Fatty acyl-CoA Ligase Research Articles

A Proteins of ANL Family FACLs and FAALs, Next Anti TB Drug Targets.

The adenylating enzymes (AEs) of the ANL family contribute to the lipid synthesis process in Mycobacterium tuberculosis (MTB). FAALs (Fatty Acyl-AMP Ligase) and FACLs (Fatty Acyl-CoA Ligase) are members of the ANL enzyme family and are closely associated with the metabolism of mycobacterial lipids responsible for successful infections. Therefore, given their role in both biosynthesis and breakdown processes within fatty acid metabolism, these two groups of proteins might be potential drug targets. The growing abundance of structural, biochemical, and genetic information on mycobacterial FAAL and FACL enzymes could provide a solid foundation for the creation of next-generation anti-tuberculosis drugs, via rational drug designing.

ACSL4 promotes malignant progression of Hepatocellular carcinoma by targeting PAK2 transcription

Long-chain fatty acyl-Coa ligase 4 (ACSL4) is an important enzyme that converts fatty acids to fatty acyl-Coa esters, there is increasing evidence for its role in carcinogenesis. However, the precise role of ACLS4 in hepatocellular carcinoma (HCC) is not clearly understood. In the present study, we provide evidence that ACSL4 expression was specifically elevated in HCC and is associated with poor clinical outcomes. ACSL4 significantly promotes the growth and metastasis of HCC both in vitro and in vivo. RNA sequencing and functional experiments showed that the effect of ACSL4 on HCC development was heavily dependent on PAK2. ACSL4 expression is well correlated with PAK2 in HCC, and ACSL4 even transcriptionally increased PAK2 gene expression mediated by Sp1. In addition, emodin, a naturally occurring anthraquinone derivative, inhibited HCC cell growth and tumor progression by targeting ACSL4. In summary, ACSL4 plays a novel oncogene in HCC development by regulating PAK2 transcription. Targeting ACSL4 could be useful in drug development and therapy for HCC.

Impact of ACSL4 on the prognosis of hepatocellular carcinoma: Association with cancer-associated fibroblasts and the tumour immune microenvironment.

Recently, the association between hepatocellular carcinoma (HCC) and ferroptosis has been the focus of much attention. The expression of long chain fatty acyl-CoA ligase 4 (ACSL4), a marker of ferroptosis, in tumour tissue is related to better prognosis in various cancers. In HCC, ACSL4 expression indicates poor prognosis and is related to high malignancy. However, the mechanism remains to be fully understood. We retrospectively enrolled 358 patients with HCC who had undergone hepatic resection. Immunohistochemistry (IHC) for ACSL4 was performed. Factors associated with ASCL4 expression were investigated by spatial transcriptome analysis, and the relationships were investigated by IHC. The association between ACSL4 and the tumour immune microenvironment was examined in a public dataset and investigated by IHC. Patients were divided into ACSL4-positive (n = 72, 20.1%) and ACSL4-negative (n = 286, 79.9%) groups. ACSL4 positivity was significantly correlated with higher α-fetoprotein (p = .0180) and more histological liver fibrosis (p = .0014). In multivariate analysis, ACSL4 positivity was an independent prognostic factor (p < .0001). Spatial transcriptome analysis showed a positive correlation between ACSL4 and cancer-associated fibroblasts; this relationship was confirmed by IHC. Evaluation of a public dataset showed the correlation between ACSL4 and exhausted tumour immune microenvironment; this relationship was also confirmed by IHC. ACSL4 is a prognostic factor in HCC patients and its expression was associated with cancer-associated fibroblasts and anti-tumour immunity.

Mechanistic understanding of bacterial FAALs and the role of their homologs in eukaryotes.

Fatty acids are used in fundamental cellular processes, such as membrane biogenesis, energy generation, post-translational modification of proteins, and so forth. These processes require the activation of fatty acids by adenosine triphosphate (ATP), followed by condensation with coenzyme-A (CoA), catalyzed by the omnipresent enzyme called Fatty acyl-CoA ligases (FACLs). However, Fatty acyl-AMP ligases (FAALs), the structural homologs of FACLs, operate in an unprecedented CoA-independent manner. FAALs transfer fatty acids to the acyl carrier protein (ACP) domain of polyketide synthases (PKS) and non-ribosomal peptide synthetases (NRPS) for the biosynthesis of various antibiotics, lipopeptides, virulent complex lipids, and so forth in bacteria. Recent structural and biochemical insights from our group provide a detailed understanding of the mode of CoA rejection and ACP acceptance by FAALs. In this review, we have discussed advances in the mechanistic, evolutionary, and functional understanding of FAALs and FAAL-like domains across life forms. Here, we are proposing a "Five-tier" mechanistic model to explain the specificity of FAALs. We further demonstrate how FAAL-like domains have been repurposed into a new family of proteins in eukaryotes with a novel function in lipid metabolism.

Structural study of medium-long chain fatty acyl-CoA ligase FadD8 from Mycobacterium tuberculosis

In mycobacteria, lipids are important components of the cell wall and play a critical role for pathogenic activities. Lipids need to be activated before participating in many biological pathways. FadD proteins are members of the adenylate-forming superfamily, catalyzing activation of fatty acids. FadD8 is one of the 34 Mycobacterium tuberculosis FadD proteins, which was reported to be a putative medium-long chain fatty acyl-CoA ligase. Previous studies showed FadD8 from Mycobacterium smegmatis exhibited higher activity with oxidized cholesterol than fatty acids. However, the catalytic mechanism of the FadD8 is still exclusive. Here, we reported the crystal structure of FadD8 from Mycobacterium tuberculosis, which forms homodimer. Structural analysis revealed presence of a relatively narrow pocket compared to other FadD proteins and a novel alternative pocket, implying distinct substrate binding preference. We propose that FadD8 plays a vital role in cholesterol utilization and metabolism by catalyzing activation of cholesterol. Collectively, our findings provide novel information for the further studies of the inhibitor and drug development.

Engineering Pseudomonas putida KT2440 for chain length tailored free fatty acid and oleochemical production

Despite advances in understanding the metabolism of Pseudomonas putida KT2440, a promising bacterial host for producing valuable chemicals from plant-derived feedstocks, a strain capable of producing free fatty acid-derived chemicals has not been developed. Guided by functional genomics, we engineered P. putida to produce medium- and long-chain free fatty acids (FFAs) to titers of up to 670 mg/L. Additionally, by taking advantage of the varying substrate preferences of paralogous native fatty acyl-CoA ligases, we employed a strategy to control FFA chain length that resulted in a P. putida strain specialized in producing medium-chain FFAs. Finally, we demonstrate the production of oleochemicals in these strains by synthesizing medium-chain fatty acid methyl esters, compounds useful as biodiesel blending agents, in various media including sorghum hydrolysate at titers greater than 300 mg/L. This work paves the road to produce high-value oleochemicals and biofuels from cheap feedstocks, such as plant biomass, using this host.

Chylomicron production is repressed by RPTOR knockdown, R-α-lipoic acid and 4-phenylbutyric acid in human enterocyte-like Caco-2 cells

Although the role of mechanistic target of rapamycin complex 1 (mTORC1) in lipid metabolism has been the subject of previous research, its function in chylomicron production is not known. In this study, we created three stable human colorectal adenocarcinoma Caco-2 cell lines exhibiting normal, low, or high mTORC1 kinase activity, and used these cells to investigate the consequences of manipulating mTORC1 activity on enterocyte differentiation and chylomicron-like particle production. Constitutively active mTORC1 induced Caco-2 cell proliferation and differentiation (as judged by alkaline phosphatase activity) but weakened transepithelial electrical resistance (TEER). Repressed mTORC1 activity due to the knockdown of RPTOR significantly decreased the expression of lipogenic genes FASN, DGAT1, and DGAT2, lipoprotein assembly genes APOB and MTTP, reduced protein expression of APOB, MTTP, and FASN, downregulated the gene expression of very long-chain fatty acyl-CoA ligase (FATP2), acyl-CoA binding protein (DBI), and prechylomicron transport vesicle-associated proteins VAMP7 (vesicle-associated membrane protein 7) and SAR1B (secretion associated Ras related GTPase 1B) resulting in the repression of apoB-containing triacylglycerol-rich lipoprotein secretion. Exposure of Caco-2 cells harboring a constitutively active mTORC1 to short-chain fatty acid derivatives, R-α-lipoic acid and 4-phenylbutyric acid, downregulated chylomicron-like particle secretion by interfering with the lipidation and assembly of the particles, and concomitantly repressed mTORC1 activity with no change to Raptor abundance or PRAS40 (Thr246) phosphorylation. R-α-lipoic acid and 4-phenylbutyric acid may be useful to mitigate intestinal lipoprotein overproduction and associated postprandial inflammation.

