3VZS

Crystal structure of PhaB from Ralstonia eutropha in complex with Acetoacetyl-CoA and NADP

3VZS の概要

• エントリーDOI: 10.2210/pdb3vzs/pdb
• 関連するPDBエントリー: 3VZP 3VZQ 3VZR
• 分子名称: Acetoacetyl-CoA reductase, SULFATE ION, NADP NICOTINAMIDE-ADENINE-DINUCLEOTIDE PHOSPHATE, ... (5 entities in total)
• 機能のキーワード: alpha/beta fold, oxidoreductase
• 由来する生物種: Cupriavidus necator (Ralstonia eutropha)
• 細胞内の位置: Cytoplasm: P14697
• タンパク質・核酸の鎖数: 4
• 化学式量合計: 117453.44

構造登録者

Ikeda, K.,Tanaka, Y.,Tanaka, I.,Yao, M. (登録日: 2012-10-15, 公開日: 2013-08-28, 最終更新日: 2023-11-08)

主引用文献

Matsumoto, K.,Tanaka, Y.,Watanabe, T.,Motohashi, R.,Ikeda, K.,Tobitani, K.,Yao, M.,Tanaka, I.,Taguchi, S.
Directed evolution and structural analysis of NADPH-dependent Acetoacetyl Coenzyme A (Acetoacetyl-CoA) reductase from Ralstonia eutropha reveals two mutations responsible for enhanced kinetics
Appl.Environ.Microbiol., 79:6134-6139, 2013

PubMed 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 β-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 kcat 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.

PubMed: 23913421
DOI: 10.1128/AEM.01768-13

実験手法

X-RAY DIFFRACTION (2.14 Å)