Staphylococcus aureus oleate hydratase produces ligands that activate host PPARα.

Commensal gut bacteria use oleate hydratase to release a spectrum of hydroxylated fatty acids using host-derived unsaturated fatty acids. These compounds are thought to attenuate the immune response, but the underlying signaling mechanism(s) remain to be established. The pathogen Staphylococcus aureus also expresses an oleate hydratase and 10-hydroxyoctadecanoic acid (h18:0) is the most abundant oleate hydratase metabolite found at Staphylococcal skin infection sites. Here, we show h18:0 stimulates the transcription of a set of lipid metabolism genes associated with the activation of peroxisome proliferator activated receptor (PPAR) in the RAW 264.7 macrophage cell line and mouse primary bone marrow-derived macrophages. Cell-based transcriptional reporter assays show h18:0 selectively activates PPARα. Radiolabeling experiments with bone marrow-derived macrophages show [1-14C]h18:0 is not incorporated into cellular lipids, but is degraded by ß-oxidation, and mass spectrometry detected shortened fragments of h18:0 released into the media. The catabolism of h18:0 was >10-fold lower in bone marrow-derived macrophages isolated from Ppara -/- knockout mice, and we recover 74-fold fewer S. aureus cells from the skin infection site of Ppara -/- knockout mice compared to wildtype mice. These data identify PPARα as a target for oleate hydratase-derived hydroxy fatty acids and support the existence of an oleate hydratase-PPARα signaling axis that functions to suppress the innate immune response to S. aureus.

The Bacillus subtilis cell envelope stress-inducible ytpAB operon modulates membrane properties and contributes to bacitracin resistance.

Antibiotics that inhibit peptidoglycan synthesis trigger the activation of both specific and general protective responses. σM responds to diverse antibiotics that inhibit cell wall synthesis. Here, we demonstrate that cell wall-inhibiting drugs, such as bacitracin and cefuroxime, induce the σM-dependent ytpAB operon. YtpA is a predicted hydrolase previously proposed to generate the putative lysophospholipid antibiotic bacilysocin (lysophosphatidylglycerol), and YtpB is the branchpoint enzyme for the synthesis of membrane-localized C35 terpenoids. Using targeted lipidomics, we reveal that YtpA is not required for the production of lysophosphatidylglycerol. Nevertheless, ytpA was critical for growth in a mutant strain defective for homeoviscous adaptation due to a lack of genes for the synthesis of branched chain fatty acids and the Des phospholipid desaturase. Consistently, overexpression of ytpA increased membrane fluidity as monitored by fluorescence anisotropy. The ytpA gene contributes to bacitracin resistance in mutants additionally lacking the bceAB or bcrC genes, which directly mediate bacitracin resistance. These epistatic interactions support a model in which σM-dependent induction of the ytpAB operon helps cells tolerate bacitracin stress, either by facilitating the flipping of the undecaprenyl phosphate carrier lipid or by impacting the assembly or function of membrane-associated complexes involved in cell wall homeostasis.IMPORTANCEPeptidoglycan synthesis inhibitors include some of our most important antibiotics. In Bacillus subtilis, peptidoglycan synthesis inhibitors induce the σM regulon, which is critical for intrinsic antibiotic resistance. The σM-dependent ytpAB operon encodes a predicted hydrolase (YtpA) and the enzyme that initiates the synthesis of C35 terpenoids (YtpB). Our results suggest that YtpA is critical in cells defective in homeoviscous adaptation. Furthermore, we find that YtpA functions cooperatively with the BceAB and BcrC proteins in conferring intrinsic resistance to bacitracin, a peptide antibiotic that binds tightly to the undecaprenyl-pyrophosphate lipid carrier that sustains peptidoglycan synthesis.

Crystal structures of the fatty acid biosynthesis initiation enzymes in Bacillus subtilis.

