O- and 7-O- glucuronida-
tion suggested that (at least) two distinct binding modes within
INTRODUCTION
Consumption of dietary flavonoids such as flavonols,
flavones and isoflavones are associated with health benefits
against ailments such as cancer and heart diseases (1,2).
However, rapid glucuronidation of flavonoids in both liver
and intestine leads to the presence of mainly phase II
conjugates such as glucuronides and sulfates rather than the
parent compound in the systemic circulation (3). As a
consequence, flavonoids such as genistein and kaempferol
have very poor (less than 5%) in vivo bioavailabilities in
animals and humans (4,5), which limit their uses as
therapeutic agents.
Electronic Supplementary Material The online version of this article
(doi:10.1007/s11095-011-0418-5) contains supplementary material,
which is available to authorized users.
:
:
B. Wu B. Xu M. Hu (*)
Department of Pharmacological and Pharmaceutical Sciences
College of Pharmacy, University of Houston
1441 Moursund Street
Houston, Texas 77030, USA
e-mail: mhu@uh.edu

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Wu, Xu and Hu
Glucuronidation is a major metabolic pathway, either as
a 3′-OH group were glucuronidated only at position 7-OH,
while those containing this group also formed 3′-O-
glucuronides and sometimes 4′-O-glucuronides (23). Con-
sistent with this observation, human intestinal UGTs
including UGT1A1 and 1A8 were especially effective in
conjugating the 3′,4′ catechol unit of flavonoids (22).
Most flavonoids bear more than one potential glucur-
onidation site (i.e., aromatic hydroxyl groups); this presents
a major challenge to the computational modeling of
substrate specificity in regard to UGT metabolism. Recent-
ly, we have shown that it is necessary to model the
regiospecific glucuronidation separately in order to predict
the glucuronidation rates of flavonoids (25). It is also
recognized that more efforts will be needed to clarify the
differences between isoform-specific flavonoid metabolism
including regioselectivity (so-called “expert knowledge”);
this can be used to guide the development of predictive
models for UGT metabolism. As a part of the continuing
efforts, this paper investigated the kinetics of six UGT1A
isoforms (UGT1A1, 1A3, 1A7, 1A8, 1A9 and 1A10) using
selected flavonoids all having both 3-OH and 7-OH groups
to evaluate regioselectivity of each UGT isoform. The six
UGT1A isoforms were chosen here because of their
significant contribution to glucuronidation of flavonoids
shown in previous studies (14,15,19).
a primary or secondary (sequential) process in the disposi-
tion of xenobiotics (6). It is catalyzed by UDP-
glucuronosyltransferases (UGTs), following a SN2 mecha-
nism (7). On the basis of amino acid sequence identity,
human UGTs are classified into four families: UGT1,
UGT2, UGT3, and UGT8 (8). The most important drug-
conjugating UGTs belong to the UGT1 and UGT2
families. The human UGT1A gene cluster, located on
chromosome 2q37, spans approximately 200 kb. It contains
13 individual promoters/first exons and shared exons 2–5.
Each exon 1 spliced to exons 2–5 is regarded as a unique
gene which translates into the corresponding active
UGT1A isoform excluding the pseudogenes (i.e.,
UGT1A2p, UGT1A11p, UGT1A12p and UGT1A13p).
Among the UGT1A family, 1A8 and 1A10 are expressed
almost exclusively in the gastrointestinal tract; 1A3, 1A4
and 1A9 are primarily present in the liver; and 1A7 is
mainly distributed in the stomach and esophagus. In
contrast, 1A1 and 1A6 are ubiquitously present in many
tissues including the liver and gastrointestinal tract (9,10).
Glucuronidation phenotyping using recombinant UGT
isoforms had been widely applied in a variety of areas to (a)
determine the major metabolic pathway of a particular drug
(11,12); (b) identify the main isoform(s) responsible for
glucuronidation of a drug (13); (c) correlate glucuronidation
between organ and isoform levels (14,15); and (d) model
substrate specificity of various UGT isoforms, unraveling the
structural requirements for a UGT substrate (16). The QSAR
regression models indicated that substrate hydrophobicity was
essential for glucuronidation, which agreed with the location
of UGT on the luminal side of the endoplasmic reticulum
(17). The pharmacophore models identified two key hydro-
phobic regions adjacent from the site of glucuronidation as
the substrate features for UGTs recognition (18).
The UGT1A subfamily (except UGT1A4 and 1A6) is
mainly responsible for glucuronidating flavonoids, and the
substrate specificity shows extensive overlaps (14,19).
UGT1A4 exclusively metabolizes amine-containing com-
pounds (20), whereas UGT1A6 exhibits limited substrate
specificity for flavonoids (21). UGT1As catalyze the
formation of conjugative derivatives (i.e., glucuronides) by
transferring glucuronic acid from the cofactor UDP-
glucuronic acid (UDPGA) to the hydroxyl oxygen(s).