Expression of Fatty Acyl-CoA Ligase Drives One-Pot De Novo Synthesis of Membrane-Bound Vesicles in a Cell-Free Transcription-Translation System.

Despite the central importance of lipid membranes in cellular organization, it is challenging to reconstitute their formation de novo from minimal chemical and biological elements. Here, we describe a chemoenzymatic route to membrane-forming noncanonical phospholipids in which cysteine-modified lysolipids undergo spontaneous coupling with fatty acyl-CoA thioesters generated enzymatically by a fatty acyl-CoA ligase. Due to the high efficiency of the reaction, we were able to optimize phospholipid formation in a cell-free transcription-translation (TX-TL) system. Combining DNA encoding the fatty acyl-CoA ligase with suitable lipid precursors enabled one-pot de novo synthesis of membrane-bound vesicles. Noncanonical sphingolipid synthesis was also possible by using a cysteine-modified lysosphingomyelin as a precursor. When the sphingomyelin-interacting protein lysenin was coexpressed alongside the acyl-CoA ligase, the in situ assembled membranes were spontaneously decorated with protein. Our strategy of coupling gene expression with membrane lipid synthesis in a one-pot fashion could facilitate the generation of proteoliposomes and brings us closer to the bottom-up generation of synthetic cells using recombinant synthetic biology platforms.

Development of small-molecule inhibitors of fatty acyl-AMP and fatty acyl-CoA ligases in Mycobacterium tuberculosis

Lipid metabolism in Mycobacterium tuberculosis (Mtb) relies on 34 fatty acid adenylating enzymes (FadDs) that can be grouped into two classes: fatty acyl-CoA ligases (FACLs) involved in lipid and cholesterol catabolism and long chain fatty acyl-AMP ligases (FAALs) involved in biosynthesis of the numerous essential and virulence-conferring lipids found in Mtb. The precise biochemical roles of many FACLs remain poorly characterized while the functionally non-redundant FAALs are much better understood. Here we describe the systematic investigation of 5′-O-[N-(alkanoyl)sulfamoyl]adenosine (alkanoyl adenosine monosulfamate, alkanoyl-AMS) analogs as potential multitarget FadD inhibitors for their antitubercular activity and biochemical selectivity towards representative FAAL and FACL enzymes. We identified several potent compounds including 12-azidododecanoyl-AMS 28, 11-phenoxyundecanoyl-AMS 32, and nonyloxyacetyl-AMS 36 with minimum inhibitory concentrations (MICs) against M. tuberculosis ranging from 0.098 to 3.13 μM. Compound 32 was notable for its impressive biochemical selectivity against FAAL28 (apparent Ki = 0.7 μM) versus FACL19 (Ki > 100 μM), and uniform activity against a panel of multidrug and extensively drug-resistant TB strains with MICs ranging from 3.13 to 12.5 μM in minimal (GAST) and rich (7H9) media. The SAR analysis provided valuable insights for further optimization of 32 and also identified limitations to overcome.

Two Functional Fatty Acyl Coenzyme A Ligases Affect Free Fatty Acid Metabolism To Block Biosynthesis of an Antifungal Antibiotic in Lysobacter enzymogenes.

