A study aimed at generating analogs of the β-amino acid-containing hitachimycins investigated the tolerability of different substituents on (S)-β-Phe in the adenylation reaction of HitB. HitB contains all the β-amino acid-specific A domain motifs discussed above, and it was found to have reasonable tolerance for a wide range of alternative substrates. Upon solving the structure of HitB in complex with (S)-β-3-bromo-Phe-AMS, it was noted that the side chain of F328 was flexible (HitB numbering), allowing for the activation of meta-substituted (S)-β-Phe analogs. Additional studies on β-amino acid activating A domains led to the reprogramming of an α-amino acid A domain and swapped its functionality. Niquille and co-workers have used fluorescence activated cell sorting (FACS) to enable rapid screening of a yeast cell surface display library of TycA mutants. They replaced the four-residue β13β14 loop with a randomized tripeptide, and at the opposite side of the pocket, A236 was randomized to account for other structural mutations (GrsA_A numbering). Of all the variants that were sequenced, the mutation A236V was 100% conserved, and the sequence of the β13β14 loop converged to the motif Xaa-Leu-Val (where Xaa is Ala, Thr, Cys, Val or Leu). Upon structural analysis, it was found that the conserved D235 interacted with the substrate β-amino group, as noted in VinN above. The randomized residues at position 236 and in the β13β14 loop were deemed essential for the α/βspecificity switch, which allowed for the measurement of a 40,000-fold increase in TycA preference for (S)-β-Phe over L-Phe.