Structural basis for disulphide-CoA Inhibition of a butyryl-CoA hexameric thioesterase

Yogesh Khandokar, Parul Srivastava, Shane Raidal, Subir Sarker, Jade K Forwood

Research output: Contribution to journalArticle

Abstract

Acyl-coenzyme A thioesterases (ACTs) catalyse the hydrolysis of thioester bonds between fatty-acyl chains and coenzyme A (CoA), producing a free fatty-acyl chain and CoA. These enzymes are expressed ubiquitously across prokaryotes and eukaryotes, and play important roles in lipid metabolism. There are 25 thioesterase families, subdivided based on their active site configuration, protein oligomerization, and substrate specificity. Understanding the mechanism of regulation within these families is important due to their roles in controlling the cell concentration of a range of fatty acids and CoA-bound compounds. Here we report a structural basis for a novel mode of inhibition of an ACT from Staphylococcus aureus. The enzyme displays a hotdog fold composed of five β-strands wrapping around a central α-helix, and an additional 30 residue α-helix located at its C-terminus. We show that the enzyme is a hexamer and has specificity towards butyryl-CoA. Structural analysis revealed putative catalytic residues, and we show through site directed mutagenesis that Asn28, Asp43, and Thr60 are critical for activity. Additionally, we show that the Asn28Ala destabilises the enzyme oligomeric state into two distinct populations. Co-crystallization of the enzyme with the substrate butyryl-CoA produced a crystal with three CoA ligands bound in the enzyme active sites: CoA, butyryl-CoA, and disulphide-CoA, the latter of which inhibits enzyme activity. Our study provides new insights into the structure and specificity of hexameric thioesterases, inhibitory feedback mechanisms, and possible biotechnological applications in short-chain fatty acid production such as biofuels, pharmaceuticals, and industrial compounds.

Original languageEnglish
Article number107477
Pages (from-to)1-15
Number of pages15
JournalJournal of Structural Biology
Volume210
Issue number1
Early online date03 Feb 2020
DOIs
Publication statusPublished - 01 Apr 2020

Fingerprint Dive into the research topics of 'Structural basis for disulphide-CoA Inhibition of a butyryl-CoA hexameric thioesterase'. Together they form a unique fingerprint.

  • Cite this