Bacteria use the fatty acid composition of membrane lipids to maintain homeostasis of the bilayer. ß-Ketoacyl-ACP synthase III (FabH) initiates fatty acid biosynthesis and is the primary determinant of the fatty acid composition. FabH condenses malonyl-acyl carrier protein with an acyl-Coenzyme A primer to form ß -ketoacyl-acyl carrier protein which is used to make substrates for lipid synthesis. The acyl-Coenzyme A primer determines whether an acyl chain in the membrane has iso, anteiso, or no branching (straight chain) and biophysical properties of the membrane. The soil bacterium Bacillus subtilis encodes two copies of FabH (BsFabHA and BsFabHB), and here we solve their crystal structures. The substrate-free 1.85 Å and 2.40 Å structures of BsFabHA and BsFabHB show both enzymes have similar residues that line the active site but differ in the architecture surrounding the catalytic residues and oxyanion hole. Branching in the BsFabHB active site may better accommodate the structure of an iso-branched acyl-Coenzyme A molecule and thus confer superior utilization to BsFabHA for this primer type. The 2.02 Å structure of BsFabHA•Coenzyme A shows how the active site architecture changes after binding the first substrate. The other notable difference is an amino acid insertion in BsFabHB that extends a cap that covers the dimer interface. The cap topology is diverse across FabH structures and appears to be a distinguishing feature. FabH enzymes have variable sensitivity to natural product inhibitors and the availability of crystal structures help clarify how nature designs antimicrobials that differentially target FabH homologs.

The carboxy terminus causes interfacial assembly of oleate hydratase on a membrane bilayer.

The soluble flavoprotein oleate hydratase (OhyA) hydrates the 9-cis double bond of unsaturated fatty acids. OhyA substrates are embedded in membrane bilayers; OhyA must remove the fatty acid from the bilayer and enclose it in the active site. Here, we show that the positively charged helix-turn-helix motif in the carboxy terminus (CTD) is responsible for interacting with the negatively charged phosphatidylglycerol (PG) bilayer. Super-resolution microscopy of Staphylococcus aureus cells expressing green fluorescent protein fused to OhyA or the CTD sequence shows subcellular localization along the cellular boundary, indicating OhyA is membrane-associated and the CTD sequence is sufficient for membrane recruitment. Using cryo-electron microscopy, we solved the OhyA dimer structure and conducted 3D variability analysis of the reconstructions to assess CTD flexibility. Our surface plasmon resonance experiments corroborated that OhyA binds the PG bilayer with nanomolar affinity and we found the CTD sequence has intrinsic PG binding properties. We determined that the nuclear magnetic resonance structure of a peptide containing the CTD sequence resembles the OhyA crystal structure. We observed intermolecular NOE from PG liposome protons next to the phosphate group to the CTD peptide. The addition of paramagnetic MnCl2 indicated the CTD peptide binds the PG surface but does not insert into the bilayer. Molecular dynamics simulations, supported by site-directed mutagenesis experiments, identify key residues in the helix-turn-helix that drive membrane association. The data show that the OhyA CTD binds the phosphate layer of the PG surface to obtain bilayer-embedded unsaturated fatty acids.

Pantothenate Kinase Activation Restores Brain Coenzyme A in a Mouse Model of Pantothenate Kinase-Associated Neurodegeneration.

Pantothenate kinase-associated neurodegeneration (PKAN) is characterized by a motor disorder with combinations of dystonia, parkinsonism, and spasticity, leading to premature death. PKAN is caused by mutations in the PANK2 gene that result in loss or reduction of PANK2 protein function. PANK2 is one of three kinases that initiate and regulate coenzyme A biosynthesis from vitamin B5, and the ability of BBP-671, an allosteric activator of pantothenate kinases, to enter the brain and elevate coenzyme A was investigated. The metabolic stability, protein binding, and membrane permeability of BBP-671 all suggest that it has the physical properties required to cross the blood-brain barrier. BBP-671 was detected in plasma, liver, cerebrospinal fluid, and brain following oral administration in rodents, demonstrating the ability of BBP-671 to penetrate the brain. The pharmacokinetic and pharmacodynamic properties of orally administered BBP-671 evaluated in cannulated rats showed that coenzyme A (CoA) concentrations were elevated in blood, liver, and brain. BBP-671 elevation of whole-blood acetyl-CoA served as a peripheral pharmacodynamic marker and provided a suitable method to assess target engagement. BBP-671 treatment elevated brain coenzyme A concentrations and improved movement and body weight in a PKAN mouse model. Thus, BBP-671 crosses the blood-brain barrier to correct the brain CoA deficiency in a PKAN mouse model, resulting in improved locomotion and survival and providing a preclinical foundation for the development of BBP-671 as a potential treatment of PKAN. SIGNIFICANCE STATEMENT: The blood-brain barrier represents a major hurdle for drugs targeting brain metabolism. This work describes the pharmacokinetic/pharmacodynamic properties of BBP-671, a pantothenate kinase activator. BBP-671 crosses the blood-brain barrier to correct the neuron-specific coenzyme A (CoA) deficiency and improve motor function in a mouse model of pantothenate kinase-associated neurodegeneration. The central role of CoA and acetyl-CoA in intermediary metabolism suggests that pantothenate kinase activators may be useful in modifying neurological metabolic disorders.

