Metabolism Of Drugs And Xenobiotics By Human UDP Glucuronosyl Transferases

Uridine diphosphate-glucuronosyl transferases?UGTs?are a superfamily of conjugation enzymes that catalyze the glucuronidation of numerous substrates,including xenobiotics and endogenous metabolites.UGTs catalyze glucuronic acid transfer from the cofactor uridine 5'-diphosphoglucuronic acid?UDPGA?to a nucleophilic group on the substrate,forming glucuronides which are water-soluble and readily excreted via urine or bile.UGT-dependent bioconversion of xenobiotics is considered to be the most important phase II reaction type in human drug metabolism.The known substrates of the human UGTs that participate in phase II metabolism are structurally very diverse and,moreover,the sugar moieties may be transferred to multiple functional sites,such as amine,carbonyl,carboxyl,hydroxyl,and thiol groups.The reason why UGTs have various substrate structure is that human UGTs are a superfamily enzymes consist of four families?UGT1A,UGT2,UGT3A and UGT8A?with 22 UGT isoenzymes.The research on UGTs can go back to nearly 60 years ago.To data,there are numerous methods have been discovered to study the functional properties of UGTs.Pooled human liver microsomes is the most commonly way used as UGTs enzymes resource,though it is hard to get and usually very expensive.Also,HLMs is not useful when studying extrahepatic UGTs.Recombinant expression of human UGTs has been done and widely used in cell culture or microbial systems.However,recombinant expression in various mammalian or insect cell lines suffers from comparatively low expression levels and stability problems.Very recently we reported the successful production of enzyme bags from recombinant fission yeast cells?Schizosaccharomyces pombe?that express human UDP glucose dehydrogenase?UGDH?and their use for the production of the cofactor UDPGA from UDP glucose.Then in this study,we successfully transferred this method to fission yeast that recombinantly express human UGTs as enzyme resource,conducting a series of experiments to investigate human UGTs function.Upon treatment of microbes with organic solvents,such as ethanol,toluene-ethanol mixtures,or detergents like Triton X-100,the cell membrane becomes permeable allowing small molecules to freely enter and leave the cell while keeping the morphology of the cell intact.The resulting permeabilized cells,which have also been called'enzyme bags',retain most cellular structures,protein-protein interactions,and most of intracellular enzymes in their original environment which is very important for metabolic processes analysis.In the beginning,comparison of HLMs activity and enzyme bags activity treated with triton X-100 showed that it is highly possible to transfer this method to UGTs.A direct comparison of the UGT1A9-dependent glucuronidation of 11?-hydroxy progesterone activity gave an almost forty-fold higher space-time yield for enzyme bag-catalyzed biotransformation as compared to whole-cell biotransformation,demonstrating the high efficiency of enzyme bags method.Then an enzyme bags based inhibition assay was conducted to test IC₅₀0 value of mefenamic acid on UGT1A9.The results got from this experiment is 4.1?M?with a95%CI of 2.5 to 6.4?M;R²=0.97?,which is close to the value on reported before.Using UGT-Glo substrates we demonstrate new probe reactions for UGT1A5,UGT2B11,and UGT2B28,and also show for the first time that UGT1A5 can catalyze an N-glucuronidation reaction.Moreover,a bioinformatics analysis of UGT1A5missense variants indicated that in addition to the wild-type gene UGT1A5*1 there are two previously undescribed polymorphic variants present in the general population,which are a common nine-fold mutant?UGT1A5*8?and a much rarer six-fold mutant?UGT1A5*9?.We recombinantly expressed both variants and show that UGT1A5*9 displayed the same activity as the wildtype,while the activity of UGT1A5*8 was significantly higher for both O-and N-glucuronidation.Molecular modeling studies indicate that the minor effects of all mutations except for Gly259Arg are due to their distance to the substrate binding site.Remarkably,extensive molecular dynamics simulations of UGT1A5*1 and UGT1A5*8 demonstrate that the Gly259Arg mutation stabilizes helix Q through a newly formed hydrogen bonding network,which in turn places the cofactor in a much more favorable geometry in UGT1A5*8 as compared to the wild-type.In addition,due to the abuse of doping compounds by athletes in sports competition and the significance of doping compounds analysis in doping control,19human UGTs have been tested to catalyzed propranolol and its phase I metabolite,4OH-propranolol,by enzyme bags method.Except for human UGT1A9,which has been reported before,we found two human UGTs,UGT1A7 and UGT1A8 enzymes can metabolize propranolol and 4OH-propranolol.For 4OH-propranolol,we even found one more UGT isoform,UGT2A1,can metabolize it.Interestingly,UGT1A9,UGT1A7 and UGT1A8 all showed stereo-selectivity on propranolol.