Multiple mono-glucuronide isomers are often generated
from a single flavonoid that bears more than one
conjugation site (22,23), because the aglycone-binding
domain might permit multiple binding modes of the
acceptor substrate (24). Some key structural features have
been uncovered to govern the glucuronidation regioselec-
tivity (or positional preference). For example, the 3′-OH
group is the major determinant of the regioselectivity of
flavonoid glucuronidation by UGT1A1. Flavonoids lacking
MATERIALS AND METHODS
Materials
Expressed human UGT isoforms (SupersomesTM, i.e.,
UGT1A1, 1A3, 1A7-1A10) were purchased from BD
Biosciences (Woburn, MA). Rabbit anti-human UGT1A
polyclonal (H-300) and rabbit anti-goat IgG-HRP antibodies
were purchased from Santa Cruz Biotechnology (Santa
Cruz, CA). Uridine diphosphoglucuronic acid (UDPGA),
alamethicin, D-saccharic-1,4-lactone monohydrate, and
magnesium chloride were purchased from Sigma-Aldrich
(St Louis, MO). Ammonium acetate was purchased from J.
T. Baker (Phillipsburg, NT). Four (4) flavonols (Fig. 1)
containing both 3-OH and 7-OH groups (i.e., 3,7-dihydrox-
yflavone (3,7DHF), 3,5,7-trihydroxyflavone, (3,5,7THF)
3,7,4′-tryhydroxyflavone (3,7,4′THF), and 3,5,7,4′-tetrahy-
droxyflavone(3,5,7,4′QHF)) were purchased from Indofine
Chemicals (Somerville, NJ). All other materials (typically
analytical grade or better) were used as received.
Immunoblotting
The recombinant UGT Supersomes™ (20 μg protein) were
analyzed by SDS-polyacrylamide gel electrophoresis (10%
acrylamide gels) and transferred onto PVDF membranes

Regioselective Glucuronidation of Flavonols by UGT1As
1907
compounds and their glucuronides. UPLC methods for each
flavonol and the glucuronide(s) were essentially the same as
our publication (26), with slight modifications. Briefly, the
mobile phase A (A) was 2.5 mM ammonium acetate in
purified water (pH=6.5). Mobile phase B (B) was 100%
acetonitrile. The mobile phase was run 5 min at a flow rate
of 0.45 ml/min with the pre-determined gradient (0 min,
10%B; 0 to 2 min, 10–20%B; 2 to 3 min, 20–40%B; 3 to
3.5 min 40–50%B; 3.5 to 4 min, 50%–90%B; 4 to 4.5 min,
90%B; 4.5 to 5 min, 90–10%). For 3,5,7,4′QHF (kaemp-
ferol), a different mobile phase B and gradient was adopted:
mobile phase B, 100% methanol; and gradient, 0 min, 10%
B; 0 to 2 min, 10–20%B; 2 to 3 min, 20–40%B; 3 to
3.5 min, 40–50%B; 3.5 to 4 min, 50–70%B; 4 to 5 min, 70–
90%B; 5 to 5.5 min, 90%B; and 5.5 to 6 min, 90–10%B.
Quantitation of the glucuronide was based on the standard
curve of the parent compound and further calibrated using
the conversion factor as described earlier (19,26). The
following is a list of the conversion factors used: 3,7DHF-3-
O-glucuronide, 1.29; 3,7DHF-7-O-glucuronide, 0.86; 3,7,4′
THF-3-O-glucuronide, 1.10; 3,7,4′THF-7-O-glucuronide,
0.92; 3,5,7THF-3-O-glucuronide, 0.71; 3,5,7THF-7-O-
glucuronide, 1.41; 3,5,7,4′QHF-3-O-glucuronide, 1.00;
3,5,7,4′QHF-7-O-glucuronide, 0.70. Representative chroma-
tograms and their UV spectra were shown in Fig. 2.
Fig. 1 Chemical structures of the model flavonols. 3-OH of C-ring and
7-OH of A-ring are the more favorable positions for glucuronidation by
UGT1As.
(Millipore, Bedford, MA). Blots were probed with anti-
UGT1A antibody (H-300) that recognize all UGT1A
isoforms (Santa Cruz Biotechnology, Santa Cruz, CA),
followed by horseradish peroxidase-conjugated rabbit anti-
goat IgG (Santa Cruz Biotechnology, Santa Cruz, CA).
Membranes were analyzed on a FluorChem FC Imaging
System (Alpha Innotech), and intensities of UGT bands
were measured by densitometry using the AlphaEase
software.
Glucuronide Structure Identification
Glucuronide structures were identified using three inde-
pendent methods in a sequential process as summarized in
our earlier publication (26). First, the glucuronides were
confirmed by its complete hydrolysis to the aglycones when
β-D-glucuronidase was used. Second, the glucuronides were
identified as mono-glucuronides which showed a mass of
[(aglycone’s mass)+176] Da using UPLC/MS/MS. 176 Da
is the mass of single glucuronic acid. Third, the sites of
glucuronidation were confirmed by the “UV spectrum
maxima (λmax) shift method.” In general, if a 3-, 5- or 4′-
OH of a flavonol is glucuronidated, the hypsochromic shifts
(i.e. to shorter wavelength) were observed in either Band I
(300–380 nm) or Band II (240–280 nm). The resulting shift
in Band I associated with the substitution of 3-OH group
was in the order of 13~30 nm. Substitution of 5-OH group
resulted in a 5~15 nm shift in Band II, and glucuronidation
of 4′-OH group produced a 5~10 nm shift in Band I. In
contrast, substitution of the hydroxyl group at position C7
had minimal or no effect on the λmax of the UV spectrum
(Fig. 2). The above three methods are considered essential
and complementary in structural elucidation of glucuronide
generated from flavonoids with multiple conjugation sites.