Acetyl-CoA biosynthesis drives resistance to histone acetyltransferase inhibition.

Histone acetyltransferases (HATs) are implicated as both oncogene and nononcogene dependencies in diverse human cancers. Acetyl-CoA-competitive HAT inhibitors have emerged as potential cancer therapeutics and the first clinical trial for this class of drugs is ongoing (NCT04606446). Despite these developments, the potential mechanisms of therapeutic response and evolved drug resistance remain poorly understood. Having discovered that multiple regulators of de novo coenzyme A (CoA) biosynthesis can modulate sensitivity to CBP/p300 HAT inhibition (PANK3, PANK4 and SLC5A6), we determined that elevated acetyl-CoA concentrations can outcompete drug-target engagement to elicit acquired drug resistance. This not only affects structurally diverse CBP/p300 HAT inhibitors, but also agents related to an investigational KAT6A/B HAT inhibitor that is currently in Phase 1 clinical trials. Altogether, this work uncovers CoA metabolism as an unexpected liability of anticancer HAT inhibitors and will therefore buoy future efforts to optimize the efficacy of this new form of targeted therapy.

Lysophosphatidylglycerol (LPG) phospholipase D maintains membrane homeostasis in Staphylococcus aureus by converting LPG to lysophosphatidic acid.

Lysophospholipids are deacylated derivatives of their bilayer forming phospholipid counterparts that are present at low concentrations in cells. Phosphatidylglycerol (PG) is the principal membrane phospholipid in Staphylococcus aureus and lysophosphatidylglycerol (LPG) is detected in low abundance. Here, we used a mass spectrometry screen to identify locus SAUSA300_1020 as the gene responsible for maintaining low concentrations of 1-acyl-LPG in S. aureus. The SAUSA300_1020 gene encodes a protein with a predicted amino terminal transmembrane α-helix attached to a globular glycerophosphodiester phosphodiesterase (GDPD) domain. We determined that the purified protein lacking the hydrophobic helix (LpgDΔN) possesses cation-dependent lysophosphatidylglycerol phospholipase D activity that generates both lysophosphatidic acid (LPA) and cyclic-LPA products and hydrolyzes cyclic-LPA to LPA. Mn2+ was the highest affinity cation and stabilized LpgDΔN to thermal denaturation. LpgDΔN was not specific for the phospholipid headgroup and degraded 1-acyl-LPG, but not 2-acyl-LPG. Furthermore, a 2.1 Å crystal structure shows that LpgDΔN adopts the GDPD variation of the TIM barrel architecture except for the length and positioning of helix α6 and sheet ß7. These alterations create a hydrophobic diffusion path for LPG to access the active site. The LpgD active site has the canonical GDPD metal binding and catalytic residues, and our biochemical characterization of site-directed mutants support a two-step mechanism involving a cyclic-LPA intermediate. Thus, the physiological function of LpgD in S. aureus is to convert LPG to LPA, which is re-cycled into the PG biosynthetic pathway at the LPA acyltransferase step to maintain membrane PG molecular species homeostasis.

