Engineering an alcohol dehydrogenase with enhanced activity and stereoselectivity toward diaryl ketones: reduction of steric hindrance and change of the stereocontrol element

Wu, Kai; Yang, Zhijun; Meng, Xiangguo; Chen, Rong; Huang, Jiankun; Shao, Lei

doi:10.1039/c9cy02444a

Cited by 46 publications

(39 citation statements)

References 44 publications

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“…3 f). This result was similar to that of other studies [ 25 , 32 ]. Subsequently, the kinetic parameters of Bb PheDH and obtained mutants with superior activity toward NADH were assessed.…”

Section: Resultssupporting

confidence: 93%

“…Directed evolution techniques are powerful tools for generating prolific sources of biocatalysts [ 22 , 23 ]. Steric hindrance of the substrate-binding pocket plays a significant role in modulating the catalytic properties of an enzyme, including enantioselectivities [ 24 , 25 ], substrate specificities [ 26 – 28 ], and catalytic activities [ 25 , 28 – 30 ]. Recent enzyme engineering studies focused on increasing or conferring enzyme activity towards non-native bulky substrates by attenuating the steric hindrance of the substrate-binding pocket.…”

Section: Introductionmentioning

confidence: 99%

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Structure-guided steric hindrance engineering of Bacillus badius phenylalanine dehydrogenase for efficient l-homophenylalanine synthesis

Xue

et al. 2021

Biotechnol Biofuels

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Background Direct reductive amination of prochiral 2-oxo-4-phenylbutyric acid (2-OPBA) catalyzed by phenylalanine dehydrogenase (PheDH) is highly attractive in the synthesis of the pharmaceutical chiral building block l-homophenylalanine (l-HPA) given that its sole expense is ammonia and that water is the only byproduct. Current issues in this field include a poor catalytic efficiency and a low substrate loading. Results In this study, we report a structure-guided steric hindrance engineering of PheDH from Bacillus badius to create an enhanced biocatalyst for efficient l-HPA synthesis. Mutagenesis libraries based on molecular docking, double-proximity filtering, and a degenerate codon significantly increased catalytic efficiency. Seven superior mutants were acquired, and the optimal triple-site mutant, V309G/L306V/V144G, showed a 12.7-fold higher kcat value, and accordingly a 12.9-fold higher kcat/Km value, than that of the wild type. A paired reaction system comprising V309G/L306V/V144G and glucose dehydrogenase converted 1.08 M 2-OPBA to l-HPA in 210 min, and the specific space–time conversion was 30.9 mmol g−1 L−1 h−1. The substrate loading and specific space–time conversion are the highest values to date. Docking simulation revealed increases in substrate-binding volume and additional degrees of freedom of the substrate 2-OPBA in the pocket. Tunnel analysis suggested the formation of new enzyme tunnels and the expansion of existing ones. Conclusions Overall, the results show that the mutant V309G/L306V/V144G has the potential for the industrial synthesis of l-HPA. The modified steric hindrance engineering approach can be a valuable addition to the current enzyme engineering toolbox.

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“…3 f). This result was similar to that of other studies [ 25 , 32 ]. Subsequently, the kinetic parameters of Bb PheDH and obtained mutants with superior activity toward NADH were assessed.…”

Section: Resultssupporting

confidence: 93%

Section: Introductionmentioning

confidence: 99%

Structure-guided steric hindrance engineering of Bacillus badius phenylalanine dehydrogenase for efficient l-homophenylalanine synthesis

Xue

et al. 2021

Biotechnol Biofuels

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“…3,25 Similarly, a recent study on LkADH emphasized the importance of halogen bonds formed between halogensubstituted ketones and the enzyme. 34 For ADHs, it is generally accepted that stereoselectivity is decided by the direction of nucleophilic attack from the hydride of the C4 of NAD(P)H. 74 In summary, the difference in the formation efficiency between two alcohol enantiomers essentially represents the stereoselectivity of an ADH and the larger the difference, the higher the stereoselectivity. Consequently, catalytic stereoselectivity is closely related to enzyme− substrate interactions at the molecular level.…”

Section: General Catalytic Mechanism Of Sdr and Mdrmentioning

confidence: 99%

“…Na SDR from Novosphingobium aromaticivorans exhibited low activity toward bulky ketone substrates, whereas the variant G141A/I195L showed high stereoselectivity toward several diaryl ketones, especially (4-chlorophenyl)(phenyl)methanone ( 11 , ee > 93%, S ), (3-chlorophenyl)(phenyl)methanone ( 10 , ee > 99%, S ), and (4-chlorophenyl)(pyridin-2-yl) methanone ( 31 , ee > 97%, R ) . Similarly, several ADHs such as TbS ADH, , Eb SDR8, and Lk ADH could not accept diaryl methanones as substrates, whereas the TbS ADH A85G/I86L , Eb SDR8 G94Q/H145F/Y188A , and Lk ADH Y190P/I144V/L199V/E145C/M206F variants could catalyze the carbonyl reduction efficiently …”

Section: Sources and Propertiesmentioning

confidence: 99%

Stereochemistry in Asymmetric Reduction of Bulky–Bulky Ketones by Alcohol Dehydrogenases

Zhou

2020

ACS Catal.

