J. Biol. Macromol., 20 (1), 3-8 (2020)
Review
Library of drug metabolizing enzymes: applications for metabolic prediction and biosynthesis ofhuman drug metabolites
Miyu Nishikawa
Department of Biotechnology, Faculty of Engineering, Toyama Prefectural University, 5180 Kurokawa,Imizu, Toyama 939-0398, Japan, TOPUBIO Research Co., Ltd, 5180 Kurokawa, Imizu, Toyama
939-0398, Japan
Received October 11, 2019; Accepted October 24, 2019
Drug metabolizing enzymes (DMEs) play a role in modifying drug activity and in modulating
the clearance of target compounds such as endo- and xenobiotics, small molecule drugs, steroid
hormones and dietary flavonoids. Thus, the evaluation of metabolites generated by DMEs isrequired for drug development, food science and environmental assessment. Here, we generated alibrary of DMEs expressed in yeast cells as a means of studying xenobiotic metabolism from earlyphase metabolic screening to structural determination and evaluation. In this review, advantages of
the DME expression system are introduced as well as applications of the DME platform forstudying drug metabolism.
Key words: Drug metabolizing enzymes, biotransformation,
Introduction

are mostly localized in the endoplasmic reticulum

Drug metabolizing enzymes (DMEs) modify(ER) and catalyze the transfer of a glucuronic aciddrug activity and influence the clearance of target

from UDP-glucuronic acid (UDP-GA) to acompounds such as endo- and xenobiotics, smallhydroxyl group of the substrate compound.

molecularandCytosolic sulfotransferases (SULTs) are also

Metabolism by DMEs ismajor phase II enzymes, but they utilize

drugs,

dietary

endogenous steroids.

polyphenols

mainly divided into two phases; oxidation,3’-phosphoadenosine-5’-phosphosulfate

reduction and hydrolysis (phase I) followed by theas a universal sulfate donor and transfer a sulfate

transfergroup to a hydroxyl moiety of the substrate.

ofhydrophilicgroups(phaseII).

(PAPS)

Cytochrome P450s (CYPs) are common phase I

DMEs comprise a gene superfamily, which

enzymes that catalyze the oxidation of a wide

enables DMEs to convert various types ofvariety of compounds. Microsomal CYPs arecompounds into metabolites. In human, more thanoften involved in the metabolism of xenobiotics.

50 CYP, 19 functional UGT, and at least 13 SULTThe reactions catalyzed by CYPs contribute 70%

isoforms have been reported [2-7].

ofthemain

pharmaceuticalmetabolicdrugs

pathways[1].

ofUsually, DMEs mediate biological inactivation

Uridineand hydrophilization of the original compounds

5'-diphospho-glucuronosyltransferase

for elimination in urine or bile. Sometimes,(UDP-glucuronosyltransferases, UGTs) are

however, chemical reactivity of the original

membrane-bound major phase II enzymes, which

compound is increased via metabolism mediatedE-mail: m-nishikawa@pu-toyama.ac.jp

by DMEs, which often becomes a risk factor due

3Library of drug metabolizing enzymes

toenhanced

toxicity.

acyl-glucuronidationisconjugation

reactionscontainingdrugs

Inmammals,various

oneof

themajorrequirement for exogenous cofactors [17, 18]. Inof

carboxylicacid

this review, advantages of the recombinant DME

non-steroidal

expression system in yeast are described and

such

as

anti-inflammatory drugs (NSAIDs). The resulting
acyl-glucuronides

areunstable

By

contrast,

without

thechemically
The DME expression system in yeast cells

often covalently modify macromolecules leading
cytotoxicity.

compounds

several applications are introduced.

reactive metabolites [8-10]. Acyl-glucuronides
tosubstrate

Usually, metabolic reactions catalyzed byseveral

DMEs require cofactors. For example, NAD(P)H

pharmaceutical drugs are specifically designed tois an electron donor in many phase I xenobiotic

be activated in the body by this reaction. For

metabolism reactions including those catalyzed byexample, morphine-6-glucuronide, which is aCYPs.

major active metabolite of morphine, is formed(CPR), NADH-cytochrome b5 reductase, andfrom morphine by the enzyme UGT2B7 [11].

