SPR was used to investigate the interactions between LsBOS and LsAAE3. Experiments were performed using the Biacore 8 K (Cytiva) with a series S sensor chip CM5 (Cytiva) and a running buffer of 10 mM Hepes pH 7.4, 150 mM NaCl and 0.05 % tween 20 and a temperature of 25 °C. Initial pH scouting experiments were run which indicated that pH 4 was the optimum pH to use for immobilisation. A standard amine coupling approach was used to activate the chip with reagents 1-ethyl−3-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS) then LsBOS, at a concentration of 20 nM in 10 mM acetate pH 4.0, was injected over Flow Cell 2 (FC2) for 210 s at 10 μL/min (leaving FC 1 blank as the reference) before blocking flow cells with ethanolamine. LsBOS was immobilised with a response of ~1700. Interaction between LsAAE3 and LsBOS was observed by injecting 1 μM LsAAE3 over FC1 and FC2 for 60 s at a flow rate of 50 μL/min before switching to buffer only flow.
A range of regeneration solutions were tested and 10 mM acetate at pH 4 was found to be the best for removing the bound LsAAE3. However, this was still not ideal, so it was decided to use a single cycle kinetics approach where no regeneration is used between analyte injections. Three start-up cycles were run using only buffer then a blank run with five zero concentration injections of LsAAE3. LsAAE3 was then injected at five increasing concentrations of 2.4, 12, 60, 300 and 1500 nM each with a contact time 120 s and a flow rate 30 µL/min. At the end of all injections buffer was flowed for 600 s to record the dissociation. A concentration dependent response could be seen confirming the interaction. The data were processed and analysed using Biacore Insight Evaluation Software with double-referenced subtraction of the data and then fitted to a simple Langmuir binding model. The association rate (kon) was determined to be 3.67 × 103 s−1M−1 dissociation rate (koff) as 3.61 × 10−4 s−1 giving an dissociation equilibrium constant (KD) of 98 nM.
AAE3 activity was determined in the absence of other enzymes and by a coupled enzyme assay47 (link). Each reaction contained: 10 µL E. coli extract, 100 mM Tris pH 8.0, 2 mM DTT, 5 mM ATP, 10 mM MgCl2, 0.5 mM CoA, 0.4 mM NADH, 1 mM phosphoenol-pyruvate, 300 µM sodium oxalate, 10 units each myokinase, pyruvate kinase and lactate dehydrogenase, deionised water to 100 µL. Activity was measured by reduction in OD 340 nm over time. LsBOS activity was measured in reactions containing 100 mM Tris pH 8.0, 2 mM DTT, 5 mM ATP, 10 mM MgCl2, 0.5 mM CoA, 300 µM sodium oxalate, 50 µM DAP, and 10 µL each of AAE3 and LsBOS E. coli expression extracts in various combinations, made up to 100 µL with deionised water. Amounts of β-L-ODAP and α-L-ODAP produced were measured using an LCMS procedure36 (link).
A range of regeneration solutions were tested and 10 mM acetate at pH 4 was found to be the best for removing the bound LsAAE3. However, this was still not ideal, so it was decided to use a single cycle kinetics approach where no regeneration is used between analyte injections. Three start-up cycles were run using only buffer then a blank run with five zero concentration injections of LsAAE3. LsAAE3 was then injected at five increasing concentrations of 2.4, 12, 60, 300 and 1500 nM each with a contact time 120 s and a flow rate 30 µL/min. At the end of all injections buffer was flowed for 600 s to record the dissociation. A concentration dependent response could be seen confirming the interaction. The data were processed and analysed using Biacore Insight Evaluation Software with double-referenced subtraction of the data and then fitted to a simple Langmuir binding model. The association rate (kon) was determined to be 3.67 × 103 s−1M−1 dissociation rate (koff) as 3.61 × 10−4 s−1 giving an dissociation equilibrium constant (KD) of 98 nM.
AAE3 activity was determined in the absence of other enzymes and by a coupled enzyme assay47 (link). Each reaction contained: 10 µL E. coli extract, 100 mM Tris pH 8.0, 2 mM DTT, 5 mM ATP, 10 mM MgCl2, 0.5 mM CoA, 0.4 mM NADH, 1 mM phosphoenol-pyruvate, 300 µM sodium oxalate, 10 units each myokinase, pyruvate kinase and lactate dehydrogenase, deionised water to 100 µL. Activity was measured by reduction in OD 340 nm over time. LsBOS activity was measured in reactions containing 100 mM Tris pH 8.0, 2 mM DTT, 5 mM ATP, 10 mM MgCl2, 0.5 mM CoA, 300 µM sodium oxalate, 50 µM DAP, and 10 µL each of AAE3 and LsBOS E. coli expression extracts in various combinations, made up to 100 µL with deionised water. Amounts of β-L-ODAP and α-L-ODAP produced were measured using an LCMS procedure36 (link).
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