In Lysobacter enzymogenes OH11, RpfB1 and RpfB2 were predicted to encode acyl coenzyme A (CoA) ligases. RpfB1 is located in the Rpf gene cluster. Interestingly, we found an RpfB1 homolog (RpfB2) outside this canonical gene cluster, and nothing is known about its functionality or mechanism. Here, we report that rpfB1 and rpfB2 can functionally replace EcFadD in the Escherichia colifadD mutant JW1794. RpfB activates long-chain fatty acids (n-C16:0 and n-C18:0) for the corresponding fatty acyl-CoA ligase (FCL) activity in vitro, and Glu-361 plays critical roles in the catalytic mechanism of RpfB1 and RpfB2. Deletion of rpfB1 and rpfB2 resulted in significantly increased heat-stable antifungal factor (HSAF) production, and overexpression of rpfB1 or rpfB2 completely suppressed HSAF production. Deletion of rpfB1 and rpfB2 resulted in increased L. enzymogenes diffusible signaling factor 3 (LeDSF3) synthesis in L. enzymogenes Overall, our results showed that changes in intracellular free fatty acid levels significantly altered HSAF production. Our report shows that intracellular free fatty acids are required for HSAF production and that RpfB affects HSAF production via FCL activity. The global transcriptional regulator Clp directly regulated the expression of rpfB1 and rpfB2 In conclusion, these findings reveal new roles of RpfB in antibiotic biosynthesis in L. enzymogenesIMPORTANCE Understanding the biosynthetic and regulatory mechanisms of heat-stable antifungal factor (HSAF) could improve the yield in Lysobacter enzymogenes Here, we report that RpfB1 and RpfB2 encode acyl coenzyme A (CoA) ligases. Our research shows that RpfB1 and RpfB2 affect free fatty acid metabolism via fatty acyl-CoA ligase (FCL) activity to reduce the substrate for HSAF synthesis and, thereby, block HSAF production in L. enzymogenes Furthermore, these findings reveal new roles for the fatty acyl-CoA ligases RpfB1 and RpfB2 in antibiotic biosynthesis in L. enzymogenes Importantly, the novelty of this work is the finding that RpfB2 lies outside the Rpf gene cluster and plays a key role in HSAF production, which has not been reported in other diffusible signaling factor (DSF)/Rpf-producing bacteria.

Efficient production of surfactin from xylose-rich corncob hydrolysate using genetically modified Bacillus subtilis 168.

As one of the most powerful biosurfactants, surfactin has extensive application prospects in numerous industrial fields. Bacillus subtilis 168 was genetically modified to produce surfactin by increasing the supply of the precursor fatty acyl-CoA by overexpressing 4' phosphopantetheinyl transferase, medium-chain acyl-acyl carrier protein (ACP) thioesterase and fatty acyl CoA ligase (encoded by sfp, bte, and yhfL, respectively), and knocking out acyl-CoA dehydrogenase (encoded by fadE). The resulting recombinant strain BSFX022 produced 2203mg/L surfactin with xylose as carbon source. The lower accumulation of organic acids with xylose as carbon source made it possible to maintain surfactin production in a non-buffered fermentation system, and the yield reached 2074mg/L. Furthermore, to reduce the costs, waste biomass such as corncob hydrolysate and monosodium glutamate wastewater (MGW) were used, and 2032mg/L of surfactin was produced in the optimal waste-based medium. To our best knowledge, this is the first report of surfactin production using genetically modified Bacillus subtilis 168 with xylose as carbon source.

Production of fluorinated fengycins in Bacillus spp.

Bacillus spp. produces lipopeptides that are of pharmaceutical and agricultural interest and include iturins, surfactins and fengycins. Their antimicrobial mechanism has been well studied but the role of the lipid chain and its biosynthesis is relatively unknown, especially in fengycin. In this project, we aim to fully understand the mechanisms of fengycin’s antimicrobial activity and generate new fengycins with fluorine in the lipid chain. We have already produced fluorinated lipopeptides via precursor-directed biosynthesis using fluorinated amino acids. The Bacillus genome was sequenced by MicrobesNG and a total of 34 gene clusters were identified, of which 19 were fully characterised using anti SMASH. Genes within the fengycin cluster were cleaved during the sequencing process and iturin biosynthetic genes were also present. Four fatty acyl-CoA ligases within the genome were identified using Artemis in an aim to heterologously express them, and carry out activity assays with fluorinated lipid tails. Lipopeptide production was optimised by altering culture conditions (pH, aeration and temperature) and it was found that 37 °C is the optimum temperature for biosynthesis.