The Phospholipase A1 Activity of Glycerol Ester Hydrolase (Geh) Is Responsible for Extracellular 2-12(S)-Methyltetradecanoyl-Lysophosphatidylglycerol Production in Staphylococcus aureus.

Phosphatidylglycerol (PG) is the major membrane phospholipid of Staphylococcus aureus and predominately consists of molecular species with ≥16-carbon acyl chains in the 1-position and anteiso 12(S)-methyltetradecaonate (a15) esterified at the 2-position. The analysis of the growth media for PG-derived products shows S. aureus releases essentially pure 2-12(S)-methyltetradecanoyl-sn-glycero-3-phospho-1'-sn-glycerol (a15:0-LPG) derived from the hydrolysis of the 1-position of PG into the environment. The cellular lysophosphatidylglycerol (LPG) pool is dominated by a15-LPG but also consists of ≥16-LPG species arising from the removal of the 2-position. Mass tracing experiments confirmed a15-LPG was derived from isoleucine metabolism. A screen of candidate secreted lipase knockout strains pinpointed glycerol ester hydrolase (geh) as the gene required for generating extracellular a15-LPG, and complementation of a Δgeh strain with a Geh expression plasmid restored extracellular a15-LPG formation. Orlistat, a covalent inhibitor of Geh, also attenuated extracellular a15-LPG accumulation. Purified Geh hydrolyzed the 1-position acyl chain of PG and generated only a15-LPG from a S. aureus lipid mixture. The Geh product was 2-a15-LPG, which spontaneously isomerizes with time to a mixture of 1- and 2-a15-LPG. Docking PG in the Geh active site provides a structural rationale for the positional specificity of Geh. These data demonstrate a physiological role for Geh phospholipase A1 activity in S. aureus membrane phospholipid turnover. IMPORTANCE Glycerol ester hydrolase, Geh, is an abundant secreted lipase whose expression is controlled by the accessory gene regulator (Agr) quorum-sensing signal transduction pathway. Geh is thought to have a role in virulence based on its ability to hydrolyze host lipids at the infection site to provide fatty acids for membrane biogenesis and substrates for oleate hydratase, and Geh inhibits immune cell activation by hydrolyzing lipoprotein glycerol esters. The discovery that Geh is the major contributor to the formation and release of a15-LPG reveals an unappreciated physiological role for Geh acting as a phospholipase A1 in the degradation of S. aureus membrane phosphatidylglycerol. The role(s) for extracellular a15-LPG in S. aureus biology remain to be elucidated.

A short-chain acyl-CoA synthetase that supports branched-chain fatty acid synthesis in Staphylococcus aureus.

Staphylococcus aureus controls its membrane biophysical properties using branched-chain fatty acids (BCFAs). The branched-chain acyl-CoA precursors, utilized to initiate fatty acid synthesis, are derived from branched-chain ketoacid dehydrogenase (Bkd), a multiprotein complex that converts α-keto acids to their corresponding acyl-CoAs; however, Bkd KO strains still contain BCFAs. Here, we show that commonly used rich medias contain substantial concentrations of short-chain acids, like 2-methylbutyric and isobutyric acids, that are incorporated into membrane BCFAs. Bkd-deficient strains cannot grow in defined medium unless it is supplemented with either 2-methylbutyric or isobutyric acid. We performed a screen of candidate KO strains and identified the methylbutyryl-CoA synthetase (mbcS gene; SAUSA300_2542) as required for the incorporation of 2-methylbutyric and isobutyric acids into phosphatidylglycerol. Our mass tracing experiments show that isobutyric acid is converted to isobutyryl-CoA that flows into the even-chain acyl-acyl carrier protein intermediates in the type II fatty acid biosynthesis elongation cycle. Furthermore, purified MbcS is an ATP-dependent acyl-CoA synthetase that selectively catalyzes the activation of 2-methylbutyrate and isobutyrate. We found that butyrate and isovalerate are poor MbcS substrates and activity was not detected with acetate or short-chain dicarboxylic acids. Thus, MbcS functions to convert extracellular 2-methylbutyric and isobutyric acids to their respective acyl-CoAs that are used by 3-ketoacyl-ACP synthase III (FabH) to initiate BCFA biosynthesis.

