Analysis of coenzyme A activated compounds in actinomycetes.
Acyl-CoAs are crucial compounds involved in essential metabolic pathways such as the Krebs cycle and lipid, carbohydrate, and amino acid metabolisms, and they are also key signal molecules involved in the transcriptional regulation of lipid biosynthesis in many organisms. In this study, we took advantage of the high selectivity of mass spectrometry and developed an ion-pairing reverse-phase high-pressure liquid chromatography electrospray ionization high-resolution mass spectrometry (IP-RP-HPLC/ESI-HRMS) method to carry on a comprehensive analytical determination of the wide range of fatty acyl-CoAs present in actinomycetes. The advantage of using a QTOF spectrometer resides in the excellent mass accuracy over a wide dynamic range and measurements of the true isotope pattern that can be used for molecular formula elucidation of unknown analytes. As a proof of concept, we used this assay to determine the composition of the fatty acyl-CoA pools in Mycobacterium, Streptomyces, and Corynebacterium species, revealing an extraordinary difference in fatty acyl-CoA amounts and species distribution between the three genera and between the two species of mycobacteria analyzed, including the presence of different chain-length carboxy-acyl-CoAs, key substrates of mycolic acid biosynthesis. The method was also used to analyze the impact of two fatty acid synthase inhibitors on the acyl-CoA profile of Mycobacterium smegmatis, which showed some unexpected low levels of C24 acyl-CoAs in the isoniazid-treated cells. This robust, sensitive, and reliable method should be broadly applicable in the studies of the wide range of bacteria metabolisms in which acyl-CoA molecules participate.
Laboratory of Physiology and Genetics of Actinomycetes, Instituto de Biología Molecular y Celular de Rosario (IBR-CONICET), Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, Rosario, (2000), Argentina.
Matías
M
Cabruja
Laboratory of Physiology and Genetics of Actinomycetes, Instituto de Biología Molecular y Celular de Rosario (IBR-CONICET), Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, Rosario, (2000), Argentina.
Bernardo Bazet
BB
Lyonnet
Laboratory of Mass Spectrometry, Centro Científico Tecnológico Rosario, CONICET, Rosario, Argentina.
Gustavo
G
Millán
Laboratory of Physiology and Genetics of Actinomycetes, Instituto de Biología Molecular y Celular de Rosario (IBR-CONICET), Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, Rosario, (2000), Argentina. gramajo@ibr-conicet.gov.ar.
Hugo
H
Gramajo
Laboratory of Physiology and Genetics of Actinomycetes, Instituto de Biología Molecular y Celular de Rosario (IBR-CONICET), Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, Rosario, (2000), Argentina. gago@ibr-conicet.gov.ar.
Gabriela
G
Gago
D039903:Actinobacteria / Q000378:metabolism*; D000214:Acyl Coenzyme A / Q000032:analysis / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D003352:Corynebacterium / Q000378:metabolism*; D064429:Fatty Acid Synthases / Q000037:antagonists & inhibitors*; D054872:Fatty Acid Synthesis Inhibitors / Q000494:pharmacology; D007538:Isoniazid / Q000494:pharmacology; D050356:Lipid Metabolism / Q000502:physiology; D020102:Mycobacterium smegmatis / Q000378:metabolism*; D021241:Spectrometry, Mass, Electrospray Ionization; D013302:Streptomyces / Q000378:metabolism*
D000214:Acyl Coenzyme A; D054872:Fatty Acid Synthesis Inhibitors; D064429:Fatty Acid Synthases; D007538:Isoniazid
Acyl-CoA; Corynebacterium; IP-RP-HPLC-ESI-HRMS; Lipid synthesis; Mycobacterium; Streptomyces