In vitro acyl-CoA synthetase activity assay
The activities of MbcS and IbuA were evaluated using a coupled and
continuous spectrophotometric assay based on NADH consumption, as
described (Crosby et al., 2012). Briefly, 100 μL reactions were composed
of 50 mM HEPES pH 7.5, 1 mM tris (2-carboxyethyl) phosphine (TCEP), 2.5
mM ATP, 5 mM MgCl2, 0.5 mM coenzyme A, 3 mM
phosphoenolpyruvate, 0.1 mM NADH, 1 unit of pyruvate kinase, 5 units of
myokinase, 1.5 units of lactate dehydrogenase, and 60 nM MbcS
(calculated for monomeric enzyme) (or 30 nM IbuA). The reactions were
initiated by adding 2 μL of the indicated carboxylic acid substrate to
the 98 μL reaction mixture. The changes in the absorbance, measured at
340 nM, were recorded over time at 37°C degrees in a computer-controlled
Synergy H1 plate reader running Gen5 software ver3.14 (Agilent/BioTek).
Each experiment was performed in triplicate. All reagents and enzymes
were purchased from Sigma, except for lactate dehydrogenase, which was
purchased from Worthington Biochemical Corporation. The specific
activity of MbcS for different carboxylic acids as substrates was
calculated using the molar extinction coefficient of NADH (6220
M-1 cm-1) and taking into account
that two moles of NADH are oxidized for every mole of AMP being produced
(Garrity et al., 2007). Specific activity was reported as μmol of AMP
per minute per milligram of MbcS.