Fig. 2: Biochemical characterization of BKACE.

A Specific activities of selected BKACE homologs with 4 mM MSA and 1 mM acetyl-CoA. Homologs were chosen from the initial high-throughput screen. Data are presented as individual and mean values ± SD. B Michaelis-Menten kinetics for BKACE15 with different malonate semialdehyde concentrations. We excluded concentrations above 5 mM MSA as we observed substrate inhibition with increasing concentrations. Data are presented as mean ± SD. C Michaelis-Menten kinetics for BKACE15 with different acetyl-CoA concentrations. Data are presented as mean ± SD. D Relative activity of BKACE15 and its inhibition. Measurements were done with 5 mM MSA, 1 mM acetyl-CoA and different concentrations of acetoacetate. Relative activities were calculated by normalizing the data to the activity with 0 mM acetoacetate. Data are presented as mean ± SD. IC₅₀: Half maximal inhibitory constant. All measurements were done in duplicates (D) or triplicates (A, B, C). Quantification of acetoacetate formation (A, B, C) or formyl-CoA formation (D) was used to determine enzyme activities. The data were analyzed using nonlinear regression (B, C, D). E) Overall structure of BKACE15. The four monomers of the tetrameric complex are indicated by different colors. F Active site entry channel of BKACE15 with bound acetyl-CoA (magenta) and acetoacetate (not visible) (PDB 9HNF). The surface representation of acetyl-CoA highlights the complete occupancy of the entry channel. G Overlay of BKACE15 active sites of PDB 8RIP (malonate and CoA bound) and PDB 9HNF (acetoacetate and acetyl-CoA bound). Malonate, CoA and the corresponding backbone are shown in dark grey, acetoacetate, acetyl-CoA and the corresponding backbone in yellow, zinc (Zn²⁺) in metal grey, polar interactions in light grey dashes. Substrate labeling is shown in yellow for acetyl-CoA (“ACO”) and acetoacetate (“AAE”) and in dark grey for CoA (“COA”) and malonate (“MLI”). Labeling of foreground residues in black, labeling of background residues in dark grey. Electron density maps of substrates in the active site are shown in Supplementary Fig. 14. Source data are provided as a Source Data file.