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Engineering potassium activation into biosynthetic thiolase
- Source :
- Biochemical Journal. 478:3047-3062
- Publication Year :
- 2021
- Publisher :
- Portland Press Ltd., 2021.
-
Abstract
- 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.
- Subjects :
- Models, Molecular
Protein Conformation
Stereochemistry
Allosteric regulation
Crystallography, X-Ray
Protein Engineering
Biochemistry
Substrate Specificity
Conserved sequence
Residue (chemistry)
Enzyme activator
Bacterial Proteins
Acetyl Coenzyme A
Acetyl-CoA C-Acetyltransferase
Binding site
Molecular Biology
Zoogloea
Thiolase
Substrate (chemistry)
Cell Biology
Protein engineering
Cations, Monovalent
Enzyme Activation
Kinetics
Mutation
Biocatalysis
Potassium
Acyl Coenzyme A
Protein Multimerization
Protein Binding
Subjects
Details
- ISSN :
- 14708728 and 02646021
- Volume :
- 478
- Database :
- OpenAIRE
- Journal :
- Biochemical Journal
- Accession number :
- edsair.doi.dedup.....3319b5d7698066ac68109b88c88b8cff