Critical roles of residues 36 and 40 in the phenol and tertiary amine aglycone substrate selectivities of UDP-glucuronosyltransferases 1A3 and 1A4

Article abstract of DOI:10.1124/mol.107.037952

Despite high sequence identity, UGT1A3 and UGT1A4 differ in terms of substrate selectivity. UGT1A3 glucuronidates the planar phenols 1-naphthol (1-NP) and 4-methylumbelliferone (4-MU), whereas UGT1A4 converts the tertiary amines lamotrigine (LTG) and trifluoperazine (TFP) to quaternary ammonium glucuronides. Residues 45 to 154 (which incorporate 21 of the 35 amino acid differences) and 45 to 535 were exchanged between UGT1A3 and UGT1A4 to generate UGT1A3-4_(45-535), UGT1A3-4_(45-154)-3, UGT1A4-3 _(45-535), and UGT1A4-3_(45-154)-4 hybrid proteins. Although differences in kinetic parameters were observed between the parent enzymes and chimeras, UGT1A4-3_(45-535) and UGT1A4-3_(45-154)-4 [but not UGT1A3-4_(45-535) and UGT1A3-4_(45-154)-3] retained the capacity to glucuronidate LTG and TFP. Likewise, UGT1A3-4_(45-535) and UGT1A3-4_(45-154)-3 retained the capacity to glucuronidate 1-NP and 4-MU, but UGT1A4-3_(45-535) and UGT1A4-3_(45-154)-4 exhibited low or absent activity. Within the first 44 residues, UGT1A3 and UGT1A4 differ in sequence at positions 36 and 40. "Reciprocal" mutagenesis was performed to generate the UGT1A3(I36T), UGT1A3(H40P), UGT1A4(T36I), and UGT1A4 (P40H) mutants. The T36I and P40H mutations in UGT1A4 reduced in vitro clearances for LTG and TFP glucuronidation by >90%. Conversely, the I36T and H40P mutations in UGT1A3 reduced the in vitro clearances for 1-NP and 4-MU glucuronidation by >90%. Introduction of the single H40P mutation in UGT1A3 conferred LTG and TFP glucuronidation, whereas the single T36I mutation in UGT1A4 conferred 1-NP and 4-MU glucuronidation. Thus, residues 36 and 40 of UGT1A3 and UGT1A4 are pivotal for the respective selectivities of these enzymes toward planar phenols and tertiary amines, although other regions of the proteins influence binding affinity and/or turnover. Copyright

Full text of DOI:10.1124/mol.107.037952

0026-895X/07/7204-1054–1062$20.00
MOLECULAR PHARMACOLOGY
Copyright © 2007 The American Society for Pharmacology and Experimental Therapeutics
Mol Pharmacol 72:1054–1062, 2007
Vol. 72, No. 4
37952/3257426
Printed in U.S.A.
Critical Roles of Residues 36 and 40 in the Phenol and
Tertiary Amine Aglycone Substrate Selectivities of
UDP-Glucuronosyltransferases 1A3 and 1A4
Takahiro Kubota,1 Benjamin C. Lewis, David J. Elliot, Peter I. Mackenzie, and
John O. Miners
Department of Clinical Pharmacology, Flinders University and Flinders Medical Centre, Adelaide, Australia
Received May 8, 2007; accepted July 16, 2007
ABSTRACT
Despite high sequence identity, UGT1A3 and UGT1A4 differ in exhibited low or absent activity. Within the first 44 residues,
terms of substrate selectivity. UGT1A3 glucuronidates the pla- UGT1A3 and UGT1A4 differ in sequence at positions 36 and
nar phenols 1-naphthol (1-NP) and 4-methylumbelliferone (4- 40. “Reciprocal” mutagenesis was performed to generate the
MU), whereas UGT1A4 converts the tertiary amines lamotrigine UGT1A3(I36T), UGT1A3(H40P), UGT1A4(T36I), and UGT1A4
(LTG) and trifluoperazine (TFP) to quaternary ammonium glu- (P40H) mutants. The T36I and P40H mutations in UGT1A4 re-
curonides. Residues 45 to 154 (which incorporate 21 of the 35 duced in vitro clearances for LTG and TFP glucuronidation by
amino acid differences) and 45 to 535 were exchanged be- Ͼ90%. Conversely, the I36T and H40P mutations in UGT1A3
tween UGT1A3 and UGT1A4 to generate UGT1A3-4(45–535)
,
reduced the in vitro clearances for 1-NP and 4-MU glucuronida-
UGT1A3-4(45–154)-3, UGT1A4-3(45–535), and UGT1A4-3(45–154)-4 tion by Ͼ90%. Introduction of the single H40P mutation in
hybrid proteins. Although differences in kinetic parameters UGT1A3 conferred LTG and TFP glucuronidation, whereas the
were observed between the parent enzymes and chimeras, single T36I mutation in UGT1A4 conferred 1-NP and 4-MU glu-
UGT1A4-3(45–535) and UGT1A4-3(45–154)-4 [but not UGT1A3- curonidation. Thus, residues 36 and 40 of UGT1A3 and UGT1A4
4(45–535) and UGT1A3-4(45–154)-3] retained the capacity to glu- are pivotal for the respective selectivities of these enzymes toward
curonidate LTG and TFP. Likewise, UGT1A3-4(45–535) and planar phenols and tertiary amines, although other regions of the
UGT1A3-4(45–154)-3 retained the capacity to glucuronidate proteins influence binding affinity and/or turnover.
1-NP and 4-MU, but UGT1A4-3(45–535) and UGT1A4-3(45–154)-4
Glucuronidation involves the covalent linkage of glucu- and hydroxysteroids (Miners and Mackenzie 1991; Radomin-
ronic acid, derived from the cofactor UDP-glucuronic acid
(UDPGA), to a substrate bearing a nucleophilic functional
group or atom, most commonly an alcohol (aliphatic or phe-
nolic), amine, or carboxylic acid. Given the widespread occur-
rence of these functional groups in synthetic and “biological”
chemicals, glucuronidation is not surprisingly an essential
clearance and detoxification mechanism for a myriad of com-
pounds that include drugs, environmental chemicals, and
endogenous compounds particularly bilirubin, fatty acids,
ska-Pandya et al., 1999). The glucuronidation reaction is
catalyzed by the UDP-glucuronosyltransferase (UGT) super-
family of enzymes. Almost 30 UGT genes have been identi-
fied in humans, and these have been classified in three sub-
families: 1A, 2A, and 2B (Mackenzie et al., 2005). UGT1A and
UGT2B enzymes, which are the largest subfamilies, are of
greatest importance in the metabolism of xenobiotics and
endogenous compounds in humans. It is well established that
the individual UGT1A and UGT2B enzymes exhibit distinct
but overlapping aglycone substrate selectivities and differ in
terms of regulation of expression (Tukey and Strassburg
2000; Radominska-Pandya et al., 1999; Miners et al., 2004;
Kiang et al., 2005). However, the structural basis of UGT
enzyme substrate selectivity has yet to be fully elucidated.
