Table 3.
Specific activity (ΔA235nm min−1 mg−1) | |||||
---|---|---|---|---|---|
Alginate | Poly-ManA | Poly-GulA | Poly-MG | Poly-GlcA | |
WT | 328.3 ± 27.0 | 661.9 ± 29.1 | 190.3 ± 8.3 | 289.3 ± 9.8 | 2.8 ± 0.4 |
Possible involvement in β-elimination mechanism | |||||
Y455F | 3.6 ± 0.3 | 4.0 ± 0.4 | 15.3 ± 1.4 | 11.5 ± 0.1 | 0.2 ± 0.1 |
Enhanced poly-GlcA activity | |||||
H208A | 48.2 ± 3.5 | 83.2 ± 4.2 | 6.3 ± 0.8 | 3.6 ± 0.4 | 3.1 ± 0.8 |
H208W | 185.3 ± 11.8 | 490.8 ± 4.1 | 48.5 ± 1.0 | 117.1 ± 7.8 | 7.1 ± 0.6 |
H208F | 88.4 ± 4.0 | 189.7 ± 4.1 | 9.5 ± 0.8 | 23.3 ± 1.0 | 77.2 ± 1.2 |
Additional putative substrate-binding residues | |||||
R143L | 224.1 ± 13.9 | 245.4 ± 15.3 | 95.5 ± 2.5 | 159.0 ± 9.2 | 1.1 ± 0.3 |
Q153A | 129.0 ± 3.5 | 456.4 ± 26.4 | 30.3 ± 3.5 | 73.6 ± 4.4 | 0.2 ± 0.1 |
Q153 N | 113.8 ± 9.9 | 323.7 ± 17.1 | 83.4 ± 2.6 | 108.7 ± 10.7 | 0.9 ± 0.2 |
L155A | 158.5 ± 9.5 | 597.6 ± 46.9 | 28.4 ± 0.9 | 89.6 ± 1.4 | 0.3 ± 0.1 |
L155 N | 125.6 ± 12.6 | 512.7 ± 23.2 | 42.4 ± 2.9 | 131.9 ± 4.8 | 0.3 ± 0.2 |
N201L | 380.1 ± 9.2 | 915.8 ± 31.9 | 138.6 ± 12.9 | 255.3 ± 9.9 | 3.1 ± 0.7 |
H206F | 73.9 ± 2.3 | 147.8 ± 3.9 | 57.0 ± 2.9 | 73.2 ± 0.2 | 0.4 ± 0.1 |
Y258F | 173.4 ± 9.1 | 346.2 ± 16.1 | 88.7 ± 6.0 | 127.7 ± 9.5 | 0.5 ± 0.1 |
Y263F | 36.1 ± 0.4 | 83.5 ± 1.7 | 5.3 ± 0.6 | 13.5 ± 1.4 | 0.2 ± 0.1 |
Purified wild-type and mutant Smlt2602 was added to 1 mg/mL of each substrate in 20 mM sodium phosphate buffer at pH 8.5 for alginate-based polysaccharides and pH 8 for poly-GlcA. Enzymatic activity was monitored by absorbance at 235 nm. In addition to the substrates listed below, each enzyme was tested against poly-GalA, heparin, heparan sulfate, and hyaluronic acid, with no detectable activity. All reactions were performed in triplicate, and error is reported as standard deviation