7LDC

Zoogloea ramigera biosynthetic thiolase Q183Y/Y218E mutant

Summary for 7LDC

• Entry DOI: 10.2210/pdb7ldc/pdb
• Related: 7LBZ 7LCA 7LCL 7LD2
• Descriptor: Acetyl-CoA acetyltransferase, SULFATE ION, COENZYME A, ... (4 entities in total)
• Functional Keywords: acetyl-coa c-acetyltransferase, acetoacetyl-coa thiolase, type ii thiolase, biosynthetic thiolase, potassium activation, transferase
• Biological source: Zoogloea ramigera
• Total number of polymer chains: 4
• Total formula weight: 170207.99

Authors

Marshall, A.C.,Bruning, J.B. (deposition date: 2021-01-13, release date: 2021-09-01, Last modification date: 2024-10-23)

Primary citation

Marshall, A.C.,Bruning, J.B.
Engineering potassium activation into biosynthetic thiolase.
Biochem.J., 478:3047-3062, 2021

PubMed 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.

PubMed: 34338286
DOI: 10.1042/BCJ20210455

Experimental method

X-RAY DIFFRACTION (2.5 Å)