UGT1A enzymes comprise a unique amino terminus do-
main of 285 to 289 residues but an identical carboxyl termi-
nus comprising 246 residues that arise from the splicing of
This work was supported by a grant from the National Health and Medical
Research Council of Australia. P.I.M. is a National Health and Medical Re-
search Council of Australia Senior Principal Research Fellow. T.K. was sup-
ported by an Overseas Study Scholarship from the Japanese Research Foun-
dation.
1 Current affiliation: Department of Drug Metabolism and Biopharmaceu-
tics, Faculty of Pharmacy, Chiba Institute of Science, Chiba, Japan.
Article, publication date, and citation information can be found at
http://molpharm.aspetjournals.org.
doi:10.1124/mol.107.037952.
ABBREVIATIONS: UDPGA, UDP-glucuronic acid; UGT, UDP-glucuronosyltransferase; LTG, lamotrigine; TFP, trifluoperazine; 4-MU, 4-methyl-
umbelliferone; 1-NP, 1-naphthol; HEK, human embryonic kidney; HPLC, high-performance liquid chromatography.
1054

Molecular Basis of UGT1A3 and UGT1A4 Substrate Selectivity
1055
individual “first” exons to four common exons (Ritter et al., this laboratory to identify the substrate binding domain of
1992; Mackenzie et al., 2005). Whereas the conserved car- UGT2B15 (Lewis et al., 2007), we used common SphI and
boxyl terminus contributes to cofactor (UDPGA) binding HpaI restriction sites in the UGT1A3 and UGT1A4 cDNAs to
(Radominska-Pandya et al., 1999), the aglycone substrate generate UGT1A3-4(45–535), UGT1A3-4(45–154)-3, UGT1A4-
selectivity of UGT1A enzymes is clearly determined by the 3(45–535) and UGT1A4-3(45–154)-4 chimeras. Substrate selec-
amino terminus. Although UGT2B enzymes are encoded by tivity was assessed using LTG, TFP, 4-MU, and 1-NP as the
unique genes and therefore differ in sequence throughout the model substrates (Fig. 2). After demonstration that substrate
polypeptide chain, studies with chimeric rat, rabbit and hu- selectivity was associated with the first 44 amino acids of
man proteins and nuclear magnetic resonance analysis sim- each enzyme, the two residues (36 and 40) that differ in this
ilarly implicate the amino terminus domains of several region of the mature protein were subsequently shown to be
UGT2B enzymes in substrate binding and selectivity (Mack- critical for the respective selectivities of UGT1A3 and
enzie 1990; Li et al., 1997; Lewis et al., 2007). Mutagenesis UGT1A4.
has further identified a limited number of amino acids that
influence the binding of UGT1A6, UGT1A10, UGT2B15, and
UGT2B17 substrates (Dubois et al., 1999; Ouzzine et al.,
Materials and Methods
Materials
2000; Senay et al., 2002; Martineau et al., 2004; Xiong et al.,
2006; Lewis et al., 2007), and mis-sense mutations arising
from genetic polymorphism of both UGT1A and UGT2B
genes are known to alter substrate binding and/or turnover
(Miners et al., 2002; Guillemette, 2003).
LTG and lamotrigine N2-glucuronide were obtained from the
Wellcome Research Laboratories (Beckenham, UK). 4-MU, 4-meth-
ylumbelliferone-␤-D-glucuronide, 1-naphthol (1-NP), 1-naphthol-␤-D-
glucuronide, TFP (dihydrochloride salt), and UDPGA (trisodium
salt) were purchased from Sigma-Aldrich (Sydney, Australia). Pfu
Ultra Polymerase was from Stratagene (La Jolla, CA), and restric-
tion enzymes were supplied by New England Biolabs (Ipswich, MA).
Dulbecco’s modified Eagle’s medium, minimal essential medium
nonessential amino acids (10 mM), and penicillin/streptomycin solu-
tion (penicillin-G, 5000 U/ml; streptomycin sulfate, 5000 mg/ml)
were purchased from Invitrogen (Carlsbad, CA). Other reagents and
organic solvents were of analytical reagent grade. The UGT1A3 and
UGT1A4 cDNAs were isolated as described previously (Mojarrabi et
al., 1996; Uchaipichat et al., 2004).
Within the UGT1A subfamily, UGT1A3 and UGT1A4 dif-
fer in sequence by only 35 amino acids (Fig. 1). Despite
sharing 93.4% sequence identity, however, UGT1A3 and
UGT1A4 differ markedly in terms of substrate selectivity. It
is noteworthy that only UGT1A4 converts tertiary amines
such as lamotrigine (LTG) and trifluoperazine (TFP) (see Fig.
2 for structures) to a quaternary ammonium glucuronide
(Green and Tephly, 1998; Green et al., 1998; Rowland et al.,
2006; Uchaipichat et al., 2006). Conversely, UGT1A4 lacks
activity toward the planar phenols 4-methylumbelliferone
(4-MU) and 1-naphthol (1-NP) (see Fig. 2 for structures),
whereas UGT1A3 and indeed most other human UGTs cat-
alyze the glucuronidation of these compounds (Green et al.,
1998; Uchaipichat et al., 2004).
The high sequence identity of UGT1A3 and UGT1A4 pro-
vides a convenient experimental model for probing the struc-
tural basis for the differing substrate selectivities of UGT
enzymes. The majority of the amino acid differences between
UGT1A3 and UGT1A4 occur between positions 47 and 127
(Fig. 1). Based on the chimeragenesis approach adopted in
Methods
Generation of Chimeric and Mutant UGT cDNAs. The
UGT1A3 and UGT1A4 cDNAs (accession numbers NM_019093 and
NM_007120, respectively) were independently subcloned into pBlue-
script-II-SK(ϩ) (Stratagene, La Jolla, CA) for DNA manipulation.
Parental templates, which are of equal length, were used to generate
four UGT1A3/1A4 chimeras, namely UGT1A3-4(45–535) (referred to
subsequently as UGT1A3-4), UGT1A3-4(45–154)-3 (referred to subse-
quently as UGT1A3-4-3), UGT1A4-3(45–535) (referred to subsequently
as UGT1A4-3), and UGT1A4-3(45–154)-4 (referred to subsequently as
UGT1A4-3-4) according to the strategy shown in Fig. 3. Digestion of
the parental templates with SphI and NotI gave a 1487-base pair
Fig. 1. Amino acid alignment of UGT1A3 and UGT1A4. Identical resi-
dues are shaded gray. Residues 285 to 534 are identical; hence, sequence
beyond position 290 is not shown. Overall, the UGT1A3 and UGT1A4
proteins share 93.4% sequence identity and 95.9% similarity.
Fig. 2. Structures of lamotrigine (LTG), trifluoperazine (TFP), 1-naph-
thol (1-NP), and 4-methylumbelliferone (4-MU). Arrows indicate the pri-
mary sites of glucuronidation.

1056
Kubota et al.
fragment (encoding residues 45–534) that was exchanged between conditions were optimized for linearity with respect to both protein
the parental templates to generate the UGT1A3-4 and UGT1A4-3 concentration and incubation time. Details of individual assays,
cDNAs. Likewise, the UGT1A3-4-3 and UGT1A4-3-4 chimeras were based on methods established in this laboratory, are provided below.
