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Dimerization of human uridine diphosphate glucuronosyltransferase allozymes 1A1 and 1A9 alters their quercetin glucuronidation activities.

Liu YQ, Yuan LM, Gao ZZ, Xiao YS, Sun HY, Yu LS, Zeng S - Sci Rep (2016)

Bottom Line: Uridine diphosphate glucuronosyltransferase 1A (UGT1A) is a major phase II drug-metabolism enzyme superfamily involved in the glucuronidation of endobiotics and xenobiotics in humans.Many polymorphisms in UGT1A genes are reported to inhibit or decrease UGT1A activity.SNPs of UGT1A altered the ability of protein-protein interaction, resulting in differential FRET efficiencies and donor-acceptor r distances.

View Article: PubMed Central - PubMed

Affiliation: Institute of Drug Metabolism and Pharmaceutical Analysis, Zhejiang Province Key Laboratory of Anti-Cancer Drug Research, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou 310058, China.

ABSTRACT
Uridine diphosphate glucuronosyltransferase 1A (UGT1A) is a major phase II drug-metabolism enzyme superfamily involved in the glucuronidation of endobiotics and xenobiotics in humans. Many polymorphisms in UGT1A genes are reported to inhibit or decrease UGT1A activity. In this study, two UGT1A1 allozymes, UGT1A1 wild-type and a splice mutant, as well as UGT1A9 wild-type and its three UGT1A9 allozymes, UGT1A9*2(C3Y), UGT1A9*3(M33T), and UGT1A9*5(D256N) were single- or double-expressed in a Bac-to-Bac expression system. Dimerization of UGT1A1 or UGT1A9 allozymes was observed via fluorescence resonance energy transfer (FRET) and co-immunoprecipitation analysis. SNPs of UGT1A altered the ability of protein-protein interaction, resulting in differential FRET efficiencies and donor-acceptor r distances. Dimerization changed the chemical regioselectivity, substrate-binding affinity, and enzymatic activity of UGT1A1 and UGT1A9 in glucuronidation of quercetin. These findings provide molecular insights into the consequences of homozygous and heterozygous UGT1A1 and UGT1A9 allozymes expression on quercetin glucuronidation.

No MeSH data available.


Enzyme kinetic, Eadie-Hofstee, and V/S−V plots for quercetin-7-glucuronidation by UGT1A9*N-HA, UGT1A9*N-CFP, and UGT1A9*N-CFP + UGT1A9*N-HA.The glucuronidation rates are presented as mean ± SD of three independent determinations. The rates were normalized according to the relative expression levels. The respective kinetic constants are presented in Tables 2 and 3.
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f5: Enzyme kinetic, Eadie-Hofstee, and V/S−V plots for quercetin-7-glucuronidation by UGT1A9*N-HA, UGT1A9*N-CFP, and UGT1A9*N-CFP + UGT1A9*N-HA.The glucuronidation rates are presented as mean ± SD of three independent determinations. The rates were normalized according to the relative expression levels. The respective kinetic constants are presented in Tables 2 and 3.

Mentions: In the context of HA-tagged constructs (Figs 5 and 6), the kinetic parameters of UGT1A9*1 were found to exhibit allosteric effects on M1 and M2 formation. However, the kinetic parameters were found to fit well with Michaelis-Menten kinetics of UGT1A9*2, 1A9*3, and 1A9*5 for M1 formation and UGT1A9*2 and 1A9*5 for M2 formation. In the context of the CFP-tagged constructs (Figs 5 and 6), the kinetic parameters of UGT1A9*2, 1A9*3, and 1A9*5 were found to fit well with Michaelis-Menten kinetics for M1 formation. Interestingly, CFP-tagged UGT1A9*2 and 1A9*3 displayed similar kinetic parameters as UGT1A9*1 for M2 formation.


Dimerization of human uridine diphosphate glucuronosyltransferase allozymes 1A1 and 1A9 alters their quercetin glucuronidation activities.

Liu YQ, Yuan LM, Gao ZZ, Xiao YS, Sun HY, Yu LS, Zeng S - Sci Rep (2016)

Enzyme kinetic, Eadie-Hofstee, and V/S−V plots for quercetin-7-glucuronidation by UGT1A9*N-HA, UGT1A9*N-CFP, and UGT1A9*N-CFP + UGT1A9*N-HA.The glucuronidation rates are presented as mean ± SD of three independent determinations. The rates were normalized according to the relative expression levels. The respective kinetic constants are presented in Tables 2 and 3.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4837415&req=5

f5: Enzyme kinetic, Eadie-Hofstee, and V/S−V plots for quercetin-7-glucuronidation by UGT1A9*N-HA, UGT1A9*N-CFP, and UGT1A9*N-CFP + UGT1A9*N-HA.The glucuronidation rates are presented as mean ± SD of three independent determinations. The rates were normalized according to the relative expression levels. The respective kinetic constants are presented in Tables 2 and 3.
Mentions: In the context of HA-tagged constructs (Figs 5 and 6), the kinetic parameters of UGT1A9*1 were found to exhibit allosteric effects on M1 and M2 formation. However, the kinetic parameters were found to fit well with Michaelis-Menten kinetics of UGT1A9*2, 1A9*3, and 1A9*5 for M1 formation and UGT1A9*2 and 1A9*5 for M2 formation. In the context of the CFP-tagged constructs (Figs 5 and 6), the kinetic parameters of UGT1A9*2, 1A9*3, and 1A9*5 were found to fit well with Michaelis-Menten kinetics for M1 formation. Interestingly, CFP-tagged UGT1A9*2 and 1A9*3 displayed similar kinetic parameters as UGT1A9*1 for M2 formation.

Bottom Line: Uridine diphosphate glucuronosyltransferase 1A (UGT1A) is a major phase II drug-metabolism enzyme superfamily involved in the glucuronidation of endobiotics and xenobiotics in humans.Many polymorphisms in UGT1A genes are reported to inhibit or decrease UGT1A activity.SNPs of UGT1A altered the ability of protein-protein interaction, resulting in differential FRET efficiencies and donor-acceptor r distances.

View Article: PubMed Central - PubMed

Affiliation: Institute of Drug Metabolism and Pharmaceutical Analysis, Zhejiang Province Key Laboratory of Anti-Cancer Drug Research, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou 310058, China.

ABSTRACT
Uridine diphosphate glucuronosyltransferase 1A (UGT1A) is a major phase II drug-metabolism enzyme superfamily involved in the glucuronidation of endobiotics and xenobiotics in humans. Many polymorphisms in UGT1A genes are reported to inhibit or decrease UGT1A activity. In this study, two UGT1A1 allozymes, UGT1A1 wild-type and a splice mutant, as well as UGT1A9 wild-type and its three UGT1A9 allozymes, UGT1A9*2(C3Y), UGT1A9*3(M33T), and UGT1A9*5(D256N) were single- or double-expressed in a Bac-to-Bac expression system. Dimerization of UGT1A1 or UGT1A9 allozymes was observed via fluorescence resonance energy transfer (FRET) and co-immunoprecipitation analysis. SNPs of UGT1A altered the ability of protein-protein interaction, resulting in differential FRET efficiencies and donor-acceptor r distances. Dimerization changed the chemical regioselectivity, substrate-binding affinity, and enzymatic activity of UGT1A1 and UGT1A9 in glucuronidation of quercetin. These findings provide molecular insights into the consequences of homozygous and heterozygous UGT1A1 and UGT1A9 allozymes expression on quercetin glucuronidation.

No MeSH data available.