Combinatorial Mutation Analysis of ω‐Transaminase to Create an Engineered Variant Capable of Asymmetric Amination of Isobutyrophenone

Abstract

ω-Transaminase (ω-TA) is an important enzyme for asymmetric synthesis of chiral amines. Rapid creation of a desirable ω-TA variant, readily available for scalable process operation, is demanded and has attracted intense research efforts. In this study, we aimed to develop a quantitative mutational analysis (i. e., Ranalysis) that enables prediction of combinatorial mutation outcomes and thereby provides reliable guidance of enzyme engineering through combination of already characterized mutations. To this end, we determined three mutatable active-site residues of ω-TA from Ochrobactrum anthropi (i. e., leucine 57, tryptophan 58 and valine 154) by examining activities of nine alanine-scanning mutants for seven substrate pairs. The R-analysis of the mutatable residues is based on assessment of changes in relative activities for a series of structurally analogous substrates. Using three sets of substrates (five α-keto acids, six arylalkylamines and three arylalkyl ketones), we found that combination of two point mutations display additive effects of each mutational outcome such as steric relaxation for bulky substrates or catalytic enhancement for amination of ketones. Consistent with the Ranalysis-based prediction, the ω-TA variant harboring triple alanine mutations, i. e. L57A, W58A and V154A, showed high activity improvements for bulky substrates, e. g. a 3.2 × 10 4 -fold activity increase for 1phenylbutylamine. The triple mutant even enabled asymmetric amination of isobutyrophenone, carrying a branched-chain alkyl substituent to be accepted in a small binding pocket that normally shows a steric limit up to an ethyl group, with > 99% ee of a resulting (S)-amine.

Transaminases for industrial biocatalysis: novel enzyme discovery

“…Advances made with protein engineering can be fed back into the discovery pipeline in the quest for novel wild-type TAms, as well as new enzymes informing which mutations may be beneficial in future protein engineering. Similar studies have improved the activities of other enzymes through protein engineering such as Oa-TAm (Kim et al 2019b) and Cv-TAm (Almahboub et al 2018;Voss et al 2018), as well as engineering a strain of yeast to contain Cv-TAm for application in whole cell biocatalysis (Braun-Galleani et al 2019).…”

Computational Redesign of an ω-Transaminase from Pseudomonas jessenii for Asymmetric Synthesis of Enantiopure Bulky Amines

“…23 Replacing Trp58 of OaTA also promoted activity with other aliphatic and aromatic ketones, as seen with mutants W58L and W58A. 23,57 In Vf TA, the corresponding Trp57 was mutated to Phe or Cys in several designs for the synthesis of hydrophobic bulky amines. 15,20 In the redesign of Vf TA for asymmetric synthesis of (1S)-1-(1,1′-biphenyl-2yl)ethanamine, the best variant-containing mutation W57F exhibited 1716-fold higher activity in comparison to the wildtype Vf TA.…”

“…In Oa TA variants designed for 2a and 3a , mutation W58L relieved steric hindrance in the large binding pocket and gave a 760-fold increase in activity with 2a (3 mU/mg) and 210-fold increase in activity with 3a (0.7 mU/mg) compared to the parent . Replacing Trp58 of Oa TA also promoted activity with other aliphatic and aromatic ketones, as seen with mutants W58L and W58A. , In Vf TA, the corresponding Trp57 was mutated to Phe or Cys in several designs for the synthesis of hydrophobic bulky amines. , In the redesign of Vf TA for asymmetric synthesis of (1 S )-1-(1,1′-biphenyl-2-yl)­ethanamine, the best variant-containing mutation W57F exhibited 1716-fold higher activity in comparison to the wild-type Vf TA . Mutation W57G was beneficial for the activity of Vf TA toward different aliphatic amines, while W57F gave improved activity with different aliphatic aldehydes and ( R )-ethyl 5-methyl 3-oxooctanoate. ,, The corresponding position in Cv TA is Trp60, and mutation W60C improved the activity in the deamination of ( S )-1-phenylethylamine and the amination of a series of ketones .…”

Protein engineering of amine transaminases

“…> 99.9%), while the wild-type OaTA only gave 23% conversion. To enhance the efficiency of combining mutations after the identification of improved single mutants, a method called R-analysis was employed to predict if adding a mutation in OaTA will have a positive (R > 1) or negative (0 < R < 1) effect on activity with a set of structurally related target substrates including α-keto acids, (S)-arylalkylamines and arylalkyl ketones (Kim et al, 2019). The cosubstrate was fixed.…”

“…Residue Phe85 (VfTA)/Phe88 (CvTA)/Phe86 (PjTA), located in the small binding pocket, is frequently targeted and mutated to smaller residues like alanine, valine or leucine to relieve the steric hindrance limiting acceptance of bulky substituents (Midelfort et al, 2013;Sirin et al, 2014;Nobili et al, 2015;Genz et al, 2016;Voss et al, 2018;Land et al, 2020;Meng et al, 2021;Novick et al, 2021;Xiang et al, 2021;Sheludko et al, 2022). Another key residue in the small binding pocket is Val153 (VfTA)/Val154 (OaTA) and two smaller residues (alanine and serine) were tested (Midelfort et al, 2013;Sirin et al, 2014;Nobili et al, 2015;Dourado et al, 2016;Genz et al, 2016;Kim et al, 2019;Novick et al, 2021;Xiang et al, 2021). A key residue from the large binding pocket often responsible for steric hindrance with substrate is Trp57 (VfTA)/Trp60 (CvTA)/Trp58 (OaTA/PjTA), and mutations to smaller residues such as glycine, alanine, leucine, phenylalanine, and cysteine have been successful in reducing steric interference (Cho et al, 2008;Cassimjee et al, 2012;Midelfort et al, 2013;Sirin et al, 2014;Han et al, 2015cHan et al, , 2017Genz et al, 2015Genz et al, , 2016Dourado et al, 2016;Kim et al, 2019;Meng et al, 2021;Novick et al, 2021;Xiang et al, 2021).…”