Structural studies reveal flexible roof of active site responsible for ω-transaminase CrmG overcoming by-product inhibition.

Amine compounds biosynthesis using ω-transaminases has received considerable attention in the pharmaceutical industry. However, the application of ω-transaminases was hampered by the fundamental challenge of severe by-product inhibition. Here, we report that ω-transaminase CrmG from Actinoalloteichus cyanogriseus WH1-2216-6 is insensitive to inhibition from by-product α-ketoglutarate or pyruvate. Combined with structural and QM/MM studies, we establish the detailed catalytic mechanism for CrmG. Our structural and biochemical studies reveal that the roof of the active site in PMP-bound CrmG is flexible, which will facilitate the PMP or by-product to dissociate from PMP-bound CrmG. Our results also show that amino acceptor caerulomycin M (CRM M), but not α-ketoglutarate or pyruvate, can form strong interactions with the roof of the active site in PMP-bound CrmG. Based on our results, we propose that the flexible roof of the active site in PMP-bound CrmG may facilitate CrmG to overcome inhibition from the by-product. Xu, Tang et al. show that ω-transaminase CrmG is insensitive to the inhibition from by-products α-ketoglutarate or pyruvate. They find that these by-products cannot interact strongly with the flexible roof of the active site in CrmG. This study provides insights into the rational design of transaminase to eliminate by-product inhibition.