The hydrolysis by β-D-glucuronidase is used to confirm β-
glucuronide without knowing the number of glucuronic acids
attached, whereas MS/MS can detect the number of
Glucuronidation Kinetics
Enzyme kinetic parameters of glucuronidation by selected
UGT1A isoforms (i.e., UGT1A1, 1A3, 1A7, 1A8, 1A9, and
1A10) were determined by measuring initial glucuronida-
tion rates of flavonols at a series of concentrations. The
experimental procedures of UGT assays were exactly the
same as our previous publications (14,19,26). Glucuronide
formation was verified to be linear with respect to
incubation time (5–60 mins) and protein concentration
(13–53 μg/ml). Glucuronidation rates were calculated as
nmol glucuronide(s) formed per Supersomes™ protein
amount per reaction time (in unit of nmol/mg protein/
min), as the actual enzyme concentration is unknown. The
aglycone substrate concentrations in the range of 0.039–
40 μM were used unless method sensitivity or substrate
solubility necessitated otherwise. All experiments were
performed in triplicates.
UPLC Analysis of Flavonols and Glucuronides
The Waters ACQUITY™ UPLC (Ultra Performance Liquid
Chromatography) system was used to analyze the parent

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Wu, Xu and Hu

Regioselective Glucuronidation of Flavonols by UGT1As
1909
Fig. 2 Representative UPLC chromatograms (left) and UV spectra (right)
ƒ
Statistical Analysis
of flavonols and their 3-O- / 7-O-glucuronides. (a–d) panels represent
3,7DHF, 3,5,7THF, 3,7,4′THF and 3,5,7,4′-QHF and their 3-O- / 7-O-
glucuronides, respectively. UPLC samples were from enzyme assays in
which each flavonol (10 μM) was incubated with 27.5 μg/ml UGT1A9 for
10 or 60 mins. For spectra, black solid line denotes the flavonols, red
dotted lines are 3-O-glucuronides, and blue dash-dotted lines are 7-O-
glucuronides. Compared to the parent compounds, band I of 3-O-
glucuronides either disappeared or had large hypsochromic shifts (to
shorter wavelengths) (e.g. 19.3~26.7 nm). In contrast, 7-O-glucuronides
had minimal or no changes in the UV spectrum (26).
Significance of mean comparison was examined base on
student’s t-test using GraphPad Prism V5 for Windows
(GraphPad Software, San Diego, CA). Statistical signifi-
cance was demonstrated with p<0.05.
RESULTS
Relative Expression Level Between the UGT1A
Enzymes
substituted glucuronic acids, but it is unable to probe the exact
conjugative position. In addition, the structure of 3,7-DHF-7-
O-glucuronide was confirmed by NMR spectra (Table S1,
Supplementary Material). NMR analysis was not performed
for glucuronides generated from other flavonols due to
serious difficulty in purifying the glucuronide isomers.
Relative expression levels of the six UGT1A isoforms were
estimated by the Western blot analysis using an anti-
UGT1A antibody (against all UGT1A proteins). Each of
the isoforms expressed had a protein of apparent molecular
mass of approximately 55 kDa (Fig. 3). The relative
expression ratios of UGT1A1 (set as 1), 1A3, 1A7, 1A8,
1A9, and 1A10 were 1:0.82:0.94:1.15:0.87:0.93. The
maximal difference was about 1.4 times.
Data Analysis
Kinetic parameters (Vmax, Km and Ksi (substrate inhibition
constant)) were estimated by fitting the Michaelis-Menten
and/or substrate inhibition equations to the substrate
concentrations and initial rates. Similar to glucuronidation
rates, Vmax values were also determined as nmol glucuro-
nide formed/mg SupersomesTM protein/min (or nmol/mg
protein/min). Eadie-Hofstee plots (Fig. S1, Supplementary
Material) were used as diagnostics for model selection. Data
analysis was performed by GraphPad Prism V5 for
Windows (GraphPad Software, San Diego, CA). The
goodness of fit was evaluated on the basis of R2 values,
RSS (residual sum of squares), RMS (root mean square)
and residual plots (27).
For better comparison between UGT isoforms, the
catalytic efficiencies (or CLint values) of various UGT
isoform-mediated glucuronidation were normalized to
UGT1A1 using the relative UGT protein expression level.
Then, the relative catalytic efficiencies were determined by
arbitrarily assigning the calibrated CLint as 1 for the 3-O-
glucuronidation of 3,7DHF by UGT1A1.
Regioselective Glucuronidation of Flavonols
by UGT1A1
Four model flavonols (3,7DHF, 3,5,7THF, 3,7,4′THF and
3,5,7,4′QHF) present four possible glucuronidation sites
(i.e., 3-OH, 5-OH, 7-OH and 4′-OH). Only 3-O- and 7-O-
glucuronides were observed at all studied concentrations for
all four compounds (Figs. 4, 5, 6, 7, 8 and 9). This is
consistent with the fact that both 4′-OH and 5-OH groups
are typically inactive for UGT1A-mediated glucuronida-
tion, when a 3-OH or a 7-OH group is also present in the
structure (19). The kinetic parameters for 3,7,4′THF
glucuronidation by UGT1A3, 1A7, 1A8 and 1A10 were
not determined, because the compound was not metabo-
lized by these UGT isoforms.
