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Directed Evolution and Structural Analysis of NADPH-Dependent Acetoacetyl Coenzyme A (Acetoacetyl-CoA) Reductase from Ralstonia eutropha Reveals Two Mutations Responsible for Enhanced Kinetics

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Title: Directed Evolution and Structural Analysis of NADPH-Dependent Acetoacetyl Coenzyme A (Acetoacetyl-CoA) Reductase from Ralstonia eutropha Reveals Two Mutations Responsible for Enhanced Kinetics
Authors: Matsumoto, Ken'ichiro Browse this author
Tanaka, Yoshikazu Browse this author
Watanabe, Tsuyoshi Browse this author
Motohashi, Ren Browse this author
Ikeda, Koji Browse this author
Tobitani, Kota Browse this author
Yao, Min Browse this author
Tanaka, Isao Browse this author
Taguchi, Seiichi Browse this author
Keywords: in vitro evolution
polyhydroxyalkanoate
biobased plastic
3-ketoacyl-ACP reductase
Issue Date: Oct-2013
Publisher: Amer soc microbiology
Journal Title: Applied and environmental microbiology
Volume: 79
Issue: 19
Start Page: 6134
End Page: 6139
Publisher DOI: 10.1128/AEM.01768-13
Abstract: NADPH-dependent acetoacetyl-coenzyme A (acetoacetyl-CoA) reductase (PhaB) is a key enzyme in the synthesis of poly(3-hydroxybutyrate) [P(3HB)], along with beta-ketothiolase (PhaA) and polyhydroxyalkanoate synthase (PhaC). In this study, PhaB from Ralstonia eutropha was engineered by means of directed evolution consisting of an error-prone PCR-mediated mutagenesis and a P(3HB) accumulation-based in vivo screening system using Escherichia coli. From approximately 20,000 mutants, we obtained two mutant candidates bearing Gln47Leu (Q47L) and Thr173Ser (T173S) substitutions. The mutants exhibited k(cat) values that were 2.4-fold and 3.5-fold higher than that of the wild-type enzyme, respectively. In fact, the PhaB mutants did exhibit enhanced activity and P(3HB) accumulation when expressed in recombinant Corynebacterium glutamicum. Comparative three-dimensional structural analysis of wild-type PhaB and highly active PhaB mutants revealed that the beneficial mutations affected the flexibility around the active site, which in turn played an important role in substrate recognition. Furthermore, both the kinetic analysis and crystal structure data supported the conclusion that PhaB forms a ternary complex with NADPH and acetoacetyl-CoA. These results suggest that the mutations affected the interaction with substrates, resulting in the acquirement of enhanced activity.
Type: article (author version)
URI: http://hdl.handle.net/2115/55134
Appears in Collections:工学院・工学研究院 (Graduate School of Engineering / Faculty of Engineering) > 雑誌発表論文等 (Peer-reviewed Journal Articles, etc)

Submitter: 松本 謙一郎

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