Relief of CoA sequestration and restoration of mitochondrial function in a mouse model of propionic acidemia.

Propionic acidemia (PA, OMIM 606054) is a devastating inborn error of metabolism arising from mutations that reduce the activity of the mitochondrial enzyme propionyl-CoA carboxylase (PCC). The defects in PCC reduce the concentrations of nonesterified coenzyme A (CoASH), thus compromising mitochondrial function and disrupting intermediary metabolism. Here, we use a hypomorphic PA mouse model to test the effectiveness of BBP-671 in correcting the metabolic imbalances in PA. BBP-671 is a high-affinity allosteric pantothenate kinase activator that counteracts feedback inhibition of the enzyme to increase the intracellular concentration of CoA. Liver CoASH and acetyl-CoA are depressed in PA mice and BBP-671 treatment normalizes the cellular concentrations of these two key cofactors. Hepatic propionyl-CoA is also reduced by BBP-671 leading to an improved intracellular C3:C2-CoA ratio. Elevated plasma C3:C2-carnitine ratio and methylcitrate, hallmark biomarkers of PA, are significantly reduced by BBP-671. The large elevations of malate and α-ketoglutarate in the urine of PA mice are biomarkers for compromised tricarboxylic acid cycle activity and BBP-671 therapy reduces the amounts of both metabolites. Furthermore, the low survival of PA mice is restored to normal by BBP-671. These data show that BBP-671 relieves CoA sequestration, improves mitochondrial function, reduces plasma PA biomarkers, and extends the lifespan of PA mice, providing the preclinical foundation for the therapeutic potential of BBP-671.

Vaginal Lactobacillus fatty acid response mechanisms reveal a novel strategy for bacterial vaginosis treatment.

Bacterial vaginosis (BV), a common syndrome characterized by Lactobacillus-deficient vaginal microbiota, is associated with adverse health outcomes. BV often recurs after standard antibiotic therapy in part because antibiotics promote microbiota dominance by Lactobacillus iners instead of Lactobacillus crispatus, which has more beneficial health associations. Strategies to promote L. crispatus and inhibit L. iners are thus needed. We show that oleic acid (OA) and similar long-chain fatty acids simultaneously inhibit L. iners and enhance L. crispatus growth. These phenotypes require OA-inducible genes conserved in L. crispatus and related species, including an oleate hydratase (ohyA) and putative fatty acid efflux pump (farE). FarE mediates OA resistance, while OhyA is robustly active in the human vaginal microbiota and sequesters OA in a derivative form that only ohyA-harboring organisms can exploit. Finally, OA promotes L. crispatus dominance more effectively than antibiotics in an in vitro model of BV, suggesting a novel approach for treatment.

A genome-wide atlas of antibiotic susceptibility targets and pathways to tolerance.

Detailed knowledge on how bacteria evade antibiotics and eventually develop resistance could open avenues for novel therapeutics and diagnostics. It is thereby key to develop a comprehensive genome-wide understanding of how bacteria process antibiotic stress, and how modulation of the involved processes affects their ability to overcome said stress. Here we undertake a comprehensive genetic analysis of how the human pathogen Streptococcus pneumoniae responds to 20 antibiotics. We build a genome-wide atlas of drug susceptibility determinants and generated a genetic interaction network that connects cellular processes and genes of unknown function, which we show can be used as therapeutic targets. Pathway analysis reveals a genome-wide atlas of cellular processes that can make a bacterium less susceptible, and often tolerant, in an antibiotic specific manner. Importantly, modulation of these processes confers fitness benefits during active infections under antibiotic selection. Moreover, screening of sequenced clinical isolates demonstrates that mutations in genes that decrease antibiotic sensitivity and increase tolerance readily evolve and are frequently associated with resistant strains, indicating such mutations could be harbingers for the emergence of antibiotic resistance.

Biochemical characterization of the first step in sulfonolipid biosynthesis in Alistipes finegoldii.