UGT1A1 consistently generated much more flavonol-7-
O-glucuronides than 3-O-glucuronides over the tested
concentration range for 3,7DHF, 3,5,7THF and 3,5,7,4′
Fig. 3 (a) Western blot analysis of UGT expression levels in recombinant human UGT Supersomes™. (b) UGT band intensities for the six UGT
isoforms.

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Wu, Xu and Hu
Fig. 4 Kinetics profiles of UGT1A1-mediated glucuronidation of four flavonols (3,7DHF, 3,5,7THF, 3,7,4′THF and 3,5,7,4′QHF). The corresponding
Eadie-Hofstee plots are presented in Fig. S1 (Supplementary Materials). For Figs. 3, 4, 5, 6, 7 and 8, solid and dashed lines denote formation rates of
flavonol 3-O-glucuronides and 7-O-glucuronide, respectively. Each data point represents the average of three replicates. Experimental details are
presented under Materials and Methods. For kinetic parameters, please see Table II (Figs. 3 and 4) or Table III (Figs. 5, 6, 7 and 8).
Fig. 5 Kinetics profiles of
UGT1A3-mediated glucuronida-
tion of three flavonols (3,7DHF,
3,5,7THF and 3,5,7,4′QHF). The
corresponding Eadie-Hofstee plots
are presented in Fig. S1
(Supplementary Material).

Regioselective Glucuronidation of Flavonols by UGT1As
1911
Fig. 6 Kinetics profiles of UGT1A7-mediated glucuronidation of three flavonols (3,7DHF, 3,5,7THF and 3,5,7,4′QHF). The corresponding Eadie-Hofstee
plots are presented in Fig. S1 (Supplementary Material).
QHF (Fig. 4). The formation ratios of 7-O- /3-O-glucur-
onides fell into the narrow ranges of 3.0-4.2 and 2.3–4.1 for
3,7DHF and 3,5,7THF, respectively (Fig. 10). For 3,5,7,4′
QHF, UGT1A1 only generated 7-O-glucuronide. In the
cases of 3,7DHF and 3,5,7THF, the Km values were similar
for 3-O- and 7-O-glucuronidation, but the differences in
Vmax values were more than 3-fold in favor of 7-O-
glucuronidation. Therefore, UGT1A1 had much higher
Fig. 7 Kinetics profiles of
UGT1A8-mediated glucuronida-
tion of three flavonols (3,7DHF,
3,5,7THF and 3,5,7,4′QHF). The
corresponding Eadie-Hofstee plots
are presented in Fig. S1
(Supplementary Material).

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Wu, Xu and Hu
Fig. 8 Kinetics profiles of UGT1A9-mediated glucuronidation of four flavonols (3,7DHF, 3,5,7THF, 3,7,4THF and 3,5,7,4′QHF). The corresponding
Eadie-Hofstee plots are presented in Fig. S1 (Supplementary Material).
catalytic efficiency (as reflected by CLint=Vmax/Km) for 7-
OH than that for 3-OH group (greater than 3.4-fold).
Together with the fact that the enzyme had the highest
binding affinity with 3,5,7,4′QHF but showed a medium Vmax
value, the results suggested that higher binding affinity was
not as necessarily associated with higher catalytic capacity
(reflected by Vmax value). For 3,7,4′THF, the formation rates
of 7-O-glucuronide were slightly higher than those of 3-O-
Fig. 9 Kinetics profiles of
UGT1A10-mediated glucuronida-
tion of three flavonols (3,7DHF,
3,5,7THF and 3,5,7,4′QHF). The
corresponding Eadie-Hofstee plots
are presented in Fig. S1 (Supple-
mentary Material).

Regioselective Glucuronidation of Flavonols by UGT1As
1913
Fig. 10 Formation rate ratios
(data transformation from Figs. 4,
5, 6, 7, 8 and 9) between 3-O-
and 7-O-glucuronides catalyzed
by UGT1A1, 1A3, 1A7, 1A8, 1A9
and 1A10. For 7-OH preferred
enzymes (UGT1A1 and 1A3), the
ratios of 7-O- over 3-O-glucuro-
nidation were plotted against the
concentration of corresponding
parent compounds, whereas the
ratios of 3-O- over 7-O-
glucuronidation were plotted for
the 3-OH preferred enzymes
(UGT1A7-1A10). Reference line
of value 1 is shown in red.
glucuronide (Figs. 4 and 10), and the derived kinetic
parameters for the positional glucuronidation were similar
between these two glucuronides (Table I).
were 8.6–11.9 and 7.2–17 for 3,7DHF and 3,5,7THF,
respectively (Fig. 10). Likewise, UGT1A3 displayed sub-
strate inhibition kinetics with the regioselective metabolism
(Fig. 5 and Table I). The substrate inhibition constants (Ksi)
lay within 7.52–100 μM, significantly larger than those of
UGT1A1 (p<0.05). The Km values for UGT1A3-mediated
glucuronidation of flavonols varied between 0.84 μM and
8.81 μM. Compared to UGT1A1, UGT1A3 seemed to
have even stronger preference for 7-OH metabolism, since
the catalytic efficiency ratios (i.e., 7-OH over 3-OH) were
up to 13.5 and 8.1 for 3,7DHF and 3,5,7THF, respectively.
