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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.

Matsumoto K, Tanaka Y, Watanabe T, Motohashi R, Ikeda K, Tobitani K, Yao M, Tanaka I, Taguchi S
Appl Environ Microbiol 79 6134-9 (2013)
Related entries: 3vzp, 3vzq, 3vzr

Cited: 21 times
EuropePMC logo PMID: 23913421

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.

Articles - 3vzs mentioned but not cited (2)

  1. Directed evolution and structural analysis of NADPH-dependent Acetoacetyl Coenzyme A (Acetoacetyl-CoA) reductase from Ralstonia eutropha reveals two mutations responsible for enhanced kinetics. Matsumoto K, Tanaka Y, Watanabe T, Motohashi R, Ikeda K, Tobitani K, Yao M, Tanaka I, Taguchi S. Appl Environ Microbiol 79 6134-6139 (2013)
  2. Assay development and inhibition of the Mt-DprE2 essential reductase from Mycobacterium tuberculosis. Batt SM, Toth S, Rodriguez B, Abrahams KA, Veerapen N, Chiodarelli G, Cox LR, Moynihan PJ, Lelievre J, Fütterer K, Besra GS. Microbiology (Reading) 169 (2023)


Reviews citing this publication (1)

  1. Beyond the outer limits of nature by directed evolution. Molina-Espeja P, Viña-Gonzalez J, Gomez-Fernandez BJ, Martin-Diaz J, Garcia-Ruiz E, Alcalde M. Biotechnol Adv 34 754-767 (2016)

Articles citing this publication (18)

  1. Anaerobic poly-3-D-hydroxybutyrate production from xylose in recombinant Saccharomyces cerevisiae using a NADH-dependent acetoacetyl-CoA reductase. de Las Heras AM, Portugal-Nunes DJ, Rizza N, Sandström AG, Gorwa-Grauslund MF. Microb Cell Fact 15 197 (2016)
  2. Improved artificial pathway for biosynthesis of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) with high C6-monomer composition from fructose in Ralstonia eutropha. Insomphun C, Xie H, Mifune J, Kawashima Y, Orita I, Nakamura S, Fukui T. Metab Eng 27 38-45 (2015)
  3. Characterization of mutants of a tyrosine ammonia-lyase from Rhodotorula glutinis. Zhou S, Liu P, Chen J, Du G, Li H, Zhou J. Appl Microbiol Biotechnol 100 10443-10452 (2016)
  4. Engineering of Escherichia coli for the biosynthesis of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) from glucose. Wang Q, Luan Y, Cheng X, Zhuang Q, Qi Q. Appl Microbiol Biotechnol 99 2593-2602 (2015)
  5. Modification of acetoacetyl-CoA reduction step in Ralstonia eutropha for biosynthesis of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) from structurally unrelated compounds. Zhang M, Kurita S, Orita I, Nakamura S, Fukui T. Microb Cell Fact 18 147 (2019)
  6. MtgA Deletion-Triggered Cell Enlargement of Escherichia coli for Enhanced Intracellular Polyester Accumulation. Kadoya R, Matsumoto K, Ooi T, Taguchi S. PLoS One 10 e0125163 (2015)
  7. Biosynthesis of Trehangelin in Polymorphospora rubra K07-0510: Identification of Metabolic Pathway to Angelyl-CoA. Inahashi Y, Shiraishi T, Palm K, Takahashi Y, Ōmura S, Kuzuyama T, Nakashima T. Chembiochem 17 1442-1447 (2016)
  8. Improved production of poly(lactic acid)-like polyester based on metabolite analysis to address the rate-limiting step. Matsumoto K, Tobitani K, Aoki S, Song Y, Ooi T, Taguchi S. AMB Express 4 83 (2014)
  9. Applying asymptotic methods to synthetic biology: Modelling the reaction kinetics of the mevalonate pathway. Dalwadi MP, Garavaglia M, Webb JP, King JR, Minton NP. J Theor Biol 439 39-49 (2018)
  10. Mechanistic insight into acrylate metabolism and detoxification in marine dimethylsulfoniopropionate-catabolizing bacteria. Wang P, Cao HY, Chen XL, Li CY, Li PY, Zhang XY, Qin QL, Todd JD, Zhang YZ. Mol Microbiol 105 674-688 (2017)
  11. With Chitosan and PLGA as the Delivery Vehicle, Toxoplasma gondii Oxidoreductase-Based DNA Vaccines Decrease Parasite Burdens in Mice. Yu Z, Cao W, Gao X, Aleem MT, Liu J, Luo J, Yan R, Xu L, Song X, Li X. Front Immunol 12 726615 (2021)
  12. Development of Clostridium saccharoperbutylacetonicum as a Whole Cell Biocatalyst for Production of Chirally Pure (R)-1,3-Butanediol. Grosse-Honebrink A, Little GT, Bean Z, Heldt D, Cornock RHM, Winzer K, Minton NP, Green E, Zhang Y. Front Bioeng Biotechnol 9 659895 (2021)
  13. Adaptive Laboratory Evolution of Halomonas bluephagenesis Enhances Acetate Tolerance and Utilization to Produce Poly(3-hydroxybutyrate). Zhang J, Jin B, Fu J, Wang Z, Chen T. Molecules 27 3022 (2022)
  14. Comparison of engineered Escherichia coli AF1000 and BL21 strains for (R)-3-hydroxybutyrate production in fed-batch cultivation. Perez-Zabaleta M, Guevara-Martínez M, Gustavsson M, Quillaguamán J, Larsson G, van Maris AJA. Appl Microbiol Biotechnol 103 5627-5639 (2019)
  15. Crystal structure and kinetic analyses of a hexameric form of (S)-3-hydroxybutyryl-CoA dehydrogenase from Clostridium acetobutylicum. Takenoya M, Taguchi S, Yajima S. Acta Crystallogr F Struct Biol Commun 74 733-740 (2018)
  16. Enhanced poly(3-hydroxybutyrate) production in transgenic tobacco BY-2 cells using engineered acetoacetyl-CoA reductase. Yokoo T, Matsumoto K, Ooba T, Morimoto K, Taguchi S. Biosci Biotechnol Biochem 79 986-988 (2015)
  17. Enhanced cellular content and lactate fraction of the poly(lactate-co-3-hydroxybutyrate) polyester produced in recombinant Escherichia coli by the deletion of σ factor RpoN. Kadoya R, Kodama Y, Matsumoto K, Taguchi S. J Biosci Bioeng 119 427-429 (2015)
  18. Engineering an acetoacetyl-CoA reductase from Cupriavidus necator toward NADH preference under physiological conditions. Olavarria K, Pijman YO, Cabrera R, van Loosdrecht MCM, Wahl SA. Sci Rep 12 3757 (2022)


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