Regioselective sulfation and glucuronidation of phenolics: insights into the structural basis

Abstract

The phase II metabolism sulfation and glucuronidation, mediated by sulfotransferases (SULTs) and UDP-glucuronosyltransferases (UGTs) respectively, are significant metabolic pathways for numerous endo-and xenobiotics. Understanding of SULT/UGT substrate specificity including regioselectivity (i.e., position preference) is of great importance in predicting contribution of sulfation/ glucuronidation to drug and metabolite disposition in vivo. This review summarizes regioselective sulfation and glucuronidation of phenolic compounds with multiple hydroxyl (OH) groups as the potential conjugation sites. The strict regioselective patterns are highlighted for several SULT and UGT isoforms towards flavonoids, a large class of natural polyphenols. To seek for a molecular-level explanation, the enzyme structures (i.e., SULT crystal structures and a homology-modeled UGT structure) combined with molecular docking are employed. In particular, the structural basis for regioselective metabolism of flavonoids by SULT1A3 and UGT1A1 is discussed. It is concluded that the regioselective nature of these phase II enzymes is determined by the size and shape of the binding pocket. While the molecular structures of the enzymes can be used to explain regioselective metabolism regarding the binding property, predicting the turnover at different positions remains a particularly difficult task.

Figure.1. Chemical structures of phenolic compounds with multiple possible conjugation sites (i.e., hydroxyl groups)

The structures are randomly numbered (in parenthesis).

Figure.2. Crystal structures of SULT1 isoforms reveal their highly conserved 3D structure

Panel A: Structure-based sequence alignment of four crystallized SULT1 isoforms, the α-helices are in cyan and β-strands in magenta. The alignment was performed by overlay of the 3D structures. The conserved catalytic residue histidine and lysine are marked with an asterix. The residues determining the regioselectivity of SULT1A3 towards dopamine and D-dopa [15] are marked with a pound sign. Residues reported to form part of the acceptor pocket are highlighted in grey. Panel B: Ribbon diagram of the SULT1A3 crystal structure [15] showing the 3D folding of elements of secondary structure in a spectrum-colored mode. Panel C: Ribbon diagram of the SULT1A3 crystal structure [15]. The regions that form the substrate-binding pocket are shown in red. The PAPS and substrate (dopamine) are shown as black stick models.

Figure.3. Surface view of the SULT1A1 crystal [39] showing the buried state of PAPS (red stick) and substrate (blue stick)

Figure.4. Conserved interaction of PAPS with SULT residues

Panel A: The conserved PAPS interacting residues from the four SULT crystals, showing their interactions with the PAPS. Dashed lines indicate hydrogen bonds. Panel B: Alignment of four SULT1 sequences showing the PAPS-interacting residues (in red) in secondary structures. Residue numbers in SULT1A1 are labeled,

Figure.5. Structural basis of regioselective sulfation of phenolics

Surface view of the substrate-binding pockets of SULT1A3 (panel A), 1A1 (panel B), and 1E1 (panel C). The size and shape of substrate-binding sites are divergent. SULT1E1 has a wider and larger pocket, compared to SULT1A1 and 1A3. SULT1A3 has a unique hump (circled area) in the pocket. Black arrow indicates a narrow corner in the binding pocket of SULT1A3. Panel D: Surface view of the interaction between SULT1A3 and kaempferol (7-OH). Panel D1: An expanded view of residues potentially involved in interactions with kaempferol in the SULT1A3 crystal. Dashed lines indicate potential hydrogen bonds. Panel E: Surface view of the substrate-binding pocket of SULT1A3 with genistein (7-OH) docked, the B-ring was fitted to the narrow corner.

Figure.6. Regioselective glucuronidation of phenolic compounds in literatures [–48,6062]

The chemical structures are randomly numbered (in parenthesis). * Percentage based on calculated intrinsic clearance (CL_int) values. HLM, human liver microsomes; HIM, human intestine microsomes; (m)ugt1a1, mouse ugt1a1.

Figure.7. Homology model of UGT1A1

Panel A: Sequence alignment between human UGT1A1 and plant UGT71G1 (PDB code: 2ACW) used for homology modeling. The aligned sequence identity is 13.7%. The full length UGT1A1 sequence (Accession: Q5DT03) was downloaded from the SwissProt database (http://www.uniprot.org/). Approximately 450 amino acids long sequence of human 1A1 (excluding N-terminal signal peptide, C-terminal transmembrane domain and cytosolic tail) was aligned to UGT71G1 with the aid of predicted secondary structures [67]. The sequence aligning approaches used in modeling human UGT was discussed [1]. Similar to an early work [25], helices Nα3-1 and Nα3-3 were not modeled in this work and were shown as random coils in the final generated structures. The best model with the lowest objective function values (DOPE) were selected for further investigation. The quality of the model was validated using PROCHECK (http://www.jcsg.org/prod/scripts/validation/sv_final.cgi). The 44-aa signature motif of UGTs is enclosed by a box. Residues which were predicted to be in contact with aglycone are highlighted in red whereas the catalytic dyad residues (His39-Asp151) are highlighted in grey. Panel B: Ribbon diagram of the structure model of human UGT1A1. Regions forming the substrate binding pocket in N-/C- terminus are shown in red. The copied UDP-glucose (in black) and co-crystallized kaempferol (in blue) from vVGT1 (PDB code: 2C1Z) are shown as stick models. Loop N2 (shown in purple) is positioned far away from the catalytic histidine, and does not participate in binding small substrates such as flavonols.

Figure.8. The substrate binding pocket of human UGT1A1 (homology model) and its interaction with quercetin

Panel A: Molecular surface diagram of the substrate binding pocket. The copied UDP-glucose is shown as stick models. Panel B–D, stereo diagrams showing interactions between quercetin in distinct (and regioselective) catalytic modes (panel B, 3’-OH; panel C, 7-OH, panel D, 3-OH) and side chains in the substrate-binding pocket. Dashed lines indicate potential hydrogen bonds.