Surprisingly, strong substrate inhibition patterns, as
characterized by small Ksi values (within in vivo achievable
concentration value) in the range of 0.60–5.4 μM, were
observed for both 3-O- and 7-O-glucuronidation (Table I).
The Km values were in the range of 0.13–1.57 μM, which
were much smaller than reported Km value (49.8 μM) for
ethinylestradiol (28). UGT1A1 kinetics towards ethinyles-
tradiol also presented a substrate inhibition profile at high
concentrations of UDPGA, which gave the Ksi/Km ratio of
1.61. By contrast, the Ksi/Km ratios for 3,7DHF were
much less than this number, at 0.38 and 1.16 for 3-O- and
7-O-glucuronidation, respectively.
Regioselective Glucuronidation of Flavonols
by UGT1A7
In contrast to UGT1A1 and 1A3, UGT1A7 more specifically
catalyzed formation of flavonol 3-O-glucuronide (Fig. 6 and
Table II). The differences in rates between 3-O- and 7-O-
glucuronidation decreased markedly with increasing concen-
tration of flavonols (Fig. 10). The formation ratios of 3-O-
over 7-O-glucuronide at 1.25 μM were, respectively, 24.7,
11.0, and 5.6 for 3,7DHF, 3,5,7THF and 3,5,7,4′QHF,
whereas those corresponding ratios were 5.9, 1.1 and 1.0 at
Regioselective Glucuronidation of Flavonols
by UGT1A3
Similar to UGT1A1, UGT1A3 also produced much more
flavonol 7-O-glucuronides than 3-O-glucuronides, and only
generated 7-O-glucuronide for 3,5,7,4′QHF (Fig. 5 and
Table I). The formation ratios of 7-O-/3-O-glucuronides

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Wu, Xu and Hu
Tab le I The Enzyme Kinetics
Parameters for UGT1A1 and
1A3 with Appropriate Flavonols
Enzymes
Substrate/Product(s) Vmax
Km
Ksi
μM
Vmax/Km (CLint)
ml/mg protein/min
nmol/mg protein /min μM
UGT1A1 3,7DHF
3-O-glucuronide
1.02 0.34
3.04 0.42
1.57 0.49 0.60 0.38
1.04 0.19 1.21 0.23
0.65 0.29
2.92 0.66
7-O-glucuronide
3,5,7THF
3-O-glucuronide
7-O-glucuronide
3,7,4′THF
0.82 0.14
4.59 0.46
0.31 0.10 2.64 0.89
0.51 0.08 1.22 0.19
2.65 0.96
9.00 1.67
3-O-glucuronide
7-O-glucuronide
3,5,7,4′QHF
0.68 0.05
1.08 0.13
0.30 0.05 5.41 0.93
0.43 0.10 3.53 0.86
2.27 0.41
2.52 0.66
7-O-glucuronide
1.38 0.14
0.13 0.03 2.81 0.67
10.6 2.67
UGT1A3 3,7DHF
3-O-glucuronide
7-O-glucuronide
3,5,7THF
0.18 0.02
1.89 0.36
8.81 2.72
100 34.8
0.02 0.01
0.27 0.15
7.02 3.76 50.0 20.2
3-O-glucuronide
7-O-glucuronide
3,5,7,4′QHF
0.17 0.01
3.44 0.84
0.84 0.18 17.9 6.33
2.12 1.24 9.95 3.16
0.20 0.04
1.62 1.02
Data are means S.E. of three
determinations.
7-O-glucuronide
0.86 0.35
3.30 1.10 7.52 9.58
0.26 0.13
40 μM. The differences in CLint values between 3-O- and 7-
O-glucuronidation hit 63.5-, 37.2- and 5.3-folds for 3,7DHF,
3,5,7THF and 3,5,7,4′QHF, respectively. The Km values for
7-O-glcuronidation (11.7–23.2 μM) were much higher than
that for 3-O-glucuronidation (0.82–1.61 μM), which sug-
gested that the UGT1A7 binding pocket might favor more
the binding mode of flavonols for producing 3-O-
glucuronides than that for generating 7-O-glucuronides.
than the Vmax value of 3,5,7THF glucuronidation
(3.89 nmol/mg protein/min for 3-O-glucuornidation).
Regioselective Glucuronidation of Flavonols
by UGT1A9
UGT1A9 also preferentially glucuronidated the 3-OH groups
of flavonols (Fig. 8 and Table II). The 3-OH preference was
more obvious at lower concentrations (Fig. 10). The
formation ratio of 3-O- over 7-O-glucuronide was typically
greater than two at concentrations of <2 μM. The catalytic
efficiencies of UGT1A9 for 3-OH were more than 14.4-fold
higher than that for 7-OH (except 3,5,7THF, which showed
a smaller difference at 2.3-fold). The Km values of 7-O-
glucuronidation were generally significantly (p<0.05) larger
than those of 3-O-glucuornidation (except for 3,5,7THF,
which had similar Km values). Interestingly, the Vmax values
appeared to be negatively correlated with the Km values, the
Km values that were less than 1 μM coincided with Vmax
values of higher than 1.9 nmol/mg protein/min (except 7-O-
glucruonidatin of 3,7DHF). Additionally, 3,7,4′THF was a
good substrate for UGT1A9, a fair substrate for UGT1A1,
but a non-substrate for 1A3, 1A7, 1A8 or 1A10.