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Biocatalyzed asymmetric reduction of ketones is one of the most cost-effective and environmentally friendly approaches for preparing chiral alcohols. However, few natural ketone reductases (KREDs) or alcohol dehydrogenase (ADHs) with high activity and stereoselectivity toward bulky–bulky ketones have yet been reported due to the strong steric hindrance of these substrates. Recent developments in protein engineering have led to biocatalysts capable of producing chiral alcohols with high efficiency and stereoselectivity. This review discusses recent advances in several approaches for the manipulation of stereoselectivity, including engineering of the binding pocket and active site loops to accommodate bulky–bulky ketones, modulating the electronic effects on attraction and repulsion between binding pocket residues and substituents of ketone substrates, and adding traceless directing groups to substrates to alter their orientation. Microbial sources of KREDs and ADHs, screening techniques, and future outlooks for the field are also discussed.

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“…Steric hindrance of the substrate-binding pocket plays a signi cant role in modulating the catalytic properties of an enzyme, including enantioselectivities [24,25], substrate speci cities [26,27], and catalytic activities [25,28,29]. Recent enzyme engineering studies focused on increasing or conferring enzyme activity towards non-native bulky substrates by attenuating the steric hindrance of the substratebinding pocket [30,31].…”

Section: Introductionmentioning

confidence: 99%

Structure-Guided Steric Hindrance Engineering of Bacillus Badies Phenylalanine Dehydrogenase for Efficient L-Homophenylalanine Synthesis

Xue

et al. 2021

Preprint

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Background: Direct reductive amination of prochiral 2-oxo-4-phenylbutyric acid (2-OPBA) catalyzed by phenylalanine dehydrogenase (PheDH) is highly attractive in the synthesis of the pharmaceutical chiral building block L-homophenylalanine (L-HPA) given that its sole expense is ammonia and that water is the only byproduct. Current issues in this field include a poor catalytic efficiency and a low substrate loading.Results: In this study, we report a structure-guided steric hindrance engineering of PheDH from Bacillus badies to create an enhanced biocatalyst for efficient L-HPA synthesis. Mutagenesis libraries based on molecular docking, double-proximity filtering, and a degenerate codon significantly increased catalytic activity. Seven superior mutants were acquired, and the optimal triple-site mutant V309G/L306V/V144G showed a 12.9-fold higher kcat/Km value than wild-type. A paired reaction system comprising V309G/L306V/V144G and glucose dehydrogenase converted 1.08 M 2-OPBA to L-HPA in 210 min, and the specific space-time conversion was 30.9 mmoL·g−1·L−1·h−1. The substrate loading and specific space-time conversion are the highest values to date. Docking simulation revealed increases in substrate-binding volume and additional degrees of freedom of the substrate 2-OPBA in the pocket. Tunnel analysis suggested the formation of new enzyme tunnels and the expansion of existing ones.Conclusions: Overall, the results show that the mutant V309G/L306V/V144G has the potential for the industrial synthesis of L-HPA. The modified steric hindrance engineering approach can be a valuable addition to the current enzyme engineering toolbox.

show abstract

Engineering an alcohol dehydrogenase with enhanced activity and stereoselectivity toward diaryl ketones: reduction of steric hindrance and change of the stereocontrol element

Abstract: Engineering an alcohol dehydrogenase with enhanced activity and stereoselectivity toward diaryl ketones: reduction of steric hindrance and change of the stereocontrol element.

Cited by 46 publications

References 44 publications

Structure-guided steric hindrance engineering of Bacillus badius phenylalanine dehydrogenase for efficient l-homophenylalanine synthesis

Structure-guided steric hindrance engineering of Bacillus badius phenylalanine dehydrogenase for efficient l-homophenylalanine synthesis

Stereochemistry in Asymmetric Reduction of Bulky–Bulky Ketones by Alcohol Dehydrogenases

Structure-Guided Steric Hindrance Engineering of Bacillus Badies Phenylalanine Dehydrogenase for Efficient L-Homophenylalanine Synthesis

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