A universal pocket in fatty acyl-AMP ligases ensures redirection of fatty acid pool away from coenzyme A-based activation

It was also used to compare the gain of function obtained from deletion of the previously annotated FAAL-specific insertion (ΔFSI) and FAAL-specific helix (ΔFSH). Wild-type FAAL, wild-type FACL, and reaction lacking any protein were used as controls. All the TLCs were marked at the origin, where the reaction mix containing 1-¹⁴C fatty acids was spotted. The products acyl-CoA band and the acyl-AMP band along with the free fatty acid band are visualized owing to the radio-labeled fatty acid. (A) A representative image of TLC showing the canonical pocket mutations leading to the gain of function in EcFAAL. (B) A representative image of TLC showing the canonical pocket mutations leading to the gain of function in MsFAAL32. (C) A representative image of TLC showing the canonical pocket mutations leading to the gain of function in RsFAAL. (D) A representative image of TLC showing the canonical pocket mutations leading to the loss of function in MtFACL13. (E) A representative image of TLC showing the canonical pocket mutations leading to the loss of function in AfFACL. (F) A representative image of TLC showing the canonical pocket mutations leading to the loss of function in EcFACL. (G) A representative image of TLC showing the gain of function by ΔFSI mutation in RsFAAL. (H) A representative image of TLC showing the gain of function by ΔFSI mutation in MsFAAL32. Several mutations were generated in this study, which had multiple issues including protein stability, poor or complete loss of biochemical activity, etc., hence were not analyzed further and such mutations are marked by a red asterisk as ‘not part of the study.’ These original uncropped images of radio-TLC are source data for Figure 2.

These values were obtained from densitometric quantification of the TLC images, which were used to plot Figure 2c.

These values were obtained from densitometric quantification of the TLC images, which were used to plot Figure 2f.

The values are then converted to fraction of the total acyl-AMP (TA) formed to the remaining acyl-AMP (A) and the acyl-AMP converted to acyl-CoA (T).

These values were obtained from densitometric quantification of the TLC images, which were used to plot Figure 2h.

These were also used to assess the impact of mutations in the alternative pocket of EcFAAL to form acyl-EcACP. Wild-type FAAL, reaction lacking ATP, and reaction containing a mutant ACP, lacking 4'-phosphopantetheine arm, were used as controls. All the radio-CS-PAGE were marked at the origin, where the reaction mix containing 1-¹⁴C fatty acids was loaded without boiling. The acyl-ACP(s), along with unreacted fatty acids or acyl-AMP(s), could only be visualized owing to the radiolabeled fatty acid. The unreacted fatty acids or acyl-AMP(s) appeared as a diffused band at the bottom of the radio-CS-PAGE. The radio-TLC images presented here were used to assess the ability of EcFAAL and its mutations in the alternate pocket to form acyl-AMP. Wild-type EcFAAL, wild-type MtFACL13, and reaction lacking any protein were used as controls. All the TLCs were marked at the origin, where the reaction mix containing 1-¹⁴C fatty acids was spotted. The products, acyl-CoA band and the acyl-AMP band, along with the free fatty acid band, were visualized owing to the radiolabeled fatty acid. (A) A representative image showing the optimization and validation of the modified radio-CS-PAGE in three pairs of FAAL-ACP systems, namely EcFAAL-EcACP, RsFAAL-RsACP, and MxFAAL-MxACP. (B) A representative TLC image showing that alternate pocket mutations of EcFAAL have minimal or no effect on the acyl-AMP formation. (C) A representative image of radio-CS-PAGE showing that acyl-EcACP formation is severely attenuated by mutations in the alternate pocket of EcFAAL. Several mutations of the alternate pocket of EcFAAL were generated in this study, which had multiple issues including protein stability, poor or complete loss of biochemical activity, etc., hence were not analyzed further, and these mutations are marked by a red asterisk as ‘not part of the study.’ These original uncropped images of radio-CS-PAGE and radio-TLC are source data for Figure 4.

The bound CoA from the crystal structures of SeACS (PDB: 1PG4), AaCBCL (PDB: 3CW9), HsFACL (PDB: 3EQ6), and NtCCL (PDB: 5BSR) were used for the starting conformation of the head group of CoA. These crystal structures were then superimposed over the crystal structures of representative FAALs in adenylation competent state (A-state), EcFAAL (PDB: 3PBK), and MsFAAL32 (PDB: 5ICR), which were also used to generate the thioesterification competent state (T-state) by superimposing the C-terminal domain separately. The number of clashes was computed for every 1° rotation of the head group and then ranked as 3 if number ≥30, as 2 if number ≥10, and as 0 if number ≥1. These ranked values were used to plot Figure 5b and Figure 5—figure supplement 2a.