Amination of bulky ketones, particularly in (
R) configuration, is an attractive chemical conversion; however, known ω-transaminases (ω-TAs) show insufficient levels of performance. By applying two screening methods, we discovered 10 amine transaminases from the class III ω-TA family that were 38% to 76% identical to homologues. We present examples of such enzymes preferring bulky ketones over keto acids and aldehydes with stringent (
S) selectivity. We also report representatives from the class III ω-TAs capable of converting (
R) and (
S) amines and bulky ketones and one that can convert amines with longer alkyl substituents. The preference for bulky ketones was associated with the presence of a hairpin region proximal to the conserved Arg414 and residues conforming and close to it. The outward orientation of Arg414 additionally favored the conversion of (
R) amines. This configuration was also found to favor the utilization of putrescine as an amine donor, so that class III ω-TAs with Arg414 in outward orientation may participate
in vivo in the catabolism of putrescine. The positioning of the conserved Ser231 also contributes to the preference for amines with longer alkyl substituents. Optimal temperatures for activity ranged from 45 to 65°C, and a few enzymes retained ≥50% of their activity in water-soluble solvents (up to 50% [vol/vol]). Hence, our results will pave the way to design, in the future, new class III ω-TAs converting bulky ketones and (
R) amines for the production of high-value products and to screen for those converting putrescine.
IMPORTANCE Amine transaminases of the class III ω-TAs are key enzymes for modification of chemical building blocks, but finding those capable of converting bulky ketones and (
R) amines is still challenging. Here, by an extensive analysis of the substrate spectra of 10 class III ω-TAs, we identified a number of residues playing a role in determining the access and positioning of bulky ketones, bulky amines, and (
R)- and (
S) amines, as well as of environmentally relevant polyamines, particularly putrescine. The results presented can significantly expand future opportunities for designing (
R)-specific class III ω-TAs to convert valuable bulky ketones and amines, as well as for deepening the knowledge into the polyamine catabolic pathways.