The recombinant expression and enzymatic charaterisation of human ACSM2 ligase.
Abstract
Glycine conjugation is a highly conserved Phase 2 biotransformation reaction for xenobiotic carboxylic acids such as benzoate, salicylate, and medium-chain fatty acids. The glycine conjugation pathway can be saturated by excess substrates and since the modern intake of xenobiotics is increasing and may exceed the capabilities of the pathway by depleting the substrates (glycine and Coenzyme A or increasing the exposure to the toxic intermediates of the pathway. Glycine conjugation is a two-step reaction, the first reaction is catalysed by mitochondrial xenobiotic/medium-chain fatty acid, CoA ligase 2 (ACSM2), and the second reaction is catalysed by glycine acyl transferase (GLYAT). While GLYAT has been characterised and the effect of variation on enzyme function has been studied and enzyme kinetic data is available, the first reaction has been largely neglected. The ACSM2B enzyme kinetics and substrate specificity were only determined in the late 1990s and the early 2000s. No characterisation information is available for the predominant transcript in the liver namely ACSM2A. Previously attempts have failed to identify a recombinant expression system for this protein however, two potential solutions were identified and investigated. Secondly, an adenosine monophosphate (AMP)-linked assay was previously investigated however, the commercially available EnzChek Pyrophosphate assay (Thermo Fischer Scientific) was a suitable alternative assay due to the significant increase in sensitivity.
This study investigated two bacterial expression systems, with the first approach using the C41(DE3)pLysS cell line which is used in the production of difficult-to-produce recombinant proteins. The expression conditions were modified to decrease the rate of transcription and translation to allow more time for the proteins to fold correctly. The second approach used the Origami B cell line for the formation of disulphide bonds in the cytoplasm and the cell line also co-expressed molecular chaperones to facilitate the correct folding of the expression proteins to prevent the aggregation into an inclusion body. The study additionally investigated the EnzChek Pyrophosphate assay to characterise the ACSM proteins. Gas chromatography–mass spectrometry was used to investigate the in vitro reaction of benzoic acid in the presence of the ACSM2 and hGLYAT enzyme with all the other substrates and co-factors included. Both the selected expression strategies successfully produced soluble and biologically active ACSM2A and ACSM2B. The activity assay with the EnzChek Pyrophosphate assay had inconclusive results. The detection of the hippuric acid product following the in vitro reaction of sodium benzoate with all the components of glycine conjugation gave conclusive evidence of biologically active ACSM2A and ACSM2B from the C41(DE3)pLysS host however, only ACSM2A from the Origami B host was able to detect hippuric acid.
This study successfully identified two expression systems that were able to produce soluble and biologically active ACSM2A and ACSM2B. From the enzyme assay results, no enzyme kinetics were determined however, after an in vitro analysis of the glycine conjugation, it was determined that ACSM2A participates in the glycine conjugation pathway.