In Silico Based Engineering Approach to Improve Transaminases for the Conversion of Bulky Substrates

Abstract

Protein engineering is often applied to tailor substrate specificity, enantioselectivity, or stability of enzymes according to the needs of a process. In rational engineering approaches, molecular docking and molecular dynamics simulations are often used to compare transition states of wild-type and enzyme variants. Besides affecting the transition state energies by mutations, the entry of the substrate and its positioning in the active site (Michaelis complex) is also often studied, and mutagenesis of residues forming the substrate entry tunnel can have a profound impact on activity and selectivity. In this study, we combine the strengths of such a tunnel approach with MD followed by semiempirical QM calculations that allow the identification of beneficial positions and an in silico screening of possible variants. We exemplify this strategy in the expansion of the substrate scope of Chromobacterium violaceum amine transaminase toward sterically demanding substrates. Two double mutants (F88L/C418­(G/L)) proposed by the modeling showed >200-fold improved activities in the conversion of 1-phenylbutylamine and enabled the asymmetric synthesis of this amine from the corresponding ketone, which was not possible with the wild-type. The correlation of interaction energies and geometrical parameters (distance of the substrate’s carbonyl carbon to the cofactor’s amino group) as obtained in the simulations suggests that this strategy can be used for in silico prediction of variants facilitating an efficient entry and placement of a desired substrate as a first requirement for catalysis. However, when choosing amino acid positions for substitution and modeling, additional knowledge of the enzymatic reaction mechanism is required, as residues that are involved in the catalytic machinery or that guarantee the structural integrity of the enzyme will not be recognized by the developed algorithm and should be excluded manually.

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

“…Other useful methodologies for predicting the catalytic potential of ω-TA variants (using a different intermediate) are computationally more expensive ( e.g. , ∼60 CPU hours per enzyme variant in the methodology described by Voss et al ( 17 )) or make use of coarser categorizations such as active vs inactive in the work of Sirin et al ( 18 ) and high- vs low-reactivity grouping used by Han et al ( 21 ) A study in which the activity toward 2-acetylbiphenyl ketone was engineered in Vf TA followed an approach in which mutations were added in a stepwise manner, without predicting activity for a large number of variants as a ranking tool. 20 …”

“… 16 The caveat of using computational design is that the methodology is case-specific, and the choice of a good strategy is dependent on the enzyme family, the type of reaction, the type of substrate, and the limiting catalytic step. For ω-TAs, computational methods for finding mutations that modify the substrate scope typically include docking of a modeled reaction intermediate 17 and molecular dynamics simulations 17 or quantum mechanical modeling 18 of such an intermediate. Docking is the fastest amongst these methods, but explicit water molecules are generally not included to avoid a drastic increase in the search space.…”

“…Moreover, ( S )-1-phenylbutylamine has been the target of previous transaminase engineering efforts. 12 , 17 , 21 , 22 , 33 Two other target amines were ( S )-(+)-1-aminoindan ( 2b ) and ( S )-(+)-1-aminotetralin ( 3b ). The activity of Pj TA-R6 for these compounds is undetectable; the same is observed with most other ω-TAs.…”

Asymmetric Synthesis of Optically Pure Aliphatic Amines with an Engineered Robust ω-Transaminase

“…In the second half reaction, the ketone acceptor binds to the PMP enzyme to form the aminated product and regenerate PLP (Scheme 1). Recent efforts to tailor the ω-TA substrate scope have focused on stabilization of the Michaelis complex of the ketone substrate [11][12][13][14] with little or no attention to reaction intermediates. The external aldimine is a key intermediate in the transamination reaction.…”