Regioselective Glucuronidation of Flavonols
by UGT1A8
The comparisons between formation of flavonol 3-O- and
7-O-glucuronides revealed that UGT1A8 preferred 3-OH
over 7-OH (Fig. 7 and Table II). The degree of
regioselectivity between 3-OH and 7-OH varied with the
concentration of flavonols. The ratios of 3-O- over 7-O-
glucuronidation were 1.5–3.8, 2.6–4.4 and 0.81–2.59 for
3,7DHF, 3,5,7THF and 3,5,7,4′QHF, respectively. The
differences in CLint values in terms of 3-OH and 7-O-
glucuronidation were >4-fold for 3,7DHF and 3,5,7THF,
and >2-fold for 3,5,7,4′QHF.
3-O-/7-O-glucuronidation was enhanced more than 5.7
times in the presence of a 5-OH group (3,7DHF vs
3,5,7THF). However, the addition of a 4′-OH group
decreased the catalytic efficiency of 3-O-glucuronidation
from 0.60 to 0.31 ml/mg protein/min (Table II). The
catalytic capacities (Vmax) for 3,7DHF and 3,5,7,4′QHF
were as low as 0.74–0.76 nmol/mg protein/min, much less
Regioselective Glucuronidation of Flavonols
by UGT1A10
UGT1A10 was another UGT1A isoform that prefers 3-O-
glucuronidation (Fig. 9 and Table II). This was observed in

Regioselective Glucuronidation of Flavonols by UGT1As
1915
Table II The Enzyme Kinetic Parameters for Glucuronidation of Flavonols by 4 UGT1As
Enzymes
Substrate /Product(s)
Vmax
Km
Ksi
Vmax/Km (CLint)
nmol/mg protein /min
μM
μM
ml/mg protein/min
UGT1A7
3,7DHF
3-O-glucuronide
7-O-glucuronide
3,5,7THF
2.04 0.07
0.44 0.07
1.61 0.14
23.2 6.96
309 127
N/A
1.27 0.12
0.02 0.01
3-O-glucuronide
7-O-glucuronide
3,5,7,4′QHF
3.35 0.11
2.46 0.34
0.82 0.08
21.4 6.09
55.8 7.27
N/A
4.09 0.42
0.11 0.03
3-O-glucuronide
7-O-glucuronide
3,7DHF
0.77 0.04
1.10 0.11
1.46 0.32
11.6 2.91
N/A
N/A
0.53 0.12
0.10 0.03
UGT1A8
3-O-glucuronide
7-O-glucuronide
3,5,7THF
0.76 0.04
0.65 0.10
8.68 1.21
27.3 7.50
N/A
N/A
0.09 0.01
0.02 0.01
3-O-glucuronide
7-O-glucuronide
3,5,7,4′QHF
3.89 0.47
0.79 0.27
6.44 1.28
4.88 1.14
37.3 10.1
N/A
0.60 0.14
0.16 0.07
3-O-glucuronide
7-O-glucuronide
3,7DHF
0.74 0.04
1.01 0.09
2.39 0.47
6.77 1.78
N/A
N/A
0.31 0.06
0.15 0.04
UGT1A9
3-O-glucuronide
7-O-glucuronide
3,5,7THF
4.60 0.28
2.04 0.09
0.22 0.04
1.50 0.26
30.1 7.11
N/A
20.9 4.01
1.36 0.24
3-O-glucuronide
7-O-glucuronide
3,7,4′THF
7.41 0.89
2.56 0.10
0.68 0.16
0.54 0.08
7.43 2.12
N/A
10.9 2.87
4.74 0.73
3-O-glucuronide
7-O-glucuronide
3,5,7,4′QHF
2.62 0.05
1.15 0.08
0.36 0.03
2.67 0.56
N/A
N/A
7.28 0.62
0.43 0.10
3-O-glucuronide
7-O-glucuronide
3,7DHF
1.92 0.03
0.87 0.05
0.32 0.03
3.87 0.59
N/A
N/A
6.00 0.57
0.22 0.04
UGT1A10
3-O-glucuronide
7-O-glucuronide
3,5,7THF
0.19 0.01
0.26 0.02
6.66 0.66
8.74 1.37
N/A
N/A
0.03 0.00
0.03 0.01
3-O-glucuronide
7-O-glucuronide
3,5,7,4′QHF
2.01 0.14
0.77 0.04
4.49 0.61
3.96 0.58
73.5 17.8
N/A
0.45 0.07
0.19 0.03
3-O-glucuronide
7-O-glucuronide
1.46 0.16
1.10 0.20
2.44 0.55
3.34 1.21
30.0 8.00
51.4 27.2
0.60 0.15
0.33 0.14
Data are means
S.E. of three determinations. N/A means that parameter Ksi was not available; this was for the cases in which data fitted better to
Michaelis-Menten equation.
