Table 1.
Amino acids predicted to be important for 2B7UGT activity.
| Residue | Putative function | Mutation | Remaining activity (%)
|
|
|---|---|---|---|---|
| HDCA | TCC | |||
| Nucleotide interacting residues | ||||
| S308 | Structural scaffold (no direct interactions) | S308A | 83 | 70 |
| Q359 | Structural scaffold, “under” UDP | Q359A | 28 | 36 |
| E382 | Hydrogen bond with O2′ of ribose | E382A | 10 | 3 |
| W356 | Helps to position UDP-GlcUA | W356A | 20 | 43 |
| W356H | 27 | 60 | ||
| Phosphate interacting residues | ||||
| T373 | α-Phosphate via H2O | T373V | 1 | 5 |
| H374 | β-Phosphate | H374A | 3 | 7 |
| H374E | 10 | 6 | ||
| N378 | α-Phosphate | N378A | 25 | 10 |
| G379 | Forms a pocket under the α-phosphate | G379D | 18 | 5 |
| G379S | 90 | 134 | ||
| Glucuronic acid interacting residues | ||||
| D398 | O2′/O3′ of sugar | D398A | 10 | 5 |
| D398N | 2 | 11 | ||
| Q399 | O3′/O4′ of sugar | Q399A | 0 | 3 |
| Residues involved in catalysis | ||||
| H35 | Deprotonates acceptor facilitating nucleophilic attack at C1 of GlcUA | H35A | 0 | 4 |
| H35D | 0 | 3 | ||
| D151 | Stabilizes deprotonated His | D151A | 0 | 1 |
| D151N | 5 | 1 | ||
| Undetermined interactions | ||||
| S34 | Predicted to hydrogen bond to β-phosphate | S34A | 80 | 45 |
| R49* | Carboxyl function of GlcUA* | — | — | — |
| R259 | Carboxyl function of GlcUA | R259A | 3 | 1 |
| R338 | Ser in plant UGTs, interacts with nucleotide | R338S | 66 | 104 |
Predicted interactions and the effect of point mutations in UGT2B7 on activity toward hyodeoxycholic acid (HDCA) and tetrachlorocatechol (TCC) are shown. Activity data expressed as percent of wild-type activity.
*
Interactions inferred from literature not UGT2B7 homology model (Senay et al., 1997).
— No mutations done.