Construction of stabilized (R)-selective amine transaminase from Aspergillus terreus by consensus mutagenesis

Xie, Dong-Fang; Yang, Jin‐yan; Lv, Changjiang; Mei, Jiaqi; Wang, Hongpeng; Hu, Sheng; Wang, Zhao; Cao, Jia-Ren; Tu, Jun-Liang; Huang, Jun; Mei, Lehe

doi:10.1016/j.jbiotec.2019.01.007

Cited by 24 publications

(23 citation statements)

References 43 publications

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“…To further study the results of the experiment, a virtual mutation model was built by Discovery Studio 2018, and recalculated by YASARA and ETSS. Intermolecular interactions, such as hydrogen bonds, hydrophobic interactions, van der Waals forces, ionic bonds and disulphide bonds play a decisive role in stabilizing the tertiary structure of proteins (Fan et al, 2018;Xie et al, 2019). MD simulation results also support the key role of substitution of positively charged amino acids (glutamine, lysine, histidine, and arginine) or neutral amino acids (alanine) in thermal stability.…”

Section: Discussionmentioning

confidence: 68%

“…Protein engineering plays a vital role in enhancing the thermal stability of (R)selective At-ATA to expand its applicability in industrial processes (Liu et al, 2019). To date, the rational design of protein engineering involves many factors, such as surface electrostatic interactions, hydrophobic interactions, B-factor values, consensus mutagenesis, disulphide bridges, coevolution networks, and hydrogen bonding interactions (Pace et al, 2011;Wang et al, 2014;Zhang et al, 2014Zhang et al, , 2020Huang et al, 2017;Xie et al, 2018Xie et al, , 2019Moon et al, 2019;Zhu et al, 2019;Cao et al, 2020). All of these have been employed to develop stable proteins.…”

Section: Introductionmentioning

confidence: 99%

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Improving the Thermostability and Activity of Transaminase From Aspergillus terreus by Charge-Charge Interaction

Cao

Fan

et al. 2021

Front. Chem.

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Transaminases that promote the amination of ketones into amines are an emerging class of biocatalysts for preparing a series of drugs and their intermediates. One of the main limitations of (R)-selective amine transaminase from Aspergillus terreus (At-ATA) is its weak thermostability, with a half-life (t1/2) of only 6.9 min at 40°C. To improve its thermostability, four important residue sites (E133, D224, E253, and E262) located on the surface of At-ATA were identified using the enzyme thermal stability system (ETSS). Subsequently, 13 mutants (E133A, E133H, E133K, E133R, E133Q, D224A, D224H, D224K, D224R, E253A, E253H, E253K, and E262A) were constructed by site-directed mutagenesis according to the principle of turning the residues into opposite charged ones. Among them, three substitutions, E133Q, D224K, and E253A, displayed higher thermal stability than the wild-type enzyme. Molecular dynamics simulations indicated that these three mutations limited the random vibration amplitude in the two α-helix regions of 130–135 and 148–158, thereby increasing the rigidity of the protein. Compared to the wild-type, the best mutant, D224K, showed improved thermostability with a 4.23-fold increase in t1/2 at 40°C, and 6.08°C increase in T5010. Exploring the three-dimensional structure of D224K at the atomic level, three strong hydrogen bonds were added to form a special “claw structure” of the α-helix 8, and the residues located at 151–156 also stabilized the α-helix 9 by interacting with each other alternately.

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Section: Discussionmentioning

confidence: 68%

Section: Introductionmentioning

confidence: 99%

Improving the Thermostability and Activity of Transaminase From Aspergillus terreus by Charge-Charge Interaction

Cao

Fan

et al. 2021

Front. Chem.

