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. 2013 Oct;79(19):6134-9. doi: 10.1128/AEM.01768-13. Epub 2013 Aug 2.


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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Acyl Coenzyme A / chemistry
  • Acyl Coenzyme A / metabolism*
  • Alcohol Oxidoreductases / chemistry
  • Alcohol Oxidoreductases / genetics*
  • Alcohol Oxidoreductases / metabolism*
  • Coenzymes / metabolism
  • Corynebacterium glutamicum / genetics
  • Corynebacterium glutamicum / metabolism
  • Crystallography, X-Ray
  • Cupriavidus necator / enzymology*
  • DNA Mutational Analysis
  • Directed Molecular Evolution*
  • Escherichia coli / genetics
  • Escherichia coli / metabolism
  • Gene Expression
  • Hydroxybutyrates / metabolism*
  • Kinetics
  • Models, Molecular
  • Mutation, Missense
  • NADP / chemistry
  • NADP / metabolism*
  • Polyesters / metabolism*
  • Polymerase Chain Reaction
  • Protein Conformation


  • Acyl Coenzyme A
  • Coenzymes
  • Hydroxybutyrates
  • Polyesters
  • acetoacetyl CoA
  • poly-beta-hydroxybutyrate
  • NADP
  • Alcohol Oxidoreductases
  • acetoacetyl-CoA reductase

Associated data

  • PDB/3VZP
  • PDB/3VZR
  • PDB/3VZS