Expanding Substrate Specificity of ω‐Transaminase by Rational Remodeling of a Large Substrate‐Binding Pocket

Abstract

Production of structurally diverse chiral amines via biocatalytic transamination is challenged by severe steric interference in a small active site pocket of ω‐transaminase (ω‐TA). Herein, we demonstrated that structure‐guided remodeling of a large pocket by a single point mutation, instead of excavating the small pocket, afforded desirable alleviation of the steric constraint without deteriorating parental activities toward native substrates. Molecular modeling suggested that the L57 residue of the ω‐TA from Ochrobactrum anthropi acted as a latch that forced bulky substrates to undergo steric interference with the small pocket. Removal of the latch by a L57A substitution allowed relocation of the small pocket and dramatically improved activities toward various arylalkylamines and alkylamines (e.g., 1100‐fold increase in kcat/KM for α‐propylbenzylamine). This approach may provide a facile strategy to broaden the substrate specificity of ω‐TAs.magnified image

Rapid and Quantitative Profiling of Substrate Specificity of ω‐Transaminases for Ketones

“…The distance between the C4′ of PLP and the nitrogen of the amine substrate (C4′‐N) was 3.0 and 3.2 Å for ( R )‐ 2 n in ARTA and ( S )‐ 2 n in PDTA, respectively. In the previous report, the C4′‐N distance was 2.9 Å in a docking model of ( S )‐ 2 n in OATA . Considering that 2 n is a typical amino donor for ω‐TAs, these results suggest that the optimal length of C4′‐N in a productive Michaelis complex is around 3 Å.…”

“…In the previous report, the C4'-N distance was 2.9 Å in a docking model of (S)-2 n in OATA. [19] Considering that 2 n is a typical amino donor for ω-TAs, these results suggest that the optimal length of C4'-N in a productive Michaelis complex is around 3 Å. Note that the optimal distance between the electrophilic carbon center and the N of the nucleophile in a transition state for a nucleophilic attack is known to be 2.5 Å.…”

“…In the conventional HPLC analysis to measure ω‐TA activity for ketones (e. g., amination of 1 n using alanine or isopropylamine as an amino donor), UV detection of the produced amine (e. g., α‐MBA ( 2 n )) usually permits reliable quantitation of the product formation higher than 10 μM . In this case, an experimental LOD in terms of a reaction rate is over 10 μM min −1 when the reaction is allowed for a minute which is usually regarded as a minimal time required to keep enzyme reactions running reliably.…”

“…Therefore, the ALDH‐based assay is at least more than 15‐fold sensitive than the HPLC method. In addition to the higher detection sensitivity, benefit of the ALDH‐based method becomes more striking when a target substrate is non‐chromogenic alkyl ketones (e. g. 1 a – m ) because UV detection of the resulting amines requires laborious derivatization with a chromogenic reagent such as a Marfey's reagent …”

Isopropylamine as Amine Donor in Transaminase‐Catalyzed Reactions: Better Acceptance through Reaction and Enzyme Engineering

“…In contrast to ω‐TA, which accept carbonylic substrates with a distal carboxylate group, ATA tolerate substrates without a carboxyl moiety and therefore a wide range of ketones and aldehydes. Hence, in the last decade ATA became very attractive targets for enzyme engineering representing an environmentally benign alternative for the chemical transition metal‐catalyzed amine synthesis in pharmaceutical and agrochemical industries . For instance, the production of imagabaline, ( S )‐rivastigmine, or ( S )‐ivabradine has been realized on larger scale using ATA.…”