4-MU and 1-NP glucuronidation. Incubation mixtures contained
UDPGA (5 mM), MgCl2 (4 mM), HEK293 cell lysate expressing each
recombinant UGT1A protein (0.5 mg/ml), phosphate buffer (0.1 M,
pH 7.4), and 4-MU or 1-NP (9–11 concentrations) in a total volume
of 0.2 ml. Incubations were conducted in air at 37°C for 75 (4-MU) or
120 (1-NP) min, after which time reactions were terminated by the
addition of perchloric acid (70%; 3 ␮l) and cooling on ice. 4-Methyl-
umbelliferone-␤-D-glucuronide and 1-naphthol-␤-D-glucuronide were
separated and quantified according to the HPLC methods reported
by Udomuksorn et al. (2007).
LTG N2-glucuronidation. The incubation mixture, in a total vol-
ume of 0.2 ml, contained UDPGA (5 mM), MgCl2 (4 mM), HEK293
cell lysate expressing each recombinant UGT1A protein (0.5 mg/ml),
and LTG (10 concentrations). Because of its limited solubility LTG
was dissolved in 1 M phosphoric acid containing 10% acetonitrile.
Phosphate buffer (0.1 M, pH 7.4) was generated in situ by the
addition of KOH (1 M, 37.2 ␮l). Incubations were performed in air at
37°C for 75 min. Reactions were terminated by the addition of 70%
perchloric acid (3 ␮l) and cooling on ice, and the LTG N2-glucuronide
was separated and quantified according to the HPLC procedure of
Rowland et al. (2006).
TFP glucuronidation. Incubations contained UDPGA (5 mM),
MgCl2 (4 mM), HEK293 cell lysate expressing each recombinant
UGT1A protein (0.25 mg/ml), 50 mM Tris-HCl buffer, pH 7.4, and
TFP (10 concentrations). Incubations were conducted in air at 37°C
for 20 min, after which time reactions were terminated by the addi-
tion of 4% acetic acid/96% methanol (0.2 ml) and cooling on ice. TFP
glucuronide was separated and quantified by HPLC as described by
Uchaipichat et al. (2006).
All incubations were performed in duplicate, and data points rep-
resent the mean of duplicate measurements (Ͻ10% difference).
Overall within-day assay reproducibility for each procedure was
assessed by measuring the rate of glucuronide formation in 8 to 10
separate incubations with either UGT1A3 or UGT1A4 as the enzyme
source. Coefficients of variation, determined for both “low” and
“high” substrate concentrations with each assay, were Ͻ7.3%. Limits
of quantification, expressed as rates of glucuronide formation, were
1.5, 1.0, 0.7, and 0.5 pmol/min/mg for the TFP, LTG, 4-MU, and 1-NP
glucuronidation assays, respectively.
generated by exchanging the 330-base pair fragment (encoding res-
idues 45–154) obtained by digestion of the parental templates with
SphI and HpaI. UGT1A3(I36T), UGT1A3(H40P), UGT1A4(T36I),
and UGT1A4(P40H) were generated by reciprocal mutagenesis at
positions 36 and 40 of the UGT1A3 and UGT1A4 cDNA templates.
Mutants were engineered using the QuikChange-II site-directed mu-
tagenesis protocol (Stratagene) with the oligonucleotide primers
shown in Table 1. After digestion with XhoI and NotI (Fig. 3), coding
sequences were subcloned into the mammalian expression vector
pEF-IRES-puro6 for transfection into HEK293 cells (Uchaipichat et
al., 2004). All DNA manipulations were confirmed on both strands by
direct sequencing (ABI PRISM 3100; Applied Biosystems, Foster
City, CA).
Expression of UGT Enzymes, Chimeras and Mutants. The
UGT1A3, UGT1A4, UGT1A3-4, UGT1A3-4-3, UGT1A4-3, UGT1A4-
3-4,
UGT1A3(I36T),
UGT1A3(H40P),
UGT1A4(T36I)
and
UGT1A4(P40H) cDNAs were stably expressed in the human embry-
onic kidney cell line, HEK293. After transfection, cells were incu-
bated in a humidified incubator with an atmosphere of 5% CO2 at
37°C in Dulbecco’s modified Eagle’s medium prepared in the pres-
ence of sodium bicarbonate (44 mM), minimal essential medium
nonessential amino acids (0.1 mM), fetal bovine serum (10%), and
penicillin/streptomycin (100 U/ml). Media were supplemented with
puromycin (1.0 mg/l) for selection. Cells were grown to no more than
90% confluence. The conditioned medium was decanted, and the
cultured cells were washed (three times) with prechilled (4°C) phos-
phate-buffered saline, pH 7.4. Cell pellets were resuspended in a
storage buffer (10 mM K2HPO4/KH2PO4, pH 7.4, and 0.1 mM EDTA)
and kept at Ϫ80°C until use. Cells were lysed using the sonication
method described by Uchaipichat et al. (2004).
Enzyme Assays. Substrate concentration ranges were deter-
mined from initial activity screening experiments (four concentra-
tions) to permit reliable identification of the glucuronidation kinetic
model and kinetic constants for each protein source. Incubation
Immunoblotting. Twenty micrograms of HEK293 cell lysate pro-
tein was subjected to 10% SDS-polyacrylamide gel electrophoresis
and electrophoretically transferred onto nitrocellulose (Bio-Rad Lab-
oratories, Hercules, CA). Blots were probed with an anti- UGT1A
antibody (diluted 1:1500; BD Gentest, Woburn, MA) that recognizes
an epitope in the conserved carboxyl-terminal domain of UGT1A
proteins followed by goat anti-rabbit IgG coupled to horseradish
peroxidase (diluted 1:3000; Southern Biotechnology Associates, Bir-
mingham, AL). Proteins were visualized by chemiluminescence (en-
hanced chemiluminescence-plus; GE Healthcare, Chalfont St. Giles,
Buckinghamshire, UK) and band intensities were determined with a
Bio-Rad model GS-700 imaging densitometer. Relative expression
levels reported under Results represent the mean of duplicate exper-
iments (Ͻ 10% variance).
TABLE 1
Oligonucleotides used for the construction of the UGT1A3 and UGT1A4
mutants at residues 36 and 40
Mutagenized positions are underlined. Accession numbers for UGT1A3 and UGT1A4
are AY435138.1 and AY435139.1, respectively.
Fig. 3. Construction of the UGT1A3-UGT1A4 chimeras. Arrow heads
show the junctions between various UGT1A3 and UGT1A4 coding se-
quences. 5Ј- to 3Ј-XhoI and NotI restriction sites flank the entire UGT1A3
and UGT1A4 coding sequences and were used in the directional transfer
of cDNAs into the mammalian expression vector pEF-IRESpuro6. SphI
and HpaI restriction sites were employed to construct chimeric cDNAs
that result in exchange of residues 45 to 154 and 45 to 535 of UGT1A3
and UGT1A4.