Cerebellar ataxia disease-associated Snx14 promotes lipid droplet growth at ER-droplet contacts.

Lipid droplets (LDs) are nutrient reservoirs used by cells to maintain homeostasis. Nascent droplets form on the endoplasmic reticulum (ER) and grow following an influx of exogenous fatty acids (FAs). The budding of LDs requires extensive ER-LD crosstalk, but how this is regulated remains poorly understood. Here, we show that sorting nexin protein Snx14, an ER-resident protein associated with the cerebellar ataxia SCAR20, localizes to ER-LD contacts following FA treatment, where it promotes LD maturation. Using proximity-based APEX technology and topological dissection, we show that Snx14 accumulates specifically at ER-LD contacts independently of Seipin, where it remains ER-anchored and binds LDs in trans. SNX14KO cells exhibit perturbed LD morphology, whereas Snx14 overexpression promotes LD biogenesis and extends ER-LD contacts. Multi-time point imaging reveals that Snx14 is recruited to ER microdomains containing the fatty acyl-CoA ligase ACSL3, where nascent LDs bud. We propose that Snx14 is a novel marker for ER-LD contacts and regulates FA-stimulated LD growth.

A Rhodococcal Transcriptional Regulatory Mechanism Detects the Common Lactone Ring of AHL Quorum-Sensing Signals and Triggers the Quorum-Quenching Response.

The biocontrol agent Rhodococcus erythropolis disrupts virulence of plant and human Gram-negative pathogens by catabolizing their N-acyl-homoserine lactones. This quorum-quenching activity requires the expression of the qsd (quorum-sensing signal degradation) operon, which encodes the lactonase QsdA and the fatty acyl-CoA ligase QsdC, involved in the catabolism of lactone ring and acyl chain moieties of signaling molecules, respectively. Here, we demonstrate the regulation of qsd operon expression by a TetR-like family repressor, QsdR. This repression was lifted by adding the pathogen quorum signal or by deleting the qsdR gene, resulting in enhanced lactone degrading activity. Using interactomic approaches and transcriptional fusion strategy, the qsd operon derepression was elucidated: it is operated by the binding of the common part of signaling molecules, the homoserine lactone ring, to the effector-receiving domain of QsdR, preventing a physical binding of QsdR to the qsd promoter region. To our knowledge, this is the first evidence revealing quorum signals as inducers of the suitable quorum-quenching pathway, confirming this TetR-like protein as a lactone sensor. This regulatory mechanism designates the qsd operon as encoding a global disrupting pathway for degrading a wide range of signal substrates, allowing a broad spectrum anti-virulence activity mediated by the rhodococcal biocontrol agent. Understanding the regulation mechanisms of qsd operon expression led also to the development of biosensors useful to monitor in situ the presence of exogenous signals and quorum-quenching activity.

Identification of Middle Chain Fatty Acyl-CoA Ligase Responsible for the Biosynthesis of 2-Alkylmalonyl-CoAs for Polyketide Extender Unit

