Active Site Engineering of ω-Transaminase Guided by Docking Orientation Analysis and Virtual Activity Screening

Sang Woo Han, Juyeon Kim, Hyun Soo Cho, jong shik shin

Research output: Contribution to journalArticle

10 Citations (Scopus)

Abstract

Creation of enzyme variants displaying desirable catalytic performance usually necessitates tedious and time-consuming procedures for library generation and selection, which may be circumvented by a computational method based on a precise understanding of the reaction mechanism in the context of active site environment. Despite the great potential of ω-transaminases (ω-TAs) for asymmetric synthesis of chiral amines from ketones, it remains elusive why ω-TAs exhibit marginal activities for most ketones in contrast to their high activities for α-keto acids and aldehydes. To address this puzzling question, crystal structure determination and molecular modeling of ω-TAs were carried out to analyze docking orientations of the amino acceptors in the Michaelis complex. We found that ketones, unlike the reactive substrates, led to nonproductive binding complexes where the bound substrate was hardly accessible to a nucleophilic attack by the pyridoxamine cofactor to initiate reductive amination of the amino acceptor. This finding led us to perform in silico mutagenesis of the S-selective ω-TA from Ochrobactrum anthropi to ameliorate the unfavorable nucleophilic attack trajectory to structurally demanding ketones. The resulting variant, carrying L57A/W58A mutations, was predicted to allow an unprecedented re-face attack on butyrophenone, leading to 105-fold activity improvement with no loss in stereoselectivity. This study is expected to provide an efficient computational strategy for creation of high-turnover ω-TA variants tailored for a target ketone by affording in silico assessment of the effect of active site mutation on an enzyme activity.

Original languageEnglish
Pages (from-to)3752-3762
Number of pages11
JournalACS Catalysis
Volume7
Issue number6
DOIs
Publication statusPublished - 2017 Jun 2

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Transaminases
Ketones
Screening
Butyrophenones
Pyridoxamine
Keto Acids
Amination
Stereoselectivity
Mutagenesis
Molecular modeling
Enzyme activity
Substrates
Computational methods
Aldehydes
Amines
Enzymes
Crystal structure
Trajectories
Acids

All Science Journal Classification (ASJC) codes

  • Catalysis

Cite this

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abstract = "Creation of enzyme variants displaying desirable catalytic performance usually necessitates tedious and time-consuming procedures for library generation and selection, which may be circumvented by a computational method based on a precise understanding of the reaction mechanism in the context of active site environment. Despite the great potential of ω-transaminases (ω-TAs) for asymmetric synthesis of chiral amines from ketones, it remains elusive why ω-TAs exhibit marginal activities for most ketones in contrast to their high activities for α-keto acids and aldehydes. To address this puzzling question, crystal structure determination and molecular modeling of ω-TAs were carried out to analyze docking orientations of the amino acceptors in the Michaelis complex. We found that ketones, unlike the reactive substrates, led to nonproductive binding complexes where the bound substrate was hardly accessible to a nucleophilic attack by the pyridoxamine cofactor to initiate reductive amination of the amino acceptor. This finding led us to perform in silico mutagenesis of the S-selective ω-TA from Ochrobactrum anthropi to ameliorate the unfavorable nucleophilic attack trajectory to structurally demanding ketones. The resulting variant, carrying L57A/W58A mutations, was predicted to allow an unprecedented re-face attack on butyrophenone, leading to 105-fold activity improvement with no loss in stereoselectivity. This study is expected to provide an efficient computational strategy for creation of high-turnover ω-TA variants tailored for a target ketone by affording in silico assessment of the effect of active site mutation on an enzyme activity.",
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Active Site Engineering of ω-Transaminase Guided by Docking Orientation Analysis and Virtual Activity Screening. / Han, Sang Woo; Kim, Juyeon; Cho, Hyun Soo; shin, jong shik.

In: ACS Catalysis, Vol. 7, No. 6, 02.06.2017, p. 3752-3762.

Research output: Contribution to journalArticle

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