Charles O Rock

• PhD
• Member at St. Jude Children's Research Hospital

Threonine deaminase (IlvA) is the first enzyme in the isoleucine branch of the branched-chain amino acid biosynthetic pathway. Kinetic studies of isoleucine feedback inhibition of Escherichia coli IlvA (EcIlvA) introduced the concept of allosteric regulation in enzymes. While the crystal structure of EcIlvA shows a tetrameric assembly of protomers...

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 as...

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 p...

Antibiotics that inhibit peptidoglycan synthesis trigger the activation of both specific and general protective responses. σ M responds to diverse antibiotics that inhibit cell wall synthesis. Here, we demonstrate that cell wall inhibiting drugs, such as bacitracin and cefuroxime, induce the σ M -dependent ytpAB operon. YtpA is a predicted hydrolas...

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 responsib...

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...

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 coenzy...

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 therapeuti...

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 identi...

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 p...

Staphylococcus aureus controls its membrane biophysical properties using branched-chain fatty acids. 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 knockout...

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 metabolis...

Many bacterial species are represented by a pan-genome, whose genetic repertoire far outstrips that of any single bacterial genome. Here we investigate how a bacterial pan-genome might influence gene essentiality and whether essential genes that are initially critical for the survival of an organism can evolve to become non-essential. By using Tran...

Histone acetyltransferases (HAT) catalyze the acylation of lysine side chains and are implicated in diverse human cancers as both oncogenes and non-oncogene dependencies ¹ . Acetyl-CoA-competitive HAT inhibitors have garnered attention as potential cancer therapeutics and the first clinical trial for this class is ongoing ( NCT04606446 ). Despite b...

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...

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 evol...

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 p...

Sulfonolipids (SL) are unusual lipids found in the outer membranes of Gram-negative bacteria in the phylum Bacteroidetes. SL and its deacylated derivative, capnine, are sulfur analogs of ceramide-1-phosphate and sphingosine-1-phosphate, respectively. The structural similarity to key mammalian signaling molecules led us to investigate the biochemist...

Staphylococcus aureus produces a combination of branched and straight chain fatty acids to maintain membrane homeostasis. Recently, we showed that the branched:straight chain ratio in fatty acids is controlled by amino acid metabolism that supplies the branched-chain acyl-CoA primers for fatty acid biosynthesis. The fatty acid composition is determ...

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 limit...

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 PAN...

Many bacterial species are represented by a pan-genome, whose genetic repertoire far outstrips that of any single bacterial genome. Here we investigate how a bacterial pan-genome might influence gene essentiality, and whether essential genes that are initially critical for the survival of an organism can evolve to become non-essential. By using Tn-...

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 p...

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...

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...

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. Poten...

Bacterial fatty acid synthesis (FASII) in Escherichia coli is initiated by the condensation of an acetyl-CoA with a malonyl-acyl carrier protein (ACP) by the β-ketoacyl-ACP synthase III enzyme, FabH. E. coli ΔfabH knockout strains are viable due to the yiiD gene that allows FabH-independent FASII initiation. However, the molecular function of the y...

Fatty acid (FA) transfer proteins extract FA from membranes and sequester their ligand to facilitate its movement through the cytosol. While detailed views of soluble protein-FA complexes are available, how FA exchange occurs at the membrane has remained unknown. Staphylococcus aureus FakB1 is a prototypical bacterial FA transfer protein that binds...

Propionic acidemia (PA) is a rare autosomal-recessive metabolic disease that arises from mutations in propionyl-CoA (C3-CoA) carboxylase. Reduced enzyme activity slows C3-CoA metabolism, leading to an elevated plasma C3:C2-carnitine ratio, the hallmark biomarker of PA. The metabolic imbalances experienced in PA are however poorly defined. Here, we...

Branched-chain amino acids (primarily isoleucine) are important regulators of virulence and are converted to precursor molecules used to initiate fatty acid synthesis in Staphylococcus aureus. Defining how bacteria control their membrane phospholipid composition is key to understanding their adaptation to different environments. Here, we used mass...

Adaptation of bacteria to their host environment is a key component of colonization and pathogenesis. As an essential component of bacterial membranes, fatty acid composition contributes to host adaptation.

FAD-dependent bacterial oleate hydratases (OhyA) catalyze the addition of water to isolated fatty acid carbon-carbon double bonds. Staphylococcus aureus uses OhyA to counteract the host innate immune response by inactivating antimicrobial unsaturated fatty acids. Mechanistic information explaining how OhyAs catalyze regio- and stereospecific hydrat...

Membrane lipid homeostasis is required for bacteria to survive in a spectrum of host environments. This homeostasis is achieved by regulation of fatty acid chain length and of the ratio of saturated to unsaturated fatty acids. In the pathogen Streptococcus pneumoniae , fatty acid biosynthesis is encoded by a cluster of fatty acid biosynthesis ( fab...

Herein is a report on the molecular exchange occurring between multilateral symbiosis partners—a tit-for-tat exchange that led to the characterization of two new metabolites, conocandin B (fungal-derived) and dentigerumycin F (bacterial-derived). The structures were determined by NMR, mass spectrometry, genomic analysis, and chemical derivatization...

Most bacteria construct their phospholipid membrane with fatty acids made from type II fatty acid synthesis system (FASII). FASII consists of dissociable enzymes that grow and modify a fatty acid chain before transferring it to the membrane. FASII produces acyl chains varying in the number of carbons and the degree of saturation which enables bacte...

FAD-dependent bacterial oleate hydratases (OhyA) catalyze the addition of water to isolated fatty acid carbon-carbon double bonds. Staphylococcus aureus uses OhyA to counteract the host innate immune response by inactivating antimicrobial unsaturated fatty acids. Mechanistic information explaining how OhyAs catalyze regio- and stereospecific hydrat...

Streptococcus pneumoniae is a major human pathogen that must adapt to unique nutritional environments in several host niches. The pneumococcus can metabolize a range of carbohydrates that feed into glycolysis ending in pyruvate, which is catabolized by several enzymes. We investigated how the pneumococcus utilizes these enzymes to metabolize differ...

The shortage of antibiotics against drug-resistant Staphylococcus aureus has led to the development of new drugs targeting the elongation cycle of fatty acid (FA) synthesis that are progressing toward the clinic. An objection to the use of FA synthesis inhibitors is that S. aureus can utilize exogenous FAs to construct its membrane, suggesting that...

Enoyl-acyl carrier protein reductase (FabI) catalyzes a rate-controlling step in bacterial fatty-acid synthesis and is a target for antibacterial drug development. A phylogenetic analysis shows that FabIs fall into four divergent clades. Members of clades 1–3 have been structurally and biochemically characterized, but the fourth clade found in memb...

Fatty acids are abundant nutrients in the gut, and this work identified the biochemical pathway for fatty acid utilization in the prominent anaerobic, Gram-negative inhabitants of the gut microbiome, the phylum Bacteroidetes. Alistipes finegoldii is a representative Bacteroidetes resident of the human gut. This organism has not been well studied un...

Fatty acid synthesis is important in both prokaryotes and eukaryotes for formation of functional membranes and dysregulation of the pathway has implications for cell growth and survival. In bacteria, fatty acid production is tightly regulated by the Type II fatty acid synthesis (FASII) pathway. FabH (3‐ketoacyl acyl carrier protein (ACP) synthase I...

The Fatty acid kinase (Fak) system is a two‐protein system allowing Gram‐positive pathogens to incorporate exogenous fatty acids (FA) for membrane phospholipid synthesis. Exogenous FA bind to a FA binding protein, FakB, are phosphorylated by a kinase, FakA, and used to construct the membrane. Phosphorylated FA can also be interconverted to acyl‐acy...

Members of the Bacteroidetes phylum, represented by Alistipes finegoldii, are prominent anaerobic, Gram‐negative inhabitants of the gut microbiome. The lipid biosynthetic pathways were analyzed using bioinformatic analyses, lipidomics, metabolic labeling and biochemistry to characterize exogenous fatty acid metabolism. A. finegoldii only produced s...

Streptococcus pneumoniae is responsible for the majority of pneumonia, motivating ongoing searches for insights into its physiology that could enable new treatments. S. pneumoniae responds to exogenous fatty acids by suppressing its de novo biosynthetic pathway and exclusively utilizing extracellular fatty acids for membrane phospholipid synthesis....

Two-component regulatory systems enable bacteria to adapt to changes in their environment during infection by altering gene expression and coordinating antihost responses. Despite the critical role of two-component systems in bacterial survival and pathogenesis, the activating signals for most of these regulators remain unidentified. This is exempl...

Oleate hydratases (OhyA) belong to a large family of bacterial proteins catalyzing the hydration or isomerization of double bonds in unsaturated fatty acids. A Staphylococcus aureus gene (Sa0102) is predicted to encode an OhyA. Here, we recombinantly expressed and purified SaOhyA and found that it forms a homodimer that requires FAD for activity. S...

Pantothenate kinase generates 4′‐phosphopantothenate in the first and rate‐determining step of coenzyme A (CoA) biosynthesis. The human genome encodes three well‐characterized and nearly identical pantothenate kinases (PANK1‐3) plus a putative bifunctional protein (PANK4) with a predicted amino‐terminal pantothenate kinase domain fused to a carboxy...

Fatty acid (FA) kinase produces acyl-phosphate for the synthesis of membrane phospholipids in Gram-positive bacterial pathogens. FA kinase consists of a kinase protein (FakA) that phosphorylates an FA substrate bound to a second module, an FA binding protein (FakB). Staphylococcus aureus expresses two distinct, but related, FakBs with different FA...

Pantothenate kinase (PANK) is a metabolic enzyme that regulates cellular coenzyme A (CoA) levels. There are three human PANK genes, and inactivating mutations in PANK2 lead to pantothenate kinase associated neurodegeneration (PKAN). Here we performed a library screen followed by chemical optimization to produce PZ-2891, an allosteric PANK activator...

The central position of coenzyme A (CoA) in intermediary metabolism and its role in human neurodegenerative disease, led us to develop chemical probes targeting pantothenate kinase (PANK), the regulatory enzyme that controls CoA biosynthesis. There are four mammalian PANK isoforms that exhibit tissue‐specific expression and differ in their sensitiv...

The human genome is predicted to encode four pantothenate kinase proteins. The PANK1, PANK2, and PANK3 proteins are established functional pantothenate kinases, but the function of the PANK4 protein is disputed. The PANK4 is predicted to encode a fusion of a pantothenate kinase domain at the N‐terminus and a phosphatase domain at the C‐terminus. Th...

Chlamydia trachomatis is an obligate intracellular pathogen with a reduced genome reflecting its host cell dependent life style. However, C. trachomatis has retained all of the genes required for fatty acid and phospholipid synthesis that are present in free-living bacteria. C. trachomatis assembles its cellular membrane using its own biosynthetic...

Staphylococcal species are a leading cause of bacterial drug-resistant infections and associated mortality. One strategy to combat bacterial drug resistance is to revisit compromised targets, and to circumvent resistance mechanisms using structure-assisted drug discovery. The folate pathway is an ideal candidate for this approach. Antifolates targe...

The SaeRS two-component system is a master activator of virulence factor transcription in Staphylococcus aureus, but the cellular factors that control its activity are unknown. Fatty acid (FA) kinase is a two-component enzyme system required for extracellular FA uptake and SaeRS activity. Here, we demonstrate the existence of an intracellular nones...

Phosphatidic acid (PA), the central intermediate in membrane phospholipid synthesis, is generated by two acyltransferases in a pathway conserved in all life forms. The second step in this pathway is catalyzed by 1-acyl-sn-glycerol-3-phosphate acyltransferase, called PlsC in bacteria. Here we present the crystal structure of PlsC from Thermotoga mar...

The G protein-coupled receptor Smoothened (Smo) is the signal transducer of the Sonic Hedgehog (Shh) pathway. Smo signals through G protein-dependent and -independent routes, with G protein-independent canonical signaling to Gli effectors requiring Smo accumulation in the primary cilium. The mechanisms controlling Smo activation and trafficking are...

Bacterial type II fatty acid synthesis (FASII) is a target for novel antibiotic development. All bacteria encode for mechanisms to incorporate exogenous fatty acids, and some bacteria can use exogenous fatty acids to bypass FASII inhibition. Bacteria encode three different mechanisms for activating exogenous fatty acids for incorporation into phosp...

Coenzyme A (CoA) is a cofactor that is central to energy metabolism and CoA synthesis is controlled by the enzyme pantothenate kinase (PanK). A transgenic mouse strain expressing human PANK2 was derived to determine the physiological impact of PANK overexpression and elevated CoA levels. The Tg(PANK2) mice expressed high levels of the transgene in...

Importance: The increasing prevalence of antibiotic-resistant bacterial pathogens is a major challenge currently facing the medical community. Such pathogens are of particular importance in immunocompromised patients as these individuals may favor emergence of novel resistance determinants due to lack of innate immune defenses and intensive antibi...

Bacteria are a model system to study membrane lipid biosynthesis and metabolism. The chapter reviews the major events in bacterial membrane lipid synthesis. The most energy intensive part of membrane lipid synthesis is the dissociated type II fatty acid synthase. The initiation module consists of the enzymes required to initiate type II fatty acid...

Membrane homeostasis in bacteria ensures survival in rapidly changing environments. Most prokaryotes utilize the essential type II fatty acid synthesis to generate lipid species required for membrane formation and adaptation. This highly conserved system consists of multiple enzymes that each catalyzes a specific reaction. FAS II is an energy-inten...

Modulation of T cell proliferation and function by immunoregulatory myeloid cells is an essential means of preventing self-reactivity and restoring tissue homeostasis. Consumption of amino acids such as arginine and tryptophan by immunoregulatory macrophages is one pathway that suppresses local T cell proliferation. Using a reduced complexity in vi...

Enoyl-acyl carrier protein reductase catalyzes the last step in each elongation cycle of type II bacterial fatty acid synthesis and is a key regulatory protein in bacterial fatty acid synthesis. The facultative intracellular pathogen Listeria monocytogenes encodes two functional enoyl-ACP isoforms based on their ability to complement the temperatur...

The pneumococcus is one of the most prodigious producers of hydrogen peroxide amongst bacterial pathogens. Hydrogen peroxide production by the pneumococcus has been implicated in antibiotic synergism, competition between other bacterial colonizers of the nasopharynx, and damage to epithelial cells. However, the role during invasive disease has been...

qRT-PCR. Mean CT values with standard deviation of oxidative stress response genes with and without LL-37. (DOCX)

Strains used in this study. (DOCX)

ATP and acetyl-phosphate steady-state levels are reduced in the spxB and double mutants. Intracellular steady-state levels of ATP (A) and acetyl-phosphate (B) were determined. Strains include TIGR4 and the spxB, lctO, and spxB lctO mutants. Values were normalized to total cell protein and then plotted as percentage of wild type. ATP and acetyl-phos...

qRT-PCR. Mean CT values with standard deviation of six genes. (DOCX)

Primers used in this study. (DOCX)

Complementation of lctO to the double mutant restores the spxB mutant phenotype. Functional complementation with pABG5-lctO was determined. Strains include TIGR4 and the spxB, lctO, and spxB lctO mutants, and the lctO mutant and double mutant complemented with pABG5-lctO (lctO-/+ and spxB-lctO-/+). Capsule production was measured using capsule blot...

Median Log values of bacterial burden. (DOCX)

Whole genome sequencing and SNP analysis comparing the spxB mutant to wild type TIGR4. spxB mutant was sequenced twice and was compared to the TIGR4 genome in the NCBI database (tab 1) and to the TIGR4 strain used in these studies (tab 2). Identified SNPs are listed: spxB mutant (columns J and K). (XLSX)

Whole genome sequencing and SNP analysis comparing multiple colonies of the spxB mutant to wild type TIGR4. Colonies of the two phenotypes of the spxB mutant and the TIGR4 strain used in these studies were sequenced and were compared to the TIGR4 genome in the NCBI database. Identified SNPs are listed: TIGR4 (columns K and L); twenty small acapsula...

Hydrogen peroxide production is reduced in the spxB mutant and is negligible in the double mutant in TIGR4 and D39. Hydrogen peroxide production was determined in two methods. Strains include wild type and the spxB, lctO, and the spxB lctO double mutant in the TIGR4 (A, C, F) and D39 (B, D, F) backgrounds, as well as the TIGR4 lctO mutant and doubl...

Pantothenate kinase is the master regulator of CoA biosynthesis, and is feedback inhibited by acetyl-CoA. Comparison of the human PANK3-acetyl-CoA complex to the structures of PANK3 in four catalytically relevant complexes, AMPPNP-Mg2+, AMPPNP-Mg2+-pantothenate, ADP-Mg2+-phosphopantothenate, and AMPPN-Mg2+ revealed a large conformational change in...

Broad-spectrum antibiotic therapy decimates the gut microbiome resulting in a variety of negative health consequences. Debio 1452 is a staphylococci-selective enoyl-acyl carrier protein reductase (FabI) inhibitor under clinical development, and was used to determine if treatment with pathogen-selective antibiotics would minimize disturbance to the...