Sulfonolipids are unusual lipids found in the outer membranes of Gram-negative bacteria in the phylum Bacteroidetes. Sulfonolipid and its deacylated derivative, capnine, are sulfur analogs of ceramide-1-phosphate and sphingosine-1-phosphate, respectively; thus, sulfonolipid biosynthesis is postulated to be similar to the sphingolipid biosynthetic pathway. Here, we identify the first enzyme in sulfonolipid synthesis in Alistipes finegoldii as the product of the alfi_1224 gene, cysteate acyl-acyl carrier protein (ACP) transferase (SulA). We show SulA catalyzes the condensation of acyl-ACP and cysteate (3-sulfo-alanine) to form 3-ketocapnine. Acyl-CoA is a poor substrate. We show SulA has a bound pyridoxal phosphate (PLP) cofactor that undergoes a spectral redshift in the presence of cysteate, consistent with the transition of the lysine-aldimine complex to a substrate-aldimine complex. Furthermore, the SulA crystal structure shows the same prototypical fold found in bacterial serine palmitoyltransferases (Spts), enveloping the PLP cofactor bound to Lys251. We observed the SulA and Spt active sites are identical except for Lys281 in SulA, which is an alanine in Spt. Additionally, SulA(K281A) is catalytically inactive but binds cysteate and forms the external aldimine normally, highlighting the structural role of the Lys281 side chain in walling off the active site from bulk solvent. Finally, the electropositive groove on the protein surface adjacent to the active site entrance provides a landing pad for the electronegative acyl-ACP surface. Taken together, these data identify the substrates, products, and mechanism of SulA, the PLP-dependent condensing enzyme that catalyzes the first step in sulfonolipid synthesis in a gut commensal bacterium.

Mining Fatty Acid Biosynthesis for New Antimicrobials.

Antibiotic resistance is a serious public health concern, and new drugs are needed to ensure effective treatment of many bacterial infections. Bacterial type II fatty acid synthesis (FASII) is a vital aspect of bacterial physiology, not only for the formation of membranes but also to produce intermediates used in vitamin production. Nature has evolved a repertoire of antibiotics inhibiting different aspects of FASII, validating these enzymes as potential targets for new antibiotic discovery and development. However, significant obstacles have been encountered in the development of FASII antibiotics, and few FASII drugs have advanced beyond the discovery stage. Most bacteria are capable of assimilating exogenous fatty acids. In some cases they can dispense with FASII if fatty acids are present in the environment, making the prospects for identifying broad-spectrum drugs against FASII targets unlikely. Single-target, pathogen-specific FASII drugs appear the best option, but a major drawback to this approach is the rapid acquisition of resistance via target missense mutations. This complication can be mitigated during drug development by optimizing the compound design to reduce the potential impact of on-target missense mutations at an early stage in antibiotic discovery. The lessons learned from the difficulties in FASII drug discovery that have come to light over the last decade suggest that a refocused approach to designing FASII inhibitors has the potential to add to our arsenal of weapons to combat resistance to existing antibiotics.

Domain architecture and catalysis of the Staphylococcus aureus fatty acid kinase.

Fatty acid kinase (Fak) is a two-component enzyme that generates acyl-phosphate for phospholipid synthesis. Fak consists of a kinase domain protein (FakA) that phosphorylates a fatty acid enveloped by a fatty acid binding protein (FakB). The structural basis for FakB function has been established, but little is known about FakA. Here, we used limited proteolysis to define three separate FakA domains: the amino terminal FakA_N, the central FakA_L, and the carboxy terminal FakA_C. The isolated domains lack kinase activity, but activity is restored when FakA_N and FakA_L are present individually or connected as FakA_NL. The X-ray structure of the monomeric FakA_N captures the product complex with ADP and two Mg2+ ions bound at the nucleotide site. The FakA_L domain encodes the dimerization interface along with conserved catalytic residues Cys240, His282, and His284. AlphaFold analysis of FakA_L predicts the catalytic residues are spatially clustered and pointing away from the dimerization surface. Furthermore, the X-ray structure of FakA_C shows that it consists of two subdomains that are structurally related to FakB. Analytical ultracentrifugation demonstrates that FakA_C binds FakB, and site-directed mutagenesis confirms that a positively charged wedge on FakB meshes with a negatively charged groove on FakA_C. Finally, small angle X-ray scattering analysis is consistent with freely rotating FakA_N and FakA_C domains tethered by flexible linkers to FakA_L. These data reveal specific roles for the three independently folded FakA protein domains in substrate binding and catalysis.