the regioselective glucuronidation of 3,5,7THF and 3,5,7,4′
QHF (Fig. 10). The ratios of 3-O- over 7-O-glucuronidation
rates were ~2.3 and 1.1–1.9 for 3,5,7THF and 3,5,7,4′
QHF, respectively. The CLint values for 3-O-/7-O-glucur-
onidation were 0.45/0.19 ml/mg protein/min for
3,5,7THF, 0.60/0.33 ml/mg protein/min for 3,5,7,4′
QHF. In the absence of 5-OH, the flavonol 3,7DHF was
a very poor substrate of UGT1A10 (with Vmax value less
than 0.26 nmol/mg protein/min and CLint value at
~0.03 ml/mg protein/min), whereas 3,7,4′THF was not a

1916
Wu, Xu and Hu
substrate at all. This indicated that 5-OH is critical for the
interaction between UGT1A10 and flavonols, although this
group itself was not glucuronidated. It is also suggested that
these model compounds bind much weaker to UGT1A10
than to UGT1A9, because UGT1A10 exhibited Km values
(ranging from 3.96 to 8.74 μM for 3-O-/7-O-glucuronida-
tion), that were significantly larger (p<0.05) than the Km
values displayed by UGT1A9 (0.22–0.68 μM for 3-O-
glucuronidation).
human UGT1A isoforms via kinetics determination utiliz-
ing a normalized expressed level. The “hallmarks” (i.e.,
distinct regioselectivity and/or substrate selectivity) as
presented by the glucuronidation patterns (Table III),
helped uncover the fact that these UGT1A isoforms do
not always have overlapping substrate specificities with
respect to regiospecific glucuronidation. The findings are
novel in contrast to previous reports that showed extensive
overlapping of UGT1A substrate specificity towards flavo-
noids (12,14,19). For example, 3-hydroxyflavone at 2.5 μM
was similarly metabolized by UGT1A1, 1A7, 1A8, 1A9 and
1A10 (19). The current approach holds great promises on
identifying those substrates (probes) that are exclusively
metabolized by a particular UGT isoform. Because of the
prevalence of UGT generic variants, understanding of
regiospecificity of UGT isoform-mediated glucuronidation
is also very important to anticipate metabolism in vivo, and
modeling/prediction of glucuronidation in silico. More-
over, results here would greatly assist in enzymatically
synthesizing flavonoid glucuronides conjugated at desired
position(s) for pharmaceutical and/or analytical purposes,
as accumulating in vitro and in vivo evidences point to the
ability of various (regiospecific) flavonoid glucuronides to
retain biological activities (29).
The above observations were made using kinetics
profiling (specificity and/or regioselectivity) over a wide
concentration range. This approach appears to be superior
to the more commonly used method of measuring the
enzyme activities at a single high substrate concentration
(>100 μM) (22,23). Although the latter approach has the
advantages of less cost and labor, this practice might
generate an erroneous conclusion, if the concentration
was not properly chosen. For example, the formation of 3-
O-glucuronide by UGT1A9 was much faster than that of 7-
O-glucuronide for 3,7DHF at concentrations of less than
20 μM (Fig. 8). However, no significant regioselectivity
between 3-OH and 7-OH was visible, if glucuronidation
rates were only determined at a concentration of over
40 μM. The similar observations were made with UGT1A7
metabolism of 3,5,7THF, as well as glucuronidation of
3,5,7,4′QHF by UGT1A7, 1A8 and 1A10. In addition, the
Relative Catalytic Efficiencies Between UGT1A
Enzymes
Relative catalytic efficiencies of the six UGT1As are summa-
rized in Table III. Based on the protein sequence identity
counting the substrate binding domain, UGT1A1 and 1A3,
on average, are only approximately 50% identical to each
other and to the polypeptides of the UGT1A7-1A10 cluster.
By contrast, the polypeptides within UGT1A7-1A10 cluster
are 75–92% identical (8) (Supplementary Material Fig. S2).
Interestingly, with fewer shared amino acids, UGT1A1 and
1A3 showed great similarity in regioselective glucuronidation
of flavonols (7-OH preference), although UGT1A3 was
generally less efficient than UGT1A1 in glucuronidating
flavonols, as evidenced by lower relative catalytic efficiencies
for each flavonol (Table III). On the other hand, the
UGT1A7-1A10 cluster which shares much higher sequence
identities commonly preferred to metabolize the 3-OH of
flavonols. Nevertheless, these isoforms exhibited divergent
catalytic efficiency towards the aglycones (Table III).
UGT1A9 showed a striking 3-OH preference ratio of >11
in CLint, whereas the rest of enzymes generally glucuroni-
dated flavonols with a moderate 3-OH preference ratio of<
2.1 in CLint with the exception of 3-O-glucuronidation of
3,5,7THF by UGT1A7.
DISCUSSION
This paper for the first time elucidated regioselective
glucuronidation of four multi-hydroxyl flavonols by six
Table III Normalized Catalytic
UGT1A
3,7DHF
3-O-G*
3,5,7THF
3-O-G*
3,7,4′THF
3,5,7,4′QHF
Efficiencies (Based on the Relative
UGT1A Expression Levels) of Six
Recombinant Human UGT1As in
Glucuronidation of Flavonoids.