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“…The products of the transamination reactions catalyzed by AT-ATA and the mutant L118T were analyzed by UPLC-MS (Waters, Milford, MA, USA) according to the method of Xie et al [10]. When 2-butylamine was used as an amino donor, the conditions for the UPLC analysis were as follows: an ACQUITY UPLC HSS T3 column (Waters, Milford, MA, USA) (2.1 × 100 mm, 1.8 μm) was used with a flow rate of 0.30 mL/min, and the mobile phase was a 35:65 ( v / v ) eluent A (0.1% formic acid in water (dH 2 O)) and eluent B (0.1% formic acid in acetonitrile).…”

Section: Methodsmentioning

confidence: 99%

“…To enhance the thermal stability of an ( R )-selective amine transaminase from Aspergillus terreus (AT-ATA), rational strategies, such as the combination of the B-factor profile and folding free energy calculations (ΔΔ G fold ), the introduction of disulfide bridges, and consensus mutagenesis, were investigated [8,9,10]. Recently, the fact that a protein can be considered as a network of interacting residues has attracted great interest [11,12,13,14].…”

Section: Introductionmentioning

confidence: 99%

A Single Mutation Increases the Thermostability and Activity of Aspergillus terreus Amine Transaminase

Zhu

et al. 2019

Molecules

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Enhancing the thermostability of (R)-selective amine transaminases (AT-ATA) will expand its application in the asymmetric synthesis of chiral amines. In this study, mutual information and coevolution networks of ATAs were analyzed by the Mutual Information Server to Infer Coevolution (MISTIC). Subsequently, the amino acids most likely to influence the stability and function of the protein were investigated by alanine scanning and saturation mutagenesis. Four stabilized mutants (L118T, L118A, L118I, and L118V) were successfully obtained. The best mutant, L118T, exhibited an improved thermal stability with a 3.7-fold enhancement in its half-life (t1/2) at 40 °C and a 5.3 °C increase in T5010 compared to the values for the wild-type protein. By the differential scanning fluorimetry (DSF) analysis, the best mutant, L118T, showed a melting temperature (Tm) of 46.4 °C, which corresponded to a 5.0 °C increase relative to the wild-type AT-ATA (41.4 °C). Furthermore, the most stable mutant L118T displayed the highest catalytic efficiency among the four stabilized mutants.

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“…While other properties such as substrate scope, enantioselectivity and inhibition have been successfully altered through enzyme engineering in a number of instances 3 , the improvement of the stability profile of transaminases has only gained momentum in the last couple of years. Although methods as diverse as semi-rational design 7 , structure-based re-design 8–11 , consensus mutagenesis 11,12 and incorporation of unnatural amino acids 13 have been successfully applied to improve the thermostability of some transaminases, we still do not have a clear understanding of the inactivation mechanism undermining the operational and storage stability of this group of enzymes. From an engineering perspective, a deeper insight into the molecular basis of transaminase inactivation could ease the identification of new mutagenesis targets for the improvement of transaminase stability.…”

Section: Introductionmentioning

confidence: 99%

Insight into the dimer dissociation process of the Chromobacterium violaceum (S)-selective amine transaminase

Ruggieri

Campillo-Brocal

Chen

et al. 2019

Sci Rep

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One of the main factors hampering the implementation in industry of transaminase-based processes for the synthesis of enantiopure amines is their often low storage and operational stability. Our still limited understanding of the inactivation processes undermining the stability of wild-type transaminases represents an obstacle to improving their stability through enzyme engineering. In this paper we present a model describing the inactivation process of the well-characterized (S)-selective amine transaminase from Chromobacterium violaceum. The cornerstone of the model, supported by structural, computational, mutagenesis and biophysical data, is the central role of the catalytic lysine as a conformational switch. Upon breakage of the lysine-PLP Schiff base, the strain associated with the catalytically active lysine conformation is dissipated in a slow relaxation process capable of triggering the known structural rearrangements occurring in the holo-to-apo transition and ultimately promoting dimer dissociation. Due to the occurrence in the literature of similar PLP-dependent inactivation models valid for other non-transaminase enzymes belonging to the same fold-class, the role of the catalytic lysine as conformational switch might extend beyond the transaminase enzyme group and offer new insight to drive future non-trivial engineering strategies.

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Construction of stabilized (R)-selective amine transaminase from Aspergillus terreus by consensus mutagenesis

Cited by 24 publications

References 43 publications

Improving the Thermostability and Activity of Transaminase From Aspergillus terreus by Charge-Charge Interaction

Improving the Thermostability and Activity of Transaminase From Aspergillus terreus by Charge-Charge Interaction

A Single Mutation Increases the Thermostability and Activity of Aspergillus terreus Amine Transaminase

Insight into the dimer dissociation process of the Chromobacterium violaceum (S)-selective amine transaminase

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