Understanding the biosynthetic mechanism of the atypical polyketide extender unit is important for the development of bioactive natural products. Reveromycin (RM) derivatives produced by Streptomyces sp. SN-593 possess several aliphatic extender units. Here, we studied the molecular basis of 2-alkylmalonyl-CoA formation by analyzing the revR and revS genes, which form a transcriptional unit with the revT gene, a crotonyl-CoA carboxylase/reductase homolog. We mainly focused on the uncharacterized adenylate-forming enzyme (RevS). revS gene disruption resulted in the reduction of all RM derivatives, whereas reintroduction of the gene restored the yield of RMs. Although RevS was classified in the fatty acyl-AMP ligase clade based on phylogenetic analysis, biochemical characterization revealed that the enzyme catalyzed the middle chain fatty acyl-CoA ligase (FACL) but not the fatty acyl-AMP ligase activity, suggesting the molecular evolution for acyl-CoA biosynthesis. Moreover, we examined the in vitro conversion of fatty acid into 2-alkylmalonyl-CoA using purified RevS and RevT. The coupling reaction showed efficient conversion of hexenoic acid into butylmalonyl-CoA. RevS efficiently catalyzed C8-C10 middle chain FACL activity; therefore, we speculated that the acyl-CoA precursor was truncated via β-oxidation and converted into (E)-2-enoyl-CoA, a RevT substrate. To determine whether the β-oxidation process is involved between the RevS and RevT reaction, we performed the feeding experiment using [1,2,3,4-(13)C]octanoic acid. (13)C NMR analysis clearly demonstrated incorporation of the [3,4-(13)C]octanoic acid moiety into the structure of RM-A. Our results provide insight into the role of uncharacterized RevS homologs that may catalyze middle chain FACL to produce a unique polyketide extender unit.

Identification and characterization of naturally occurring DSF-family quorum sensing signal turnover system in the phytopathogen Xanthomonas.

Molecules of the diffusible signal factor (DSF)-family are a class of quorum sensing (QS) signals used by the phytopathogens Xanthomonas. Studies during the last decade have outlined how Xanthomonas cells enter the QS phase. However, information on the mechanism underlying its exit from the QS phase is limited. RpfB has recently been reported as a fatty acyl-CoA ligase (FCL) that activates a wide range of fatty acids to their CoA esters in vitro. Here, we establish an improved quantification assay for DSF-family signals using liquid chromatography-mass spectrometry in X. campestris pv. campestris (Xcc). We first demonstrated that RpfB represents a naturally occurring DSF-family signal turnover system. RpfB effectively turns over DSF-family signals DSF and BDSF in vivo. RpfB FCL enzymatic activity is required for DSF and BDSF turnover. Deletion of rpfB slightly increased Xcc virulence in the Chinese radish and overexpression of rpfB significantly decreased virulence. We further showed that the expression of rpfB is growth phase-dependent, and its expression is significantly enhanced when Xcc cells enter the stationary phase. DSF regulates rpfB expression in a concentration-dependent manner. rpfB expression is also negatively regulated by the DSF signalling components RpfC, RpfG and Clp. The global transcription factor Clp directly binds to the AATGC-tgctgc-GCATC motif in the promoter region of rpfB to repress its expression. Finally, RpfB-dependent signal turnover system was detected in a wide range of bacterial species, suggesting that it is a conserved mechanism.

All‐trans retinoic acid promotes fatty acid oxidation in human hepatoma (HepG2) cells (LB612)

It has recently emerged that retinoic acid (RA) acts as a ligand for peroxisome proliferator activated receptor (PPAR), a master nuclear receptor of lipid metabolism, in addition to the retinoic acid receptor. The role of all‐trans‐RA (atRA) on regulating lipid metabolism is, however, not clearly understood. In the present study, we examined the effect of atRA on fatty acid oxidation (FAO) in human hepatoma (HepG2) cells. HepG2 cells were cultured in presence or absence of atRA for 24 or 72 hrs, after which, cells were harvested to isolate RNA/protein to check gene/protein expression. Proteomics analysis performed on isolated protein after 72hrs of incubation with atRA showed that atRA significantly increased expression of key enzymes involved in FAO pathway such as acyl CoA dehydrogenase, long chain fatty acyl CoA ligase 3 and 4, delta (3, 5)‐delta (2, 4)‐dienoyl‐CoA isomerase and mitochondrial ATP synthase subunit alpha. Additionally, treatment with atRA significantly increased the mRNA of PPARδ, PPARα and its co‐activator PGC1α, and PPARα/δ target genes: CPT1α, PDK4 and FGF19, which all have role in FAO. Collectively, these results indicate that atRA promotes FAO via PPARα/δ induction in a dose dependent manner. Thus, our findings improve our understanding of RA role in lipid metabolism and signaling in liver.Grant Funding Source: Supported by NIH R01 GM081569 and GM081569‐S1

Structures of Mycobacterium tuberculosis FadD10 Protein Reveal a New Type of Adenylate-forming Enzyme

