A chiral high-performance liquid chromatography–tandem mass spectrometry method for the stereospecific analysis of enoyl-coenzyme A hydratases/isomerases

• Published in: Journal of Chromatography A Vol. 1306; pp. 37 - 43
• Main Authors: Abdel-Mawgoud, Ahmad M., Lépine, François, Déziel, Eric
• Format: Journal Article
• Language: English
• Published: Amsterdam Elsevier B.V 06.09.2013; Elsevier
• Subjects: 3,5-Dimethylphenyl isocyanate; 3-Hydroxy fatty acids; Bacteria; Bacterial Proteins - chemistry; Bacteriology; Biological and medical sciences; Chromatography; Chromatography, High Pressure Liquid - methods; Derivatives; Dodecenoyl-CoA Isomerase - chemistry; Enantiomers; Enoyl-CoA Hydratase - chemistry; Escherichia coli; Fatty acids; Fundamental and applied biological sciences. Psychology; General aspects; High performance liquid chromatography; Life Sciences; Mass spectrometry; Microbiology; Monitoring; Protein Conformation; Pseudomonas aeruginosa; Pseudomonas aeruginosa - chemistry; Pseudomonas aeruginosa - enzymology; Rhamnolipids; Tandem Mass Spectrometry - methods; Urethanes; Pseudomonas aeruginosa; 3,5-Dimethylphenyl isocyanate; Rhamnolipids; Enantiomers; 3-Hydroxy fatty acids; Escherichia coli; Pseudomonadales; Tandem mass spectrometry; Separation; HPLC chromatography; Lipids; Lyases; Derivatization; Organic carbamate; Electrospray; Biosurfactant; Enoyl-CoA hydratase; Enantiomer; Bacteria; Pseudomonadaceae; Enterobacteriaceae; Lysate; Hydration; Enzyme; Coupled method; Reaction product; Chiral stationary phase; Glycolipid; Hydrolysis; Hydroxyacid; Cellulose derivatives; Carbon-oxygen lyases; Hydro-lyases; Enzymatic reaction
• ISSN: 0021-9673; 1873-3778
• DOI: 10.1016/j.chroma.2013.07.049

•We develop a novel chiral HPLC–MS/MS direct activity assay for enoyl-CoA hydratases (ECH).•We employ a new and effective derivatization of ECH product, 3-hydroxyalkanoates.•The method selectively identifies R/S enantiomers of 3-hydroxyalkanoates (C6–C16).•The method ensures high detection sensitivities of 3-hydroxyalkanoates (2.5μM).•The method identifies and quantifies ECH-1 and -2 activities in biological samples. The enzymes catalyzing the stereospecific hydration of 2-enoyl-CoA into the corresponding S- or R-3-hydroxyacyl-CoA are named enoyl-CoA hydratases (ECH), where the S-specific is called ECH-1 and the R-specific is called ECH-2. Current ECH assays are mostly based on spectrophotometric methods. Amongst many limitations, these methods do not directly measure the 3-hydroxyacyl-CoA produced, neither do they allow determination of its stereospecific configuration. We have developed a chiral HPLC method coupled with tandem mass spectrometry (MS) for the sensitive, direct, stereospecific and quantitative analysis of ECH-1/-2 reaction products, or R-/S-3-hydroxyalkanoates in general. The method is based on the reaction of the 3-hydroxyl group on the chiral carbon with 3,5-dimethylphenyl isocyanate, creating a urethane derivative which is then chirally resolved on a chiral HPLC column having 3,5-dimethylphenyl carbamate-derivatized cellulose as the chiral stationary phase. The resolved urethane derivatives are detected using tandem MS in the multiple reactions monitoring (MRM) negative electrospray ionization mode by monitoring the free hydroxy fatty acid fragment ion liberated from its parent urethane derivative. The method resolves the R-/S-enantiomers of 3-hydroxy fatty acid homologues ranging from C6 to C16. Using this method, the net ECH activity present in clarified cell lysates of the bacterium Pseudomonas aeruginosa cultivated in a rich medium was found to be of both ECH-1 and ECH-2. Interestingly, the clarified cell lysate of Escherichia coli cultivated also in a rich medium displayed mainly an ECH-1 (S-specific) activity. This method will facilitate the quantification and stereospecific characterization of ECHs, as well as the chiral lipid profiling of bacterial secondary metabolites containing hydroxyalkanoic acid moieties.