Of note, M415 is a conserved proline in α-amino acid activating A domains, and the M415P mutant abolished adenylation activity of StsA_A. Mutation of this residue in linear gramicidin synthetase A (LgrA) to either Met or Ala (P483M, P483A) led to a small but significant increase in adenylation activity towards α-ketoisovaleric acid (α-Kiv), the α-keto equivalent of its native substrate L-Val. This work further highlights the importance of the shape and backbone atom orientations in A domain binding pockets.