Mutant
Primers
UGT1A3(I36T)
UGT1A3(H40P)
UGT1A4(T36I)
UGT1A4(P40H)
5Ј-GTGTTGGTGGTGCCCACTGATGGCAGCCACTGG-3Ј
5Ј-CCCATTGATGGCAGCCCCTGGCTCAGCATGCGG-3Ј
5Ј-GGTGTTGGTGGTGCCCATTGATGGCAGCCCCTG-3Ј
5Ј-CCCACTGATGGCAGCCACTGGCTCAGCATGCGG-3Ј

Molecular Basis of UGT1A3 and UGT1A4 Substrate Selectivity
1057
Data Analysis. Kinetic constants for 4-MU, 1-NP, LTG, and TFP relative to UGT1A3 to permit comparison of Vmax values and
glucuronidation by each recombinant protein, including chimeras
and mutants, were obtained by fitting untransformed data to the
following models using Enzfitter (Biosoft, Cambridge, UK).
Michaelis-Menten equation.
in vitro clearances (CLint and CLmax).
Consistent with previous reports, UGT1A4, but not
UGT1A3, glucuronidated LTG and TFP. Of the chimeras,
UGT1A4-3 and UGT1A4-3-4 glucuronidated LTG and TFP,
whereas UGT1A3-4 and UGT1A3-4-3 lacked activity toward
these substrates. Kinetic constants for LTG and TFP glucu-
ronidation by UGT1A4, UGT1A4-3, and UGT1A4-3-4 are
shown in Tables 2 and 3, respectively, whereas kinetic plots
for LTG (as the “model” tertiary amine substrate) glucu-
ronidation by UGT1A4 and these chimeras are illustrated in
Fig. 5. In vitro clearances for LTG glucuronidation by
UGT1A4, UGT1A4-3, and UGT1A4-3-4 varied approximately
2-fold (0.70–1.34 ␮l/min/mg), although it should be noted
that CLint and CLmax are not equivalent parameters. In
contrast to the Michaelis-Menten kinetics observed with
UGT1A4 and UGT1A4-3, LTG glucuronidation by UGT1A4-
3-4 exhibited sigmoidal kinetics (modeled by the Hill equa-
tion) (Fig. 5); Km (or S50) and Vmax values differed between
each protein (Table 2). TFP glucuronidation by UGT1A4-3
and UGT1A4-3-4 also exhibited sigmoidicity in contrast to
the substrate inhibition kinetics observed for TFP metabo-
lism by UGT1A4 (Table 3). Vmax values for TFP glucuronida-
tion by the chimeras were lower than that observed for
UGT1A4 resulting in lower in vitro clearances (Table 3).
Again, however, caution should be exercised in comparing
CLint and CLmax values for TFP glucuronidation by UGT1A4
and the chimeras. The Michaelis-Menten and substrate in-
hibition kinetics observed for UGT1A4 catalyzed glucu-
ronidation of LTG and TFP, respectively, are consistent with
published data from this laboratory (Rowland et al., 2006;
Uchaipichat et al., 2006).
Vmax ϫ ͓S͔
Km ϩ ͓S͔
v ϭ
(1)
where v is the rate of reaction, Vmax is the maximum velocity, Km is
the Michaelis-Menten constant (substrate concentration at 0.5 Vmax),
and [S] is the substrate concentration.
Substrate inhibition.
Vmax
v ϭ
(2)
1 ϩ ͑Kmր͓S͔͒ ϩ ͓͑S͔րKsi͒
where Ksi is the constant describing the substrate inhibition inter-
action.
The Hill equation, which describes sigmoidal kinetics:
nH
Vmax ϫ ͓S͔
S50 ϩ ͓S͔
v ϭ
nH
where S50 is the substrate concentration resulting in 50% of Vmax
(analogous to Km in previous equations) and nH is the Hill coefficient.
For reactions exhibiting Michaelis-Menten and substrate inhibi-
tion kinetics, intrinsic clearance (CLint) was calculated as Vmax/Km.
Autoactivation is characterized by the dependence of clearance on
substrate concentration. For reactions exhibiting sigmoidal kinetics,
maximum clearance provides an alternative estimate of the highest
clearance attained [that is, when the enzyme is fully activated before
saturation occurs (Houston and Kenworthy 2000)]. CLmax was deter-
mined as:
Vmax
S50
͑nH Ϫ 1͒
nH͑nH Ϫ 1͒
CLmax
ϭ
ϫ
(3)
1րnH
UGT1A3, but not UGT1A4, glucuronidated 1-NP and
4-MU, and the substrate inhibition kinetics observed with
both substrates are consistent with previous data from this
laboratory (Uchaipichat et al., 2004). Of the chimeras, 1-NP
was metabolized only by UGT1A3-4 and UGT1A3-4-3. How-
ever, UGT1A3-4, UGT1A3-4-3, UGT1A4-3, and UGT1A4-3-4
all glucuronidated 4-MU, although the in vitro clearances of
the latter two chimeras were low compared with UGT1A3,
UGT1A3-4, and UGT1A3-4-3. Kinetic constants for 1-NP and
Goodness of fit to kinetic and inhibition models was assessed from
the F statistic, r2 values, parameter standard error estimates, and
95% confidence intervals. Each kinetic constant is presented as the
value Ϯ S.E. of the parameter estimate. Kinetic curves are shown as
Eadie-Hofstee plots from fitting to the Michaelis-Menten, substrate
inhibition, and Hill equations.
Results
Expression of UGT1A3, UGT1A4 and the UGT1A3/4 chi-
meras is shown in Fig. 4. Relative expression levels of
UGT1A3, UGT1A3-4, UGT1A3-4-3, UGT1A4, UGT1A4-3,
and UGT1A4-3-4 were 1, 0.74, 0.73, 0.69, 1.02, and 1.18,
respectively. Given the differences in expression of the chi-
meras relative to the parent enzymes, activities reported
throughout this article have been normalized for expression
TABLE 2
Derived kinetic constants for LTG glucuronidation by UGT1A3 and
UGT1A4 chimeras and mutants
Data shown as parameters Ϯ S.E. of parameter fit.
e
CLint or
a,b
c
d
Enzyme
Km or S50
Vmax
f
CLmax
␮M
pmol/min/mg
␮l/min. mg
UGT1A3
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
0.86
0.70
1.34
0.98
N.A.
0.06
UGT1A3–4
UGT1A3–4-3
UGT1A3(I36T)
UGT1A3(H40P)
UGT1A4
N.A.
N.A.
N.A.
N.A.
235 Ϯ 8a
1162 Ϯ 44a
341 Ϯ 4a
1422 Ϯ 21c
N.A.
203 Ϯ 1
803 Ϯ 9
460 Ϯ 2
2520 Ϯ 26
N.A.
UGT1A4–3
UGT1A4–3-4
UGT1A4(T36I)
UGT1A4(P40H)
1105 Ϯ 10b
63 Ϯ 0.3
N.A., no activity.
a From fitting to the Michaelis Menten equation.
Fig. 4. Immunoblot analysis of the parent, chimeric, and mutant
UGT1A3 and UGT1A4 recombinant proteins. Twenty micrograms of cell
lysate protein was resolved by SDS-polyacrylamide gel electrophoresis,
transferred to nitrocellulose, and probed with anti-UGT1A antiserum.
