Expression of chimeric cDNAs in cell culture defines a region of UDP glucuronosyltransferase involved in substrate selection.

Mackenzie, Peter

doi:10.1016/s0021-9258(19)39785-6

Cited by 116 publications

(7 citation statements)

References 14 publications

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“…However, the rigorous structural studies of UGTs and their active sites necessary to understand the mechanistic aspects of UGT substrate specificity, inhibition, and drug-drug interactions have been hampered by a lack of purified protein. This lack of structural knowledge has resulted in the prediction of UGT binding site structures by utilizing selective inhibitors, amino acid-specific chemical modification reagents, and amino acid alignments ( − ). There are only a few reports of computer-aided molecular modeling being applied to this system ( , ), and site-directed mutagenesis studies are limited ( 13 , 17−20 ).…”

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confidence: 99%

“…Biochemistry 2006, 45 of structural knowledge has resulted in the prediction of UGT binding site structures by utilizing selective inhibitors, amino acid-specific chemical modification reagents, and amino acid alignments (9)(10)(11)(12)(13)(14)(15). There are only a few reports of computeraided molecular modeling being applied to this system (14,16), and site-directed mutagenesis studies are limited (13,(17)(18)(19)(20).…”

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confidence: 99%

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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

et al. 2006

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4-Azido-2-hydroxybenzoic acid (4-AzHBA), a novel photoactive benzoic acid derivative, has been synthesized and used as a photoprobe to identify the phenol binding site of UDP-glucuronosyltransferases (UGTs). Analysis of recombinant His-tag UGTs from the 1A family for their ability to glucuronidate p-nitrophenol (pNP) and 4-methylumbelliferone (4-MU) revealed that UGT1A10 shows high activity toward phenols and phenol derivatives. Purified UGT1A10 was photolabeled with 4-AzHBA, digested with trypsin, and analyzed by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF)-mass spectrometry. A single modified peak corresponding to amino acid residues 89-98 (EFMVFHAQWK) of UGT1A10 was identified. The attachment site of the 4-AzHBA probe was localized to the quadruplet Phe(90)-Met(91)-Val(92)-Phe(93) using ESI LC-MS/MS. Sequence alignment revealed that the Phe(90) and Phe(93) are conserved in UGT1A7-10. Site-directed mutagenesis of these two amino acids was then followed by kinetic analysis of the mutants with two phenolic substrates, pNP and 4-MU, containing one and two planar rings, respectively. Using the combination of photoaffinity labeling, enzymatic digestion, MALDI-TOF and LC-MS mass spectrometry, and site-directed mutagenesis, we have determined for the first time that Phe(90) and Phe(93) are directly involved in the catalytic activity of UGT1A10 toward 4-MU and pNP.

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confidence: 99%

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confidence: 99%

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

et al. 2006

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“…To investigate the structurefunction relationship of UGT2B enzymes, site-directed mutagenesis and the formation of chimeric proteins have identified putative substrate binding domains. Via exchange of the amino and carboxyl regions between different UGT2B enzymes, the aglycone specificity domain is proposed to be in the amino-terminal half of the protein, whereas the conserved carboxyl-terminal domain binds the common cofactor UDPGA (17,18). The conserved region between residues 350 and 400 has been postulated to be an important component of the UDPGA-binding site (2).…”

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confidence: 99%

N-Glycosylation and Residue 96 Are Involved in the Functional Properties of UDP-Glucuronosyltransferase Enzymes^,