Mycobacterium tuberculosis has a group of 34 FadD proteins that belong to the adenylate-forming superfamily. They are classified as either fatty acyl-AMP ligases (FAALs) or fatty acyl-CoA ligases based on sequence analysis. FadD10, involved in the synthesis of a virulence-related lipopeptide, was mis-annotated as a fatty acyl-CoA ligase; however, it is in fact a FAAL that transfers fatty acids to an acyl carrier protein (Rv0100). In this study, we have determined the structures of FadD10 in both the apo-form and the complexed form with dodecanoyl-AMP, where we see for the first time an adenylate-forming enzyme that does not adopt a closed conformation for catalysis. Indeed, this novel conformation of FadD10, facilitated by its unique inter-domain and intermolecular interactions, is critical for the enzyme to carry out the acyl transfer onto Rv0100 rather than coenzyme A. This contradicts the existing model of FAALs that rely on an insertion motif for the acyltransferase specificity and thus makes FadD10 a new type of FAAL. We have also characterized the fatty acid preference of FadD10 through biological and structural analyses, and the data indicate long chain saturated fatty acids as the biological substrates of the enzyme.

DarR, a TetR-like Transcriptional Factor, Is a Cyclic Di-AMP-responsive Repressor in Mycobacterium smegmatis

Cyclic dinucleotides, including cyclic di-AMP (c-di-AMP), are known to be ubiquitous second messengers involved in bacterial signal transduction. However, no transcriptional regulator has been characterized as a c-di-AMP receptor/effector to date. In the present study, using a c-di-AMP/transcription factor binding screen, we identified Ms5346, a TetR family regulator in Mycobacterium smegmatis, as a c-di-AMP receptor in bacteria. Ms5346 could specifically bind c-di-AMP with K(d) of 2.3 ± 0.5 μM. Using EMSA and DNase I footprinting assays, c-di-AMP was found to stimulate the DNA binding activity of Ms5346 and to enhance its ability to protect its target DNA sequence. A conserved 14-bp palindromic motif was identified as the DNA-binding site for Ms5346. Further, Ms5346 was found to negatively regulate expression of three target genes including Ms5347 (encoding a major facilitator family transporter), Ms5348 (encoding a medium chain fatty acyl-CoA ligase), and Ms5696 (encoding a cold shock protein, CspA). Ms5346 is the first cyclic di-AMP receptor regulator to be identified in bacteria, and we have designated it as DarR. Our findings enhance our understanding of the function and regulatory mechanism of the second messenger c-di-AMP in bacteria.

Adenylating Enzymes in Mycobacterium tuberculosis as Drug Targets

Adenylation or adenylate-forming enzymes (AEs) are widely found in nature and are responsible for the activation of carboxylic acids to intermediate acyladenylates, which are mixed anhydrides of AMP. In a second reaction, AEs catalyze the transfer of the acyl group of the acyladenylate onto a nucleophilic amino, alcohol, or thiol group of an acceptor molecule leading to amide, ester, and thioester products, respectively. Mycobacterium tuberculosis encodes for more than 60 adenylating enzymes, many of which represent potential drug targets due to their confirmed essentiality or requirement for virulence. Several strategies have been used to develop potent and selective AE inhibitors including highthroughput screening, fragment-based screening, and the rationale design of bisubstrate inhibitors that mimic the acyladenylate. In this review, a comprehensive analysis of the mycobacterial adenylating enzymes will be presented with a focus on the identification of small molecule inhibitors. Specifically, this review will cover the aminoacyl tRNAsynthetases (aaRSs), MenE required for menaquinone synthesis, the FadD family of enzymes including the fatty acyl- AMP ligases (FAAL) and the fatty acyl-CoA ligases (FACLs) involved in lipid metabolism, and the nonribosomal peptide synthetase adenylation enzyme MbtA that is necessary for mycobactin synthesis. Additionally, the enzymes NadE, GuaA, PanC, and MshC involved in the respective synthesis of NAD, guanine, pantothenate, and mycothiol will be discussed as well as BirA that is responsible for biotinylation of the acyl CoA-carboxylases.