Immunoreactive bands occur at 56 to 58 kDa.
b From fitting to the substrate inhibition equation (Ksi ϭ 7695 Ϯ 267 ␮M).
c From fitting to the Hill equation (n ϭ 1.38 Ϯ 0.02).
d Normalized for expression relative to UGT1A3.
e Calculated as Vmax/Km.
f Calculated according to eq. 4.

1058
Kubota et al.
4-MU glucuronidation by UGT1A3 and the chimeras are CLint values for 4-MU glucuronidation by UGT1A3,
shown in Tables 4 and 5, respectively, whereas kinetic plots
for 1-NP (as the model phenolic substrate) glucuronidation
by UGT1A3 and chimeras are illustrated in Fig. 6. Substrate
inhibition kinetics were observed for 1-NP and 4-MU metab-
olism by the chimeras, except for 1-NP glucuronidation by
UGT1A3-4 (Michaelis-Menten) and 4-MU glucuronidation by
UGT1A4-3 (sigmoidal) (Tables 4 and 5, Fig. 6). Whereas
UGT1A3-4, and UGT1A3-4-3 were similar (0.87–1.06 ␮l/min/
mg) (Table 5), CLint values for 1-NP glucuronidation by the
respective chimeras were 8.5- and 3-fold higher than the
CLint determined for UGT1A3 (Table 4). However, Km values
for both substrates were lower for the chimeras compared
with UGT1A3.
The first 25 residues constitute a signal peptide that is
cleaved upon insertion of the UGT protein into the mem-
brane of the endoplasmic reticulum. UGT1A3 and UGT1A4
differ by only two amino acids between positions 26 and 44
(Fig. 1); UGT1A3 has Ile and His at positions 36 and 40,
respectively, whereas UGT1A4 has Thr and Pro at these
positions. Thus, “reciprocal” mutagenesis was performed,
and the capacity of the UGT1A3(I36T), UGT1A3(H40P),
UGT1A4(T36I), and UGT1A4(P40H) mutants to glucu-
ronidate LTG, TFP, 1-NP, and 4-MU was determined. Ex-
pression of the mutants is shown in Fig. 4; relative expres-
sion levels of UGT1A3, UGT1A3(I36T), UGT1A4(H40P),
UGT1A4(T36I), and UGT1A4(P40H) were 1, 0.59, 0.49, 0.75,
and 0.41.
TABLE 3
Derived kinetic parameters for TFP glucuronidation by UGT1A3 and
UGT1A4 chimeras and mutants
Data shown as parameters Ϯ S.E. of parameter fit.
Hill
d
CLint or
a
b
c
Enzyme
Km or S50
Coefficient
Vmax
e
CLmax
b
(nH
)
␮M
pmol/min/mg ␮l/min/mg
UGT1A3
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
2.21
28
0.50
0.71
N.A.
1.75
UGT1A3–4
UGT1A3–4-3
UGT1A3(I36T)
N.A.
N.A.
N.A.
1.34 Ϯ 0.04
N.A.
UGT1A3(H40P) 31 Ϯ 1b
121 Ϯ 2
1119 Ϯ 7
46 Ϯ 1
72 Ϯ 4
N.A.
UGT1A4
40 Ϯ 1a
58 Ϯ 1b
52 Ϯ 3b
N.A.
UGT1A4–3
UGT1A4–3-4
UGT1A4(T36I)
1.58 Ϯ 0.04
1.62 Ϯ 0.07
N.A.
The T36I mutation in UGT1A4 abolished activity toward
LTG and TFP, whereas the P40H mutation reduced the in
vitro clearances for the glucuronidation of these substrates
by an order of magnitude and altered the kinetic model for
LTG/TFP glucuronidation (Tables 2 and 3, Fig. 5). Likewise,
the I36T mutation in UGT1A3 reduced the in vitro clear-
ances for 1-NP and 4-MU glucuronidation by 92 to 94%,
UGT1A4(P40H) 28 Ϯ 0.8b 1.28 Ϯ 0.03
83 Ϯ 2
N.A., no activity.
a From fitting to the substrate inhibition equation (Ksi ϭ 241 Ϯ 18 ␮M).
b From fitting to the Hill equation.
c Normalized for expression relative to UGT1A3.
d Calculated as Vmax/Km.
e Calculated according to eq. 4.
Fig. 5. Eadie-Hofstee plots for LTG glucuronidation by UGT1A4, UGT1A4-3, UGT1A4-3-4, UGT1A4(P40H), and UGT1A3(H40P). Points show
experimentally determined values (mean of duplicate estimates), whereas curves are the computer-generated curves of best fit.

Molecular Basis of UGT1A3 and UGT1A4 Substrate Selectivity
1059
whereas the H40P mutation essentially abolished the metab- particularly the capacity to glucuronidate tertiary amines
olism of these compounds (Tables 4 and 5). Substitution of and phenols. Amino acid sequence analysis and studies
Pro for His at position 40 of UGT1A3, but not Thr for Ile at
position 36, conferred tertiary amine glucuronidation activ-
ity. The CLint values for LTG glucuronidation by
UGT1A3(H40P) and UGT1A4 were close in value, although
both the Km and Vmax were lower for the mutant (Table 2).
Like UGT1A4, LTG glucuronidation by UGT1A3(H40P) fol-
lowed Michaelis-Menten kinetics, whereas TFP glucuronida-
tion by this mutant exhibited sigmoidal kinetics (compared
with substrate inhibition observed for UGT1A4). The CLint
value for TFP glucuronidation by UGT1A3(H40P) was only
8% of that of UGT1A4, largely because of a decrease in Vmax
(Table 3). Conversely, substitution of Ile for Thr at position
36 of UGT1A4, but not His for Pro at position 40, conferred
phenolic glucuronidation activity. The CLint for 1-NP glucu-
ronidation by UGT1A4(T36I) was 6.5-fold higher than that of
UGT1A3 (Table 4), whereas the CLint for 4-MU glucuronida-
tion by this mutant was only 17% of that of UGT1A3 as a
result of a markedly lower Vmax (Table 5).
with chimeric UGT proteins are consistent with the hy-
pothesis that amino-terminal domains are responsible for
the unique aglycone substrate selectivities of UGTs (Mack-
enzie 1990; Li et al., 1999; Radominska-Pandya et al.,
1999; Lewis et al., 2007). Recent work from this laboratory
demonstrated that UGT2B7-UGT2B15 chimeras that in-
corporated residues 61 to 194 of UGT2B15 exhibited
UGT2B15-like substrate selectivity and glucuronidation
kinetics (Lewis et al., 2007). Like UGT2B subfamily pro-
teins, greatest sequence dissimilarity occurs between res-
idues 60 and 190 of UGT1A proteins. Indeed, 21 of the 35
amino acid differences between UGT1A3 and UGT1A4 oc-
cur between positions 47 and 127, and we reasoned that
this region was responsible for the differing substrate se-
lectivities of the two enzymes.
Thus, residues 45 to 154 and 45 to 535 were exchanged
between UGT1A3 and UGT1A4 to generate UGT1A3-4,
UGT1A3-4-3, UGT1A4-3, and UGT1A4-3-4 hybrid proteins.
