Lysine lactoylation is a recently described protein post-translational modification (PTM). However, the biochemical pathways responsible for this acylation remain unclear. Two metabolite-dependent mechanisms have been proposed: enzymatic histone lysine lactoylation derived from lactoyl-coenzyme A (lactoyl-CoA, also termed lactyl-CoA), and non-enzymatic lysine lactoylation resulting from acyl-transfer via lactoyl-glutathione. While the former has precedent in the form of enzyme-catalysed lysine acylation, the lactoyl-CoA metabolite has not been previously quantified in mammalian systems. Here, we use liquid chromatography–high-resolution mass spectrometry (LC-HRMS) together with a synthetic standard to detect and validate the presence of lactoyl-CoA in cell and tissue samples. Conducting a retrospective analysis of data from previously analysed samples revealed the presence of lactoyl-CoA in diverse cell and tissue contexts. In addition, we describe a biosynthetic route to generate 13 C 3 15 N 1 -isotopically labelled lactoyl-CoA, providing a co-eluting internal standard for analysis of this metabolite. We estimate lactoyl-CoA concentrations of 1.14 × 10 −8 pmol per cell in cell culture and 0.0172 pmol mg −1 tissue wet weight in mouse heart. These levels are similar to crotonyl-CoA, but between 20 and 350 times lower than predominant acyl-CoAs such as acetyl-, propionyl- and succinyl-CoA. Overall our studies provide the first quantitative measurements of lactoyl-CoA in metazoans, and provide a methodological foundation for the interrogation of this novel metabolite in biology and disease.
Edited by Jeffrey E. Pessin Acyl-CoA thioesterases (Acots) hydrolyze fatty acyl-CoA esters. Acots in the mitochondrial matrix are poised to mitigate -oxidation overload and maintain CoA availability. Several Acots associate with mitochondria, but whether they all localize to the matrix, are redundant, or have different roles is unresolved. Here, we compared the suborganellar localization, activity, expression, and regulation among mitochondrial Acots (Acot2,-7,-9, and-13) in mitochondria from multiple mouse tissues and from a model of Acot2 depletion. Acot7,-9, and-13 localized to the matrix, joining Acot2 that was previously shown to localize there. Mitochondria from heart, skeletal muscle, brown adipose tissue, and kidney robustly expressed Acot2,-9, and-13; Acot9 levels were substantially higher in brown adipose tissue and kidney mitochondria, as was activity for C4:0-CoA, a unique Acot9 substrate. In all tissues, Acot2 accounted for about half of the thioesterase activity for C14:0-CoA and C16:0-CoA. In contrast, liver mitochondria from fed and fasted mice expressed little Acot activity, which was confined to long-chain CoAs and due mainly to Acot7 and Acot13 activities. Matrix Acots occupied different functional niches, based on substrate specificity (Acot9 versus Acot2 and-13) and strong CoA inhibition (Acot7,-9, and-13, but not Acot2). Interpreted in the context of -oxidation, CoA inhibition would prevent Acotmediated suppression of -oxidation, while providing a release valve when CoA is limiting. In contrast, CoA-insensitive Acot2 could provide a constitutive siphon for long-chain fatty acyl-CoAs. These results reveal how the family of matrix Acots can mitigate -oxidation overload and prevent CoA limitation. Acyl-CoA thioesterases (Acots) 2 hydrolyze acyl-CoA into CoA and an acyl chain and are classified into two families based on functional domain. Type I Acots are members of the superfamily of ␣/-hydrolases, are found only in mammals, and have a high degree of similarity (1). Humans and rodents possess Type I Acots residing within the cytoplasm (Acot1), mitochondria (Acot2), and peroxisomes (Acot3-6 in rodents, Acot3-4 in humans) (1). In contrast, Type II Acots (Acot7-15) share little similarity beyond a hotdog fold domain (2). Some possess StAR-related lipid transfer domains (2) or can interact with a Start domain protein (3) and are found in the cytoplasm (Acot7-14), mitochondria (Acot7-13 and Acot15), and peroxisomes (Acot8) (2). Dual localization is also possible (Acot7,-11, and-13). Type I and II Acots have a signature substrate specificity that includes saturated and unsaturated fatty acyl-CoAs of different chain lengths and, in fewer cases, other CoA esters (3-5). Acots are predicted to have high biological relevance because their substrates, acyl-CoA esters, are also substrates for other enzymes that serve major metabolic pathways, such as mitochondrial -oxidation, and can serve as allosteric or covalent regulators. In fact, genetic manipulation in mice of Type I or II Acots is associated ...
We previously identified the aur1 gene cluster which produces the angucycline antibiotic auricin. Preliminary characterisation of auricin revealed that it is modified by a single aminodeoxysugar, D-forosamine. Here we characterise the D-forosamine-specific genes. The four close tandem genes, aur1TQSV, encoding enzymes involved in the initial steps of the deoxysugar biosynthesis, were located on a large operon with other core auricin biosynthetic genes. Deleting these genes resulted in the absence of auricin and the production of deglycosylated auricin intermediates. The two final D-forosamine biosynthetic genes, sa59, an NDP-hexose aminotransferase, and sa52, an NDP-aminohexose N-dimethyltransferase, are located in a region rather distant from the core auricin genes. A deletion analysis of these genes confirmed their role in D-forosamine biosynthesis. The Δsa59 mutant had a phenotype similar to that of the cluster deletion mutant, while the Δsa52 mutant produced an auricin with a demethylated D-forosamine. Although auricin contains a single deoxyhexose, two glycosyltransferase genes were found to participate in the attachment of D-forosamine to the auricin aglycon. An analysis of the expression of the D-forosamine biosynthesis genes revealed that the initial D-forosamine biosynthetic genes aur1TQSV are regulated together with the other auricin core genes by the aur1Ap promoter under the control of the auricin-specific activator Aur1P. The expression of the other D-forosamine genes, however, is governed by promoters differentially dependent upon the two SARP family auricin-specific activators Aur1PR3 and Aur1PR4. These promoters contain direct repeats similar to the SARP consensus sequence and are involved in the interaction with both regulators.
Streptomyces lavendulae subsp. lavendulae CCM 3239 produces the angucycline antibiotic auricin and was thought to be the type strain of Streptomyces aureofaciens. We report the complete genome sequence of this strain, which consists of a linear chromosome and the linear plasmid pSA3239, and demonstrate it to be S. lavendulae subsp. lavendulae.
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