Biomedical subjects

Charles O Rock

Publications and source records attributed to Charles O Rock.

At least 19 recordsLinked to original sources

PubMed2024

Vaginal Lactobacillus fatty acid response mechanisms reveal a metabolite-targeted 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 lactobacilli, including an oleate hydratase (ohyA) and putative fatty acid efflux pump (farE). FarE mediates OA resistance, while OhyA is robustly active in the vaginal microbiota and enhances bacterial fitness by biochemically sequestering OA in a derivative form only ohyA-harboring organisms can exploit. OA promotes L. crispatus dominance more effectively than antibiotics in an in vitro BV model, suggesting a metabolite-based treatment approach.

Vaginosis, Bacterial↗

PubMed2006

Acyl-phosphates initiate membrane phospholipid synthesis in Gram-positive pathogens.

It is not known how Gram-positive bacterial pathogens carry out glycerol-3-phosphