Comprehensive profiling and quantitation of amine group containing metabolites

Primary and secondary amines, including amino acids, biogenic amines, hormones, neurotransmitters, and plant siderophores, are readily derivatized with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate using easily performed experimental methodology. Complex mixtures of these amine derivatives can be fractionated and quantified using liquid chromatography–electrospray ionization-mass spectrometry (LC–ESI-MS). Upon collision induced dissociation (CID) in a quadrupole collision cell, all derivatized compounds lose the aminoquinoline tag. With the use of untargeted fragmentation scan functions, such as precursor ion scanning, the loss of the aminoquinoline tag (Amq) can be monitored to identify derivatized species; and the use of targeted fragmentation scans, such as multiple reaction monitoring, can be exploited to quantitate amine-containing molecules. Further, with the use of accurate mass, charge state, and retention time, identification of unknown amines is facilitated. The stability of derivatized amines was found to be variable with oxidatively labile derivatives rapidly degrading. With the inclusion of antioxidant and reducing agents, tris(2-carboxyethyl)-phosphine (TCEP) and ascorbic acid, into both extraction solvents and reaction buffers, degradation was significantly decreased, allowing reproducible identification and quantification of amine compounds in large sample sets

Cell wall integrity is linked to mitochondria and phospholipid homeostasis in Candida albicans through the activity of the post-transcriptional regulator Ccr4-Pop2

Peer reviewed: YesNRC publication: Ye

Synthesis, Structural Elucidation, And Biochemical Analysis of Immunoactive Glucuronosyl Diacylglycerides of Mycobacteria and Corynebacteria

Glucuronosyl diacylglycerides (GlcAGroAc2) are functionally important glycolipids and membrane anchors for cell wall lipoglycans in the Corynebacteria. Here we describe the complete synthesis of distinct acyl-isoforms of GlcAGroAc2 bearing both acylation patterns of (R)-tuberculostearic acid (C19:0) and palmitic acid (C16:0) and their mass spectral characterization. Collision-induced fragmentation mass spectrometry identified characteristic fragment ions that were used to develop “rules” allowing the assignment of the acylation pattern as C19:0 (sn-1), C16:0 (sn-2) in the natural product from Mycobacterium smegmatis, and the structural assignment of related C18:1 (sn-1), C16:0 (sn-2) GlcAGroAc2 glycolipids from M. smegmatis and Corynebacterium glutamicum. A synthetic hydrophobic octyl glucuronoside was used to characterize the GDP-mannose-dependent mannosyltransferase MgtA from C. glutamicum that extends GlcAGroAc2. This enzyme is an Mg2+/Mn2+-dependent metalloenzyme that undergoes dramatic activation upon reduction with dithiothreitol

Acetylation of Trehalose Mycolates Is Required for Efficient MmpL-Mediated Membrane Transport in Corynebacterineae

Pathogenic species of Mycobacteria and Corynebacteria, including Mycobacterium tuberculosis and Corynebacterium diphtheriae, synthesize complex cell walls that are rich in very long-chain mycolic acids. These fatty acids are synthesized on the inner leaflet of the cell membrane and are subsequently transported to the periplasmic space as trehalose monomycolates (TMM), where they are conjugated to other cell wall components and to TMM to form trehalose dimycolates (TDM). Mycobacterial TMM, and the equivalent Corynebacterium glutamicum trehalose corynomycolates (TMCM), are transported across the inner membrane by MmpL3, or NCgl0228 and NCgl2769, respectively, although little is known about how this process is regulated. Here, we show that transient acetylation of the mycolyl moiety of TMCM is required for periplasmic export. A bioinformatic search identified a gene in a cell wall biosynthesis locus encoding a putative acetyltransferase (M. tuberculosis Rv0228/C. glutamicum NCgl2759) that was highly conserved in all sequenced Corynebacterineae. Deletion of C. glutamicum<i> NCgl2759</i> resulted in the accumulation of TMCM, with a concomitant reduction in surface transport of this glycolipid and syntheses of cell wall trehalose dicorynomycolates. Strikingly, loss of NCgl2759 was associated with a defect in the synthesis of a minor, and previously uncharacterized, glycolipid species. This lipid was identified as trehalose monoacetylcorynomycolate (AcTMCM) by mass spectrometry and chemical synthesis of the authentic standard. The <i>in vitro</i> synthesis of AcTMCM was dependent on acetyl-CoA, whereas <i>in vivo</i> [¹⁴C]-acetate pulse–chase labeling showed that this lipid was rapidly synthesized and turned over in wild-type and genetically complemented bacterial strains. Significantly, the biochemical and TMCM/TDCM transport phenotype observed in the Δ<i>NCgl2759</i> mutant was phenocopied by inhibition of the activities of the two C. glutamicum MmpL3 homologues. Collectively, these data suggest that NCgl2759 is a novel <u>T</u>MCM <u>m</u>ycolyl <u>a</u>cetyl<u>t</u>ransferase (TmaT) that regulates transport of TMCM and is a potential drug target in pathogenic species

Synthesis, Structural Elucidation, And Biochemical Analysis of Immunoactive Glucuronosyl Diacylglycerides of Mycobacteria and Corynebacteria

Glucuronosyl diacylglycerides (GlcAGroAc₂) are functionally important glycolipids and membrane anchors for cell wall lipoglycans in the Corynebacteria. Here we describe the complete synthesis of distinct acyl-isoforms of GlcAGroAc₂ bearing both acylation patterns of (<i>R</i>)-tuberculostearic acid (C_19:0) and palmitic acid (C_16:0) and their mass spectral characterization. Collision-induced fragmentation mass spectrometry identified characteristic fragment ions that were used to develop “rules” allowing the assignment of the acylation pattern as C_19:0 (<i>sn</i>-1), C_16:0 (<i>sn</i>-2) in the natural product from <i>Mycobacterium smegmatis</i>, and the structural assignment of related C_18:1 (<i>sn</i>-1), C_16:0 (<i>sn</i>-2) GlcAGroAc₂ glycolipids from <i>M. smegmatis</i> and <i>Corynebacterium glutamicum</i>. A synthetic hydrophobic octyl glucuronoside was used to characterize the GDP-mannose-dependent mannosyltransferase MgtA from <i>C. glutamicum</i> that extends GlcAGroAc₂. This enzyme is an Mg²⁺/Mn²⁺-dependent metalloenzyme that undergoes dramatic activation upon reduction with dithiothreitol