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Hokkaido University Collection of Scholarly and Academic Papers >
Graduate School of Engineering / Faculty of Engineering >
Peer-reviewed Journal Articles, etc >
Directed Evolution and Structural Analysis of NADPH-Dependent Acetoacetyl Coenzyme A (Acetoacetyl-CoA) Reductase from Ralstonia eutropha Reveals Two Mutations Responsible for Enhanced Kinetics
| 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)
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Submitter: 松本 謙一郎
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