7-O-G*
7-O-G*
3-O-G*
7-O-G*
3-O-G*
7-O-G*
1A1
1A3
1A7
1A8
1A9
1A10
1.0
0.0
4.5
0.5
0.0
0.0
2.4
0.0
4.1
0.4
13.8
3.0
0.2
0.2
8.4
0.3
3.5
0
3.9
0
0.0
0.0
16.3
0.5
0.2
0.3
0.4
0.5
Relative Catalytic Efficiency (Vmax
/
Km=1.0) was Arbitrarily Assigned
for the 3-O-glucuronidation of
3,7DHF by UGT1A1
2.1
6.7
0
0
0.9
0.1
0.8
0
0
0.4
36.8
0.0
19.2
0.7
12.8
0
0.8
0
10.6
1.0
*3-O-G: 3-O-glucuronidation;
7-O-G: 7-O-glucuronidation

Regioselective Glucuronidation of Flavonols by UGT1As
1917
kinetics profiling included substrate concentrations less than
1 μM, which is close to the in vivo plasma concentration of
flavonoids after they are taken orally (4). Therefore, the
data might have greater potential to correlate with in vivo
especially hepatic metabolism.
a -OH group at the C4′ or C5 had major but opposite
effects on glucuronidation at the 3 or 7-OH position. The
addition of 5-OH generally enhanced the UGT1A-
mediated conjugation (excluding UGT1A9), as evidenced
by the comparisons between 3,7DHF and 3,5,7THF, or
between 3,7,4′THF and 3,5,7,4′QHF in catalytic efficiency
(Table III). The enhancement was largely ascribed to more
efficient formation of the regioselective conjugates (7-O-
glucuronide for UGT1A1 and 1A3; 3-O-glucuronide for
UGT1A7, 1A8 and 1A10). The significance of 5-OH in
isoform-specific glucuronidation at the 3-OH or 7-OH
positions were also observed for compounds such as
chrysin, wogonin, oroxylin A, and 3,5-dihydroxyflavone
(15,19). On the contrary, the addition of 4′-OH compro-
mised UGT1A3-, 1A7-, 1A8-, and 1A9-mediated glucur-
onidation by substantially reducing their respective
positional glucuronidation (Table III). The fact that 3-O-
or 7-O-glucuronidation was reduced in the presence of 4′-
OH was also supported by the observation that 3-O-
glucuronidation of 3,4′-dihydroxyflavone was slower than
3-hydroxyflavone at three different concentrations (19).
In conclusion, among the six UGT1As (UGT1A1, 1A3,
1A7, 1A8, 1A9 and 1A10), UGT1A1 and UGT1A9 were
the most efficient conjugating enzymes with the smallest Km
values (≤1 μM) and highest intrinsic clearance values.
Regardless of their distinctive substrate specificities towards
the flavonols, UGT1A1 and 1A3 favored 7-O-
glucuornidation, whereas UGT1A7, 1A8, 1A9 and 1A10
preferred 3-O-glucuronidation. The highly different kinetic
parameters between 3-O- and 7-O- glucuronidation sug-
gested that the (at least) two distinct binding modes within
the catalytic domain were responsible for the formation of
these two glucuronide isomers, which should be considered
as useful “expert knowledge” for modeling and predicting
UGT1A-mediated glucuronidation. Studies are ongoing to
explore these binding modes using homology-based
approaches and plant UGT crystal structures.
The kinetics profiling studies indicate that all UGT1A
isoforms have more than one binding site for flavonoids,
since their glucuronidation of flavonols displayed strong
substrate inhibition patterns. To explain this pattern, one
model was proposed by Hutzler and Tracy, who advocate a
hypothetical two-site binding model, in which one binding
site is productive, whereas the other site is inhibitory and
operable only at high substrate concentrations (30). Anoth-
er group of investigators proposed a compulsory ordered bi
bi (two substrates and two products) kinetic model to
explain substrate inhibition of UGT1As (28). We used a
similar approach and showed that UGT1A9 catalyzed
glucuronidation of 3,7,4′-trihydroxyflavone indeed followed
the identical kinetic model (Supplementary Materials Fig.
S3). Therefore, one reasonable explanation of the substrate
inhibition was that binding of the aglycone substrate to the
enzyme-UDP complex led to a nonproductive dead-end
complex that slows the completion of the catalytic cycle
(31).
The hypothesis that there are at least two distinct
binding modes present in UGT1As is also strongly
supported by the fact that positional (3-O- or 7-O-)
glucuronidation always has different kinetic parameters
(24). The differentiated Km (binding affinity) or Vmax
(reflecting turnover (Kcat)) values in production of the
regiospecific glucuronides indicate that the enzymes pro-
vide two divergent interacting environments within which a
single flavonol may orient differently. This most likely
resulted from one very large active site, instead of two
separate small active sites, as supported by the use of
UGT1A1 homology models (32,33). Taken together, this
body of evidences is in strong support of the theorem that
there are at least two binding modes for UGT1A-mediated
glucuronidation of flavonols, which can be used as the
“expert knowledge” for in silico modeling of UGT1A-
mediated glucuronidation.
ACKNOWLEDGMENTS
This work was supported by grants from the National
Institutes of Health (GM070737) to MH.
Structure of flavonols could significantly impact the
regioselective glucuronidation rates, regardless of how
many modes of bindings are present in UGT1As. In
general, the preference order was 3-OH or 7-OH>4′-
OH>5-OH among the model flavonols, where the 5-OH
and 4′-OH groups were relatively inactive for glucuronida-
tion. These two positions remain poorly active even in the
absence of more active -OH groups, as reaction rates of
eight flavones with only 5 or 4′-OH available for
conjugation was much slower than those shown in this
paper (Supplementary Materials Table S2). Even though 5-
OH or 4′-OH was not glucuronidated itself, the presence of
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