Table 1. Enzymatic activities of A-PGS543-881 variants under in vivo and in vitro conditions.
Standard in vivo and in vitro A-PGS543-881 assays were performed as described under “Experimental Procedures”. The specific activity observed in the in vitro activity assay for wild type A-PGS543-881 was set as 100 % and all other values of mutant proteins were related to this value (detection limit: 0.5 %). A-PG formation in the in vivo assay was classified as: + + +, activity comparable to wild type enzyme; +, decreased activity when compared to wild type enzyme; -, no detectable A-PGS activity. The observed activity in the in vivo assay for wild type A-PGS543-881 and mutants K654S (+ + +), Y732A (+) and D765N (-) can be found visualized in supplemental figure S2.
| mutation | In vivo activity | Relative in vitro activity ± standard deviation [%] |
|---|---|---|
| Wild type A-PGS543-881 | + + + | 100 ± 0 |
| D579A | + | 3 ± 1 |
| D579N | + | 5 ± 2 |
| K580S | - | 0 |
| K580Q | - | 0 |
| Y635A | - | 0 |
| K654S | + + + | 46 ± 2 |
| K654Q | + + + | 65 ± 1 |
| E657D | + | 4 ± 1 |
| E657Q | + + + | 21 ± 4 |
| E658D | + + + | 28 ± 7 |
| E658Q | + + + | 19 ± 3 |
| S709A | + + + | 5 ± 1 |
| S709N | + + + | 17 ± 3 |
| D710A | + + + | 26 ± 3 |
| D710N | + + + | 30 ± 3 |
| E720Q | + + + | 10 ± 2 |
| S724A | + | 7 ± 2 |
| S724N | - | 0 |
| Y732A | + | 3 ± 1 |
| S763A | + + + | 23 ± 5 |
| S763N | + + + | 11 ± 1 |
| D765A | - | 0 |
| D765N | - | 0 |
| R768S | - | 0 |
| R768Q | - | 0 |
| M778A | + + + | 50 ± 2 |
| Y831A | - | 0 |
| R837S | + + + | 65 ± 8 |
| R837Q | + + + | 100 ± 8 |
| K840S | - | 0 |
| K840Q | - | 0 |
| K842S | + + + | 50 ± 2 |
| K842Q | + + + | 100 ± 3 |