Identification of structural transitions in bacterial fatty acid binding proteins that permit ligand entry and exit at membranes.

Fatty acid (FA) transfer proteins extract FA from membranes and sequester them to facilitate their movement through the cytosol. Detailed structural information is available for these soluble protein-FA complexes, but the structure of the protein conformation responsible for FA exchange at the membrane is unknown. Staphylococcus aureus FakB1 is a prototypical bacterial FA transfer protein that binds palmitate within a narrow, buried tunnel. Here, we define the conformational change from a "closed" FakB1 state to an "open" state that associates with the membrane and provides a path for entry and egress of the FA. Using NMR spectroscopy, we identified a conformationally flexible dynamic region in FakB1, and X-ray crystallography of FakB1 mutants captured the conformation of the open state. In addition, molecular dynamics simulations show that the new amphipathic α-helix formed in the open state inserts below the phosphate plane of the bilayer to create a diffusion channel for the hydrophobic FA tail to access the hydrocarbon core and place the carboxyl group at the phosphate layer. The membrane binding and catalytic properties of site-directed mutants were consistent with the proposed membrane docked structure predicted by our molecular dynamics simulations. Finally, the structure of the bilayer-associated conformation of FakB1 has local similarities with mammalian FA binding proteins and provides a conceptual framework for how these proteins interact with the membrane to create a diffusion channel from the FA location in the bilayer to the protein interior.

Proton magnetic resonance spectroscopy detects cerebral metabolic derangement in a mouse model of brain coenzyme a deficiency.

BACKGROUND: Pantothenate kinase (PANK) is the first and rate-controlling enzymatic step in the only pathway for cellular coenzyme A (CoA) biosynthesis. PANK-associated neurodegeneration (PKAN), formerly known as Hallervorden-Spatz disease, is a rare, life-threatening neurologic disorder that affects the CNS and arises from mutations in the human PANK2 gene. Pantazines, a class of small molecules containing the pantazine moiety, yield promising therapeutic effects in an animal model of brain CoA deficiency. A reliable technique to identify the neurometabolic effects of PANK dysfunction and to monitor therapeutic responses is needed. METHODS: We applied 1H magnetic resonance spectroscopy as a noninvasive technique to evaluate the therapeutic effects of the newly developed Pantazine BBP-671. RESULTS: 1H MRS reliably quantified changes in cerebral metabolites, including glutamate/glutamine, lactate, and N-acetyl aspartate in a neuronal Pank1 and Pank2 double-knockout (SynCre+ Pank1,2 dKO) mouse model of brain CoA deficiency. The neuronal SynCre+ Pank1,2 dKO mice had distinct decreases in Glx/tCr, NAA/tCr, and lactate/tCr ratios compared to the wildtype matched control mice that increased in response to BBP-671 treatment. CONCLUSIONS: BBP-671 treatment completely restored glutamate/glutamine levels in the brains of the mouse model, suggesting that these metabolites are promising clinically translatable biomarkers for future therapeutic trials.

Oleate Hydratase (OhyA) Is a Virulence Determinant in Staphylococcus aureus.