et al. 2000

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The recent cloning of several human and monkey UDP-glucuronosyltransferase (UGT) 2B proteins has allowed the characterization of these steroid metabolic enzymes. However, relatively little is known about the structure-function relationship, and the potential post-translational modifications of these proteins. The mammalian UGT2B proteins contain at least one consensus asparagine-linked glycosylation site NX(S/T). Endoglycosidase H digestion of the human and monkey UGT2B proteins demonstrates that only UGT2B7, UGT2B15, UGT2B17, and UGT2B20 are glycosylated. Although UGT2B15 and UGT2B20 contain three and four potential glycosylation sites, respectively, site-directed mutagenesis revealed that both proteins are glycosylated at the same first site. In both proteins, abolishing glycosylation decreased glucuronidation activity; however, the K(m) values and the substrate specificities were not affected. Despite the similarities between UGT2B15 and UGT2B20, UGT2B20 is largely more labile than UGT2B15. Treating HK293 cells stably expressing UGT2B20 with cycloheximide for 2 h decreased the enzyme activity by more than 50%, whereas the activity of UGT2B15 remained unchanged after 24 h. The UGT2B20 protein is unique in having an isoleucine at position 96 instead of an arginine as found in all the other UGT2B enzymes. Changing the isoleucine in UGT2B20 to an arginine stabilized enzyme activity, while the reciprocal mutation in UGT2B15 R96I produced a more labile enzyme. Secondary structure predictions of UGT2B proteins revealed a putative alpha-helix in this region in all the human and monkey proteins. This alpha-helix is shortest in UGT2B20; however, the helix is lengthened in UGT2B20 I96R. Thus, it is apparent that the length of the putative alpha-helix between residues 84 and 100 is a determining factor in the stability of UGT2B enzyme activity. This study reveals the extent and importance of protein glycosylation on UGT2B enzyme activity and that the effect of residue 96 on UGT2B enzyme stability is correlated to the length of a putative alpha-helix.

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“…However, a putative nucleotide binding motif was found to reside within the C-terminal region of UrdGT1b and UrdGT1c. This motif is found among human UDP-glucuronosyltransferases, with a smaller degree of conservation it is also present in the £avonol O(3)-glucosyltransferase from Zea mays, Streptomyces lividans macrolide GT, other CAZy family 1 members and other families [12,13]. Data from the crystal structure of MurG, an Escherichia coli peptidoglycan GT of CAZy family 28, con¢rm this motif to be involved in nucleotide binding [14].…”

Section: Expression Of the Chimeric Genes In Mutant Ax: Conversion Of Urdgt1b Into Urdgt1cmentioning

confidence: 97%

Two sequence elements of glycosyltransferases involved in urdamycin biosynthesis are responsible for substrate specificity and enzymatic activity

Hoffmeister

Ichinose

Bechthold

2001

Chemistry & Biology

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A nucleotide sugar binding motif is present in the C-terminal moiety of UrdGT1b and UrdGT1c from S. fradiae. We could demonstrate that it is an N-terminal section that determines specificity for the nucleotide sugar and also the acceptor substrate. This finding directs the way towards engineering this class of streptomycete enzymes for antibiotic derivatization applications. Amino acids 226 and 227, located outside the putative substrate binding site, might be part of a larger protein structure, perhaps a solvent channel to the catalytic center. Therefore, they could play a role in substrate accessibility to it.

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Expression of chimeric cDNAs in cell culture defines a region of UDP glucuronosyltransferase involved in substrate selection.

Cited by 116 publications

References 14 publications

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

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

N-Glycosylation and Residue 96 Are Involved in the Functional Properties of UDP-Glucuronosyltransferase Enzymes^,

Two sequence elements of glycosyltransferases involved in urdamycin biosynthesis are responsible for substrate specificity and enzymatic activity

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Expression of chimeric cDNAs in cell culture defines a region of UDP glucuronosyltransferase involved in substrate selection.

Cited by 116 publications

References 14 publications

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

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

N-Glycosylation and Residue 96 Are Involved in the Functional Properties of UDP-Glucuronosyltransferase Enzymes,

Two sequence elements of glycosyltransferases involved in urdamycin biosynthesis are responsible for substrate specificity and enzymatic activity

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N-Glycosylation and Residue 96 Are Involved in the Functional Properties of UDP-Glucuronosyltransferase Enzymes^,