Although differences in kinetic parameters were observed
between the parent enzymes and chimeras (discussed below),
UGT1A4-3 and UGT1A4-3-4 retained the characteristic ca-
pacity of UGT1A4 to glucuronidate the tertiary amines LTG
and TFP, whereas UGT1A3-4 and UGT1A3-4-3 lacked activ-
ity toward these compounds. Likewise, UGT1A3-4 and
UGT1A3-4-3 retained the UGT1A3-like capacity to glucu-
ronidate the phenolic substrates 1-NP and 4-MU. UGT1A4-3
and UGT1A4-3-4 also glucuronidated 4-MU (but not 1-NP),
although in vitro clearances for 4-MU glucuronidation by
these chimeras were an order of magnitude or more lower
than for UGT1A3.
Discussion
Despite sharing 93.4% sequence identity UGT1A3 and
UGT1A4 differ in terms of aglycone substrate selectivity,
TABLE 4
Derived kinetic constants for 1-NP glucuronidation by UGT1A3 and
UGT1A4 chimeras and mutants
Data shown as parameters Ϯ S.E. of parameter fit.
a,b
a
c
d
Enzyme
Km
Ksi
Vmax
CLint
␮M
␮M
pmol/min/mg ␮l/min/mg
UGT1A3
2330 Ϯ 24a
314 Ϯ 2b
342 Ϯ 18a
3399 Ϯ 360a
N.A.
916 Ϯ 13
300 Ϯ 2
351 Ϯ 1
127 Ϯ 3
57 Ϯ 3
N.A.
0.13
1.12
0.37
0.01
N.A.
N.A.
N.A.
N.A.
0.84
N.A.
Taken together, these data indicate that the domain span-
ning residues 1 to 44 is critical for the unique substrate
selectivities of UGT1A3 and UGT1A4. When it is considered
that the first 25 residues constitute a signal peptide that is
cleaved upon insertion of the UGT protein into the mem-
brane of the endoplasmic reticulum (Radominska-Pandya et
al., 1999), UGT1A3 and UGT1A4 differ by only two amino
acids in this domain; Ile (UGT1A3) and Thr (UGT1A4) at
position 36 and His (UGT1A3) and Pro (UGT1A4) at position
40 (Fig. 1). To gain insight into the contributions of each of
these amino acids to UGT1A3 and UGT1A4 substrate selec-
UGT1A3–4
UGT1A3–4-3
UGT1A3(I36T)
UGT1A3(H40P)
UGT1A4
2207 Ϯ 125
437 Ϯ 42
N.A.
N.A.
N.A.
N.A.
UGT1A4–3
N.A.
N.A.
N.A.
N.A.
116 Ϯ 1
N.A.
UGT1A4–3-4
UGT1A4(T36I)
UGT1A4(P40H)
N.A.
N.A.
137 Ϯ 2b
N.A.
-
N.A.
N.A., no activity.
a From fitting to the substrate inhibition equation.
b From fitting to the Michaelis Menten equation.
c Normalized for expression relative to UGT1A3.
d Calculated as Vmax/Km.
TABLE 5
Derived kinetic constants for 4-MU glucuronidation by UGT1A3 and UGT1A4 chimeras and mutants
Data shown as parameters Ϯ S.E. of parameter fit.
a
b
a
b
c
d
e
Enzyme
Km or S50
Ksi
Hill coefficient (nH
)
Vmax
CLint or CLmax
␮M
␮M
pmol/min/mg
␮l/min/mg
UGT1A3
1205 Ϯ 37a
781 Ϯ 10a
702 Ϯ 71a
994 Ϯ 39b
1493 Ϯ 93b
N.A.
916 Ϯ 30
1931 Ϯ 35
1496 Ϯ 193
1273 Ϯ 41
767 Ϯ 2
613 Ϯ 2
115 Ϯ 1
15 Ϯ 0.5
N.A.
1.06d
0.98d
0.87d
0.06e
0.01e
N.A.
0.02e
0.10d
0.18d
N.A.
UGT1A3–4
UGT1A3–4-3
UGT1A3(I36T)
UGT1A3(H40P)
UGT1A4
1.57 Ϯ 0.06
2.12 Ϯ 0.15
N.A.
N.A.
UGT1A4–3
1012 Ϯ 29b
848 Ϯ 102a
537 Ϯ 6a
N.A.
2.29 Ϯ 0.14
24 Ϯ 0.2
87 Ϯ 11
98 Ϯ 1
UGT1A4–3-4
UGT1A4(T36I)
UGT1A4(P40H)
2393 Ϯ 160
1680 Ϯ 32
N.A.
N.A.
N.A.
N.A., no activity.
a From fitting to the substrate inhibition equation.
b From fitting to the Hill equation.
c Normalized for expression relative to UGT1A3.
d Calculated as Vmax/Km.
e Calculated according to eq. 4.

1060
Kubota et al.
tivity, reciprocal mutagenesis was performed to generate the catalytic base necessary for the SN2 glucuronidation
the UGT1A3(I36T), UGT1A3(H40P), UGT1A4(T36I), and reaction (Radominska-Pandya et., 1999). This histidine is
UGT1A4(P40H) mutants. The T36I mutation in UGT1A4 conserved in all UGT1A enzymes except UGT1A4 and, con-
abolished LTG and TFP glucuronidation, whereas the P40H sistent with the mutagenesis data reported here for
substitution reduced the in vitro intrinsic clearances for LTG UGT1A3, substitution of His38 in UGT1A6 with alanine and
and TFP glucuronidation by more than 90%. Conversely, the glutamine abolished 4-MU glucuronidation (Ouzzine et al.,
H40P mutation in UGT1A3 essentially abolished 1-NP and 2000). It is noteworthy that the glucuronidation of tertiary
4-MU glucuronidation, whereas the I36T substitution re- amines does not require aglycone deprotonation for the SN2
duced the in vitro intrinsic clearances for the glucuronidation mechanism to proceed, although UGT1A4 does catalyze the
of these substrates by approximately 95%. Consistent with glucuronidation of substrates other than tertiary amines
the critical roles of both positions, the double I36T/H40P (e.g., primary/secondary amines and aliphatic alcohols, in-
mutation (i.e., UGT1A4-3) in the mature UGT1A3 protein cluding hydroxysteroids) (Green and Tephly, 1996; Bowal-
essentially abolished 1-NP and 4-MU glucuronidation, and gaha et al., 2007). The identity of the residue that acts as the
the double T36I/P40H mutation (i.e., UGT1A3-4) in UGT1A4 catalytic base in these reactions is unknown. It is notewor-
abolished LTG and TFP glucuronidation. It is intriguing, thy, however, that the single T36I (but not P40H) mutation
however, that introduction of the single H40P (but not I36T) in UGT1A4 conferred 1-NP and 4-MU glucuronidation in the
mutation in UGT1A3 conferred LTG and TFP glucuronida- present study, and this would seem consistent with the role
tion, whereas the single T36I (but not P40H) mutation in of an alternate catalytic base to histidine.