Staphylococcus aureus is an important pathogen that relies on a variety of mechanisms to evade and counteract the immune system. We show that S. aureus uses oleate hydratase (OhyA) to convert host cis-9 unsaturated fatty acids to their 10-hydroxy derivatives in human serum and at the infection site in a mouse neutropenic thigh model. Wild-type and ΔohyA strains were equally infective in the neutropenic thigh model, but recovery of the ΔohyA strain was 2 orders of magnitude lower in the immunocompetent skin infection model. Despite the lower bacterial abundance at the infection site, the levels of interleukin 6 (IL-6), monocyte chemoattractant protein 1 (MCP-1), IL-1ß, and tumor necrosis factor alpha (TNF-α) elicited by the ΔohyA strain were as robust as those of either the wild-type or the complemented strain, indicating that the immune system was more highly activated by the ΔohyA strain. Thus, OhyA functions to promote S. aureus virulence. IMPORTANCE The oleate hydratase protein family was discovered in commensal bacteria that utilize host unsaturated fatty acids as the substrates to produce a spectrum of hydroxylated products. These hydroxy fatty acids are thought to act as signaling molecules that suppress the inflammatory response to create a more tolerant environment for the microbiome. S. aureus is a significant human pathogen, and defining the mechanisms used to evade the immune response is critical to understanding pathogenesis. S. aureus expresses an OhyA that produces at least three 10-hydroxy fatty acids from host unsaturated fatty acids at the infection site, and an S. aureus strain lacking the ohyA gene has compromised virulence in an immunocompetent infection model. These data suggest that OhyA plays a role in immune modulation in S. aureus pathogenesis similar to that in commensal bacteria.

Malonyl-acyl carrier protein decarboxylase activity promotes fatty acid and cell envelope biosynthesis in Proteobacteria.

Bacterial fatty acid synthesis in Escherichia coli is initiated by the condensation of an acetyl-CoA with a malonyl-acyl carrier protein (ACP) by the ß-ketoacyl-ACP synthase III enzyme, FabH. E. coli ΔfabH knockout strains are viable because of the yiiD gene that allows FabH-independent fatty acid synthesis initiation. However, the molecular function of the yiiD gene product is not known. Here, we show the yiiD gene product is a malonyl-ACP decarboxylase (MadA). MadA has two independently folded domains: an amino-terminal N-acetyl transferase (GNAT) domain (MadAN) and a carboxy-terminal hot dog dimerization domain (MadAC) that encodes the malonyl-ACP decarboxylase function. Members of the proteobacterial Mad protein family are either two domain MadA (GNAT-hot dog) or standalone MadB (hot dog) decarboxylases. Using structure-guided, site-directed mutagenesis of MadB from Shewanella oneidensis, we identified Asn45 on a conserved catalytic loop as critical for decarboxylase activity. We also found that MadA, MadAC, or MadB expression all restored normal cell size and growth rates to an E. coli ΔfabH strain, whereas the expression of MadAN did not. Finally, we verified that GlmU, a bifunctional glucosamine-1-phosphate N-acetyl transferase/N-acetyl-glucosamine-1-phosphate uridylyltransferase that synthesizes the key intermediate UDP-GlcNAc, is an ACP binding protein. Acetyl-ACP is the preferred glucosamine-1-phosphate N-acetyl transferase/N-acetyl-glucosamine-1-phosphate uridylyltransferase substrate, in addition to being the substrate for the elongation-condensing enzymes FabB and FabF. Thus, we conclude that the Mad family of malonyl-ACP decarboxylases supplies acetyl-ACP to support the initiation of fatty acid, lipopolysaccharide, peptidoglycan, and enterobacterial common antigen biosynthesis in Proteobacteria.

LipE guided discovery of isopropylphenyl pyridazines as pantothenate kinase modulators.

Pantothenate kinase (PANK) is the critical regulator of intracellular levels of coenzyme A and has emerged as an attractive target for treating neurological and metabolic disorders. This report describes the optimization, synthesis, and full structure-activity relationships of a new chemical series of pantothenate competitive PANK inhibitors. Potent drug-like molecules were obtained by optimizing a high throughput screening hit, using lipophilic ligand efficiency (LipE) derived from human PANK3 IC50 values to guide ligand development. X-ray crystal structures of PANK3 with index inhibitors from the optimization were determined to rationalize the emerging structure activity relationships. The analysis revealed a key bidentate hydrogen bonding interaction between pyridazine and R306' as a major contributor to the LipE gain observed in the optimization. A tractable series of PANK3 modulators with nanomolar potency, excellent LipE values, desirable physicochemical properties, and a well-defined structural binding mode was produced from this study.