Engineering potassium activation into biosynthetic thiolase

Biochem J. 2021 Aug 13;478(15):3047-3062. doi: 10.1042/BCJ20210455.


Activation of enzymes by monovalent cations (M+) is a widespread phenomenon in biology. Despite this, there are few structure-based studies describing the underlying molecular details. Thiolases are a ubiquitous and highly conserved family of enzymes containing both K+-activated and K+-independent members. Guided by structures of naturally occurring K+-activated thiolases, we have used a structure-based approach to engineer K+-activation into a K+-independent thiolase. To our knowledge, this is the first demonstration of engineering K+-activation into an enzyme, showing the malleability of proteins to accommodate M+ ions as allosteric regulators. We show that a few protein structural features encode K+-activation in this class of enzyme. Specifically, two residues near the substrate-binding site are sufficient for K+-activation: A tyrosine residue is required to complete the K+ coordination sphere, and a glutamate residue provides a compensating charge for the bound K+ ion. Further to these, a distal residue is important for positioning a K+-coordinating water molecule that forms a direct hydrogen bond to the substrate. The stability of a cation-π interaction between a positively charged residue and the substrate is determined by the conformation of the loop surrounding the substrate-binding site. Our results suggest that this cation-π interaction effectively overrides K+-activation, and is, therefore, destabilised in K+-activated thiolases. Evolutionary conservation of these amino acids provides a promising signature sequence for predicting K+-activation in thiolases. Together, our structural, biochemical and bioinformatic work provide important mechanistic insights into how enzymes can be allosterically activated by M+ ions.

Keywords: acetyltransferases; enzyme activation; metalloenzymes; molecular mechanisms; protein engineering.

MeSH terms

  • Acetyl Coenzyme A / chemistry
  • Acetyl Coenzyme A / metabolism
  • Acetyl-CoA C-Acetyltransferase / chemistry
  • Acetyl-CoA C-Acetyltransferase / genetics
  • Acetyl-CoA C-Acetyltransferase / metabolism*
  • Acyl Coenzyme A / chemistry
  • Acyl Coenzyme A / metabolism
  • Bacterial Proteins / chemistry
  • Bacterial Proteins / genetics
  • Bacterial Proteins / metabolism*
  • Biocatalysis
  • Cations, Monovalent / chemistry
  • Cations, Monovalent / metabolism*
  • Crystallography, X-Ray
  • Enzyme Activation*
  • Kinetics
  • Models, Molecular
  • Mutation
  • Potassium / chemistry
  • Potassium / metabolism*
  • Protein Binding
  • Protein Conformation
  • Protein Engineering
  • Protein Multimerization
  • Substrate Specificity
  • Zoogloea / enzymology
  • Zoogloea / genetics
  • Zoogloea / isolation & purification*


  • Acyl Coenzyme A
  • Bacterial Proteins
  • Cations, Monovalent
  • acetoacetyl CoA
  • Acetyl Coenzyme A
  • Acetyl-CoA C-Acetyltransferase
  • Potassium

Supplementary concepts

  • Zoogloea ramigera