UGT1A4 conferred 1-NP and 4-MU glucuronidation despite
the apparent importance of both positions to UGT1A3 and model predicts that residue 40 of UGT1A proteins is appar-
UGT1A4 substrate selectivity. ently located within a conserved ␣-helix (Ouzzine et al., 2000;
Secondary structure analysis and the UGT1A1 homology
Mechanistic interpretation of these data are problematic in Locuson and Tracy, 2007). Proline is known to distort ␣-he-
the absence of a UGT X-ray crystal structure. However, a lical structure, and it is possible that Pro40 of UGT1A4 alters
homology model for UGT1A1, which shares approximately the architecture of the active site to permit binding of tertiary
72% sequence identity with UGT1A3 and UGT1A4, was gen- amines in a catalytically favorable orientation. It is notewor-
erated recently using a plant glycosyltransferase, UGT71G1, thy that the H40P substitution in UGT1A3 conferred LTG
as the template (Locuson and Tracy, 2007). The homology and TFP glucuronidation activity to UGT1A3. However, sub-
model identified His39, which aligns to His40 of UGT1A3, as stitution of threonine at residue 36, which is critical for
Fig. 6. Eadie-Hofstee plots for 1-NP glucuronidation by UGT1A3, UGT1A3-4, UGT1A3-4-3, UGT1A3(I36T), and UGT1A4(T36I). Points show
experimentally determined values (mean of duplicate estimates), whereas curves are the computer-generated curves of best fit.

Molecular Basis of UGT1A3 and UGT1A4 Substrate Selectivity
1061
tertiary amine glucuronidation by UGT1A4, did not confer affinity and/or turnover. It was further shown that reciprocal
LTG and TFP glucuronidation activity to UGT1A3. mutagenesis at position 40 of UGT1A3 and position 36 of
Reciprocal mutagenesis has previously been used to ex- UGT1A4 resulted in reversal of aglycone substrate selectiv-
plore the differing hydroxysteroid glucuronidation substrate ity. This is the first demonstration of reversal of substrate
profiles of UGT2B15 and UGT2B17, which share 95% selectivity between UGT enzymes by single amino acid sub-
sequence identity (Dubois et al., 1999). UGT2B15 prefer- stitutions.
entially glucuronidates 17␤-hydroxysteroids, whereas
UGT2B17 metabolizes both 3␣- and 17␤- hydroxysteroids.
References
Bowalgaha K, Elliot DJ, Mackenzie PI, Knights KM, and Miners JO (2007) The
Mutation of Ser121 of UGT2B17 to tyrosine, the correspond-
ing residue at this position in UGT2B15, abolished andros-
terone 3␣-glucuronidation without affecting the 17␤-glucu-
ronidation of dihydrotestosterone. In parallel with certain of
the observations in the present work, the reverse mutation
(Y121S) in UGT2B15 did not confer 3␣-glucuronidation ac-
tivity.
glucuronidation of ⌬4-3-keto C19 and C21- hydroxysteroids by human liver micro-
somal and recombinant UDP-glucuronosyltransferases (UGTs): 6␣- and 21- hy-
droxyprogesterone are selective substrates for UGT2B7. Drug Metab Dispos 35:
363–370.
Chen Y, Chen S, Li X, Wang X, and Zeng S (2006) Genetic variants of human
UGT1A3: functional characterization and frequency distribution in a Chinese Han
population. Drug Metab Dispos 34:1462–1467.
Coffman BL, Kearney WR, Goldsmith S, Knosp BM, and Tephly TR (2003) Opioids
bind to the amino acids 84 to 118 of UDP-glucuronosyltransferase UGT2B7. Mol
Pharmacol 63:283–288.
Genetic variants of UGT1A3 and UGT1A4 that alter glu-
Dubois SG, Beaulieu M, Levesque E, Hum DW, and Belanger A (1999) Alteration of
curonidation activity have been reported (Ehmer et al., 2004;
Iwai et al., 2004; Mori et al., 2005; Chen et al., 2006). Several
of these mutations are in close proximity to residues 36 and
40: R45W and V47A of UGT1A3 and L48V of UGT1A4. Al-
though enzyme activity was variably increased or decreased,
none of the mutations abolished glucuronidation by UGT1A3
or UGT1A4 (including tertiary amine glucuronidation in the
case of the UGT1A4 L48V substitution).
human UDP-glucuronosyltransferase UGT2B17 regio-specificity by a single amino
acid substitution. J Mol Biol 289:29–39.
Ehmer U, Vogel A, Schutte JK, Krone B, Manns MP, and Strassburg CP (2004)
Variation of hepatic glucuronidation: novel functional polymorphisms of the UDP-
glucuronosyltransferase UGT1A4. Hepatology 39:970–977.
Green MD, King CD, Mojarrabi B, Mackenzie PI, and Tephly TR (1998) Glucuronida-
tion of amines and other xenobiotics catalyzed by expressed human UDP-
glucuronosyltransferase 1A3. Drug Metab Dispos 26:507–512.
Green MD and Tephly TR (1996) Glucuronidation of amines and hydroxylated
xenobiotics and endobiotics catalyzed by expressed human UGT1.4 protein. Drug
Metab Dispos 24:356–363.
Green MD and Tephly TR (1998) Glucuronidation of amine substrates by purified
and expressed UDP-glucuronosyltransferase proteins. Drug Metab Dispos 26:860–
867.
Guillemette C (2003) Pharmacogenomics of human UDP-glucuronosyltransferase
enzymes. Pharmacogenomics J 3:136–158.
Houston JB and Kenworthy KE (2000) In vitro-in vivo scaling of CYP kinetic data
not consistent with the classical Michaelis-Menten model. Drug Metab Dispos
28:246–254.
Iwai M, Maruo Y, Ito M, Yamamoto K, Sato H, and Takeuchi Y (2004) Six novel
UDP-glucuronosyltransferase 1A3 (UGT1A3) polymorphisms with varying activ-
ity. J Hum Genet 49:123–128.
Kiang TKL, Ensom MHH, and Chang TKH (2005) UDP-Glucuronosyltransferases
and clinical drug-drug interactions. Pharmacol Ther 106:97–132.
Lewis BC, Mackenzie PI, Elliot DJ, Burchell B, Bhasker CR, and Miners JO (2007)
Amino terminal domains of human UDP-glucuronosyltransferases (UGT) 2B7 and
2B15 associated with substrate selectivity and autoactivation. Biochem Pharmacol
73:1463–1473.
Li Q, Lou XJ, Peyronneau MA, Straub PO, and Tukey RH (1997) Expression and
functional domains of rabbit liver UDP-glucuronosyltransferase 2B16 and 2B13.
J Biol Chem 272:3272–3279.
Locuson CW and Tracy TS (2007) Comparative modelling of the human UDP-
glucuronosyltransferases: insights into structure and mechanism. Xenobiotica 37:
155–168.
Mackenzie PI (1990) Expression of chimeric cDNAs in cell culture defines a region of
UDP glucuronosyltransferase involved in substrate selection. J Biol Chem 265:
3432–3435.
Mackenzie PI, Bock KW, Burchell B, Guillemette C, Ikushiro S, Iyanagi T, Miners
JO, Owens IS, and Nebert DW (2005) Nomenclature update for the mammalian
UDP glycosyltransferase (UGT) gene superfamily. Pharmacogenet Genomics 15:
677–685.
Martineau I, Tchernof A, and Belanger A (2004) Amino acid residue Ile211 is
essential for the enzymatic activity of human UDP-glucuronosyltransferase 1A10
(UGT1A10). Drug Metab Dispos 32:455–459.
Miners JO and Mackenzie PI (1991) Drug glucuronidation in humans. Pharmacol
Ther 51:347–369.
Miners JO, McKinnon RA, and Mackenzie PI (2002) Genetic polymorphisms of
UDP-glucuronosyltransferases and their functional significance. Toxicology 181:
453–456.
Miners JO, Smith PA, Sorich MJ, McKinnon RA, and Mackenzie PI (2004) Predicting
human drug glucuronidation parameters: application of in vitro and in silico
modeling approaches. Annu Rev Pharmacol Toxicol 44:1–25.
Mojarrabi B, Butler R, and Mackenzie PI (1996) cDNA cloning and characterization
of the human UDP glucuronosyltransferase, UGT1A3. Biochem Biophys Res Com-
mun 225:785–790.
Atypical, or non–Michaelis-Menten, kinetics has been re-
ported for several glucuronidation reactions (e.g., Stone et
al., 2003; Uchaipichat et al., 2004). Here, only LTG glucu-
ronidation by UGT1A4 followed Michaelis-Menten kinetics.
1-NP and 4-MU glucuronidation by UGT1A3 and TFP glu-
curonidation by UGT1A4 exhibited kinetics characteristic of
substrate inhibition. However, kinetic models and derived
parameters (Km, S50, Vmax, CLint, CLmax) for the same sub-
strate tended to vary between the parent enzyme, chimeras,
and mutants. For example, Michaelis-Menten kinetics were
observed for LTG glucuronidation by UGT1A4, UGT1A4-3,
and UGT1A3(H40P), whereas the same reaction catalyzed by
UGT1A4(P40H) and UGT1A4-3-4 exhibited substrate inhibi-
tion and sigmoidal kinetics, respectively. Despite retention of
substrate selectivity, these data indicate that differences in
active site architecture that affect substrate binding and/or
turnover, including the ability to bind multiple substrate
molecules, occur between the parent enzymes, chimeras, and
mutants. Data presented here do not preclude catalytically
unfavorable binding of LTG/TFP and 4-MU/1-NP in the re-
spective UGT1A3 and UGT1A4 active sites (or to the chime-
ras and mutants that lack either of these glucuronidation
activities). However, this study aimed to identify residues in
UGT1A3 and UGT1A4 that are critical for the glucuronida-
tion of planar phenols and tertiary amines, respectively, and
hence inhibition studies were not conducted.
Previous studies (e.g., Dubois et al., 1999; Coffman et al.,
2003; Xiong et al., 2006; Lewis et al., 2007) have generally
linked the aglycone substrate selectivity of individual UGT
enzymes with residues or domains spanning positions 60 to
194. The present study demonstrates that residues 36 and 40
of UGT1A3 and UGT1A4, which are close to the amino ter-
minus of the mature UGT protein, are pivotal for the respec-
tive selectivities of these enzymes toward planar phenols and
tertiary amines. As might be expected, however, changes in
the kinetics of aglycone glucuronidation observed between
the parent enzymes and the active UGT1A3/4 chimeras in-
dicate that other regions of the proteins influence binding
Mori A, Maruo Y, Iwai M, Sato H, and Takeuchi Y (2005) UDP-glucuronosyltrans-
ferase 1A4 polymorphisms in a Japanese population and kinetics of clozapine
glucuronidation. Drug Metab Dispos 33:672–675.
Ouzzine M, Antonio L, Burchell B, Netter P, Fournel-Gigleux S, and Magdalou J
(2000) Importance of histidine residues for the function of the human liver UDP-
glucuronosyltransferase UGT1A6: evidence for the catalytic role of histidine 370.
Mol Pharmacol 58:1609–1615.
Radominska-Pandya A, Czernik PJ, Little JM, Battaglia E, and Mackenzie PI (1999)
Structural and functional studies of UDP-glucuronosyltransferases. Drug Metab
Rev 31:817–899.
Ritter JK, Chen F, Sheen YY, Tran HM, Kimura S, Yeatman MT, and Owens IS
(1992) A novel complex locus UGT1 encodes human bilirubin, phenol, and other

1062
Kubota et al.
UDP-glucuronosyltransferase isozymes with identical carboxyl termini. J Biol
Chem 267:3257–3261.
and Miners JO (2004) Human UDP-glucuronosyltransferases: isoform selectivity
and kinetics of 4-methylumbelliferone and 1-naphthol glucuronidation, effects of
organic solvents, and inhibition by diclofenac and probenecid. Drug Metab Dispos
32:413–423.
Udomuksorn W, Elliot DJ, Lewis BC, Mackenzie PI, Yoovathaworn K and Miners JO
(2007) Influence of mutations associated with Gilbert and Crigler-Najjar type II
syndromes on the glucuronidation kinetics of bilirubin and other UDP-
glucuronosyltransferase 1A (UGT1A) substrates. Pharmacogenet Genomics, in
press.
Xiong Y, Bernardi D, Bratton S, Ward MD, Battaglia E, Finel M, Drake RR, and
Radominska-Pandya A (2006) Phenylalanine 90 and 93 are localized within the
phenol binding site of human UDP-glucuronosyltransferase 1A10 as determined
by photoaffinity labeling, mass spectrometry, and site-directed mutagenesis. Bio-
chemistry 45:2322–2332.
Rowland A, Elliot DJ, Williams JA, Mackenzie PI, Dickinson RG, and Miners JO
(2006) In vitro characterization of lamotrigine N2-glucuronidation and the lam-
otrigine-valproic acid interaction. Drug Metab Dispos 34:1055–1062.
Senay C, Jedlitschky G, Terrier N, Burchell B, Magdalou J, and Fournel-Gigleux S
(2002) The importance of cysteine 126 in the human liver UDP-glucuronosyltrans-
ferase UGT1A6. Biochim Biophys Acta 1597:90–96.
Stone AN, Mackenzie PI, Galetin A, Houston JB, and Miners JO (2003) Isoform
selectivity and kinetics of morphine 3-and 6-glucuronidation by human UDP-
glucuronosyltransferases: evidence for atypical glucuronidation kinetics by
UGT2B7. Drug Metab Dispos 31:1086–1089.
Tukey RH and Strassburg CP (2000) Human UDP-glucuronosyltransferases: metab-
olism, expression, and disease. Annu Rev Pharmacol Toxicol 40:581–616.
Uchaipichat V, Mackenzie PI, Elliot DJ, and Miners JO (2006) Selectivity of sub-
strate (trifluoperazine) and inhibitor (amitriptyline, androsterone, canrenoic acid,
hecogenin, phenylbutazone, quinidine, quinine, and sulfinpyrazone) “probes” for
human UDP- glucuronosyltransferases. Drug Metab Dispos 34:449–456.
Uchaipichat V, Mackenzie PI, Guo XH, Gardner-Stephen D, Galetin A, Houston JB,
Address correspondence to: Professor John Miners, Department of Clinical
Pharmacology, Flinders Medical Centre, Bedford Park, SA 5042, Australia.
E-mail: john.miners@flinders.edu.au