cytochrome b5, which act as an electron transfer

Morphine-6-glucuronide elicits a more potent

partner, is also essential for microsomal CYP

analgesic effect than the parent compound

mediated reactions [19]. To reconstitute thismorphine [12, 13].

electron transfer system in vitro, -NADH+,

Recently, DMEs have also become the focus ofNADPH-cytochrome

glucose-6-phosphateand

P450

reductaseglucose-6-phosphate

research in the field of food science. For example,

dehydrogenase are often added to the reaction as apolyphenolic compounds are a diverse group ofNADPH-generating system. UGT and SULT also

phytochemicals that are commonly found in therequire a more expensive cofactor UDP-GA orhuman diet. A number of reports suggest phase IIPAPS, which are donors of glucuronic acid in themetabolites of polyphenols act as the delivery

UGT reaction and sulfate in the SULT reaction,

form in plasma [14-16].

respectively [20, 21].

The preparation of metabolites converted byTo achieve enzymatic synthesis of xenobiotic

DMEs can be a bottleneck in studying themetabolites without exogenous cofactors, wemetabolism of drugs or nutrients such as

applied whole-cell dependent biosynthesis using

polyphenols. Moreover, it is often difficult to

the DME expression system in yeast. Mammalianprepare authentic standards of the metabolites inDMEs, including some from human, werethe correct regio- or stereo-selective form byheterologously

conventional organic synthesis. An enzymatic

cerevisiae AH22 cells. In order to construct theapproach to generating these compounds isCYP expression system in yeast cells, Sakaki et al.

therefore

usedanattractive

alternativestrategy.

theexpressed

CYP/CPRinfusion

proteinsuccessfully

is a serious disadvantage for this enzymaticcompounds to their corresponding metabolites [22,synthesis at an industrial scale.

23]. We also constructed a co-expression systemofUGT

withUDP-glucose

theandHowever, the requirement for expensive cofactors

We previously constructed a heterologous

biotransformed

Saccharomyces

substrate

dehydrogenase

DME expression system for CYP, UGT and SULT(UGDH) to supply endogenous UDP-GA. Thisin the budding yeast Saccharomyces cerevisiae

was necessary because yeast strain AH22 lacks

AH22 strain. In addition, biotransformation of thethe native UGDH, which is the carboxyl

metabolites was successfully achieved using

dehydrogenase to produce UDP-GA (Fig. 1).

4

M. Nishikawa
successfully constructed an expression system forCYP, UGT and SULT. Moreover, for performing
the reactions we can use stocks of recombinant
yeast cells immediately after thawing, without theneed to generate an enzyme preparation.
Screening assay using DME panels
Fig. 1. Scheme of glucuronide biosynthesis byPredicting the metabolic fate of a candidate

recombinant UGT and UGDH expressed incompound is an important step during the drugyeast.

development process. More recently, a similar
question is often also addressed in the area ofUsing this system, we successfully produced
severalhundredglucuronide

CYP, 35 UGT, and 10 SULT isoforms ofmetabolites per liter without exogenous addition

mammalian origin, some of which are human.

of UDP-GA [18]. As well as CYP and UGT, weThis variation enables us not only to examine thealso constructed a SULT expression system inenzymes that contribute to the metabolism butyeast. Heterologous expression of SULT wasalso to compare the metabolism between humansufficientdependent

and several experimental animals. We thensulfoconjugation without exogenous addition of

performed screening assays of drugs and dietary

PAPS, suggesting that native PAPS can act as apolyphenols

sulfate donor for the recombinant SULT [17]. As

Specifically, the library includes a variety ofwell as studying the metabolism of drug

DME isoforms from human, mouse, rat, rabbit

candidates brought about by CYP mediatedand pig. In UGT screening of mefenamic acid andmodification, it is also important to screen for theflufenamic acid, human UGT1A7, 1A8, 1A9 andeffect of other DMEs such as UGT. Thus,

1A10 showed relatively high levels of activity,

establishment of a DME platform will be

whereas the activity of UGT2B family enzymes

invaluable for studying xenobiotic metabolism.

was quite low. In contrast, rat and mouse Ugt2bfor

milligramsoffood science research. We have constructed 19

whole-cellusingthepanelofDMEs.

Several host cells have been used as afamily enzymes showed high activity towardheterologous expression system of DMEs bythese compounds (Fig. 2). In human SULT

different groups, which include prokaryotic

screening of polyphenols, SULT1E1 showed amicrobes such as E. coli and Salmonella

low level of regio-selectivity toward resveratroltyphimurium, insect cells and mammalian celland quercetin (Fig. 3) [17].

lines in addition to yeast [24-27]. In terms of large
scalecultivation,prokaryotic

microbes

are

Scale up for the preparation of metabolites

attractive hosts for heterologous expression.

Using a variety of enzymes can also increase

However, functional expression of CYPs andthe success rate of metabolite preparation. ForUGTs in prokaryotic microbes is often difficult toexample, rat Ugt2b1 is a useful isozyme toachieve because these are membrane-boundprepare acyl-glucuronides from several drug

enzymes mostly localized in the ER. Thus, wecompounds (Table 1). These glucuronides are also

selected yeast cells S. cerevisiae AH22 tosynthesized by human UGTs, but the yield wasconstruct the DME library, which includes

significantly

membrane-bound enzymes. Using this host we

acyl-glucuronides is required by FDA guidelines

5lower.

Theevaluationof

Library of drug metabolizing enzymes

Fig.2: Whole-cell UGT screening of mefenamic acid and flufenamic acid.
*: Not-detected by HPLC-UV., bm: recombinant UGT biomass
metabolism by UGTs and SULTs. Polyphenols
have

more

targetsites

forDMEs

thanpharmaceutical drugs because of the phenolichydroxyl groups. Thus, an enzymatic approach ispreferableinterms

stereo-selectivity.

ofWe

bothregio-successfullyandpreparedglucuronides and sulfoconjugates with high levelsof purity (>95% by HPLC-UV) and determined
their chemical structure by NMR. Quercetin (Q)is a major dietary polyphenol that elicits various
biological activities including anti-inflammation,anti-oxidant and anti-obesity. It has been reported
Fig. 3. Whole-cell human SULT screening ofthat Q-3-O-glucuronide is a major metabolite inresveratrol and quercetin.

human, but other regio-isomers of glucuronide are*: Not-detected by HPLC-UV., bm: recombinantalso generated by human UGTs [29, 30]. We

SULT biomass

preparedfour

regio-isomersofquercetin-O-glucuronides (-3, -7, -3’ and -4’
in some cases [28]. Thus, recombinant ratposition)

UGT2B1chemical properties and biological activities ofisoften

usedforglucuronidepreparation.
Dietarythesepolyphenols

preferably

undergoandareregio-isomers(unpublished data).

6

currently
invitroevaluatingandin

thevivoM. Nishikawa
Table 1. Productivity of acyl-glucuronide in scale-up reaction.

Substrate
Mefenamic acid
Flufenamic acidDiclofenac
Zomepirac

UGT source
rat 2b1
rat 2b1
rat 2b1
porcine 1A3

Yield (mg/L/day)
60132
260
47

Fig. 4 Application of the recombinant DME platform during drug development.
Conclusion.

Weinshilboum, R. M., Coughtrie, M. W.

The DME platform can be used to study the(2004) Pharmacogenetics. 14, 199–211.

metabolism of drugs or nutrients. The technology

6.

Freimuth. R. R., Wiepert, M., Chute, C. G.,

is useful for screening to large scale preparation

Wieben, E. D., Weinshilboum, R. M. (2004)

of analytical standards, structure determination

Pharmacogenomics J. 4, 54–65.

and even for their evaluation in vitro and in vivo

7.

Hildebrandt, M. A., Carrington, D. P.,(Fig. 4). Furthermore, the DME platform mightThomae, B. A., Eckloff, B. W., Schaid,also be useful in the area of analytical science andJ., Yee, V. C., Weinshilboum, R. M.

in the evaluation of environmental pollutants.

Wieben, E. D. (2007) Pharmacogenomics J.

Wearecurrentlyconstructing

additionalD.

7, 133–143.

expression systems for DMEs, which will aid the8.

detailed study of metabolism.

Iwamura, A., Nakajima, M., Oda, S., Yokoi,T. (2017) Drug Metab. Pharmacokinet. 32,2-11.

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