Table 2.
LpxB-specific activities
| LpxB | Specific activity ± SE (μmol min−1 μmol−1) | 95% Confidence interval | Percent of wild type |
|---|---|---|---|
| Wild-type | 6.17 ± 5.3*10−1 | 4.99 to 7.34 | 100 |
| L72SL75S | 1.08*10−1 ± 2.3*10−2 | 5.62*10−2 to 1.59*10−1 | 1.74 |
| L72SL76 S | 1.99 ± 3.3*10−1 | 1.21 to 2.76 | 32.2 |
| L75SL76S | 1.89 ± 2.3*10−1 | 1.35 to 2.42 | 30.6 |
| V66SV68SL69S (VVL) | 3.26*10−3 ± 5.5*10−4 | 2.02*10−3 to 4.49*10−3 | 5.28*10−2 |
| L72SL75SL76S (LLL) | 6.34*10−5 ± 1.04*10−5 | 4.03*10−5 to 8.66*10−5 | 1.03*10−3 |
| 6S | Below detection limit | ND | ND |
| N316A (NA) | 2.11*10−2 ± 1.5*10−3 | 1.78*10−2 to 2.45*10−2 | 3.42*10−1 |
| F298AN316A (FN) | 4.80*10−4 ± 6.2*10−5 | 3.33*10−4 to 6.28*10−4 | 7.79*10−3 |
| R201A (RA) | 3.16*10−4 ± 2.3*10−5 | 2.66*10−4 to 3.66*10−4 | 5.12*10−3 |
| FN + RA (50% FN) | 1.06*10−2 ± 9*10−4 | 8.50*10−3 to 1.27*10−2 | 1.72*10−1 |
| FN + RA (37% FN) | 3.63*10−3 ± 3.7*10−4 | 2.76*10−3 to 4.50*10−3 | 5.89*10−2 |
| FN + RA (24% FN) | 2.91*10−3 ± 1.6*10−4 | 2.55*10−3 to 3.27*10−3 | 4.71*10−2 |
| FN + RA (15% FN) | 2.35*10−3 ± 2.6*10−4 | 1.74*10−3 to 2.95*10−3 | 3.81*10−2 |
| FN + RA (7.3% FN) | 1.03*10−3 ± 1.5*10−4 | 6.70*10−4 to 1.38*10−3 | 1.66*10−2 |
| NA + RA1:1 | 1.06*10−2 ± 1.3*10−3 | 7.56*10−3 to 1.35*10−2 | 1.71*10−1 |
| Wild-type (NDSB-201) | 3.42 ± 6.0*10−1 | 1.994 to 4.84 | 55.4 |
| 6S (NDSB-201) | Below detection limit | ND | ND |
Reactions were performed in triplicate with 1 nM to 10 μM LpxB (as appropriate per variant), 31 μM UDP-DAG, ~ 0.13 mM lipid X, 1 mg ml−1 BSA, 0.1 M Tris-HCl pH 8.0, and 0.1% Triton X-100 at ambient temperature (21 °C). Reactions were quenched and UDP was quantified using a UDP-Glo Glycosyltransferase Assay kit (Promega), which quantifies free UDP by a luciferase coupled reaction. Specific activities with standard errors and 95% confidence intervals were calculated by linear regression of UDP concentrations at three or four time points for each LpxB variant in Graphpad Prism v7.03
ND, insufficient data