Extended Data Fig. 3: Aat promiscuity and inhibition of the condensation reaction by free Coenzyme A.
From: Synthetic anaplerotic modules for the direct synthesis of complex molecules from CO2
a Promiscuity of Aat assayed with acetyl-CoA and propionyl-CoA. Dark gray bars display EIC peak areas obtained for each product of condensation, reduction and dehydration reactions in an assay containing all three pathway enzymes (Aat, Aar, Bbd), acetyl-CoA and propionyl-CoA. All products for acetyl- and propionyl-CoA combinations were detectable, in contrast to the condensation of two propionyl-CoA. When combining modules 1-2-3-4d, only 3-hydroxybutyryl-CoA and crotonyl-CoA were detected (light gray). Measurements were performed in technical duplicates and are displayed as mean. b Aat activity coupled to Aar with varying amounts of free CoA. Obtained activities were set relative to the measurement containing no additional free CoA. To ensure Aar was not inhibited by free CoA, a control where CoA was added to the Aat reaction after 20 minutes followed by addition of Aar, was performed and showed no differences to the corresponding setup using all enzymes (data not shown). Measurements were performed in technical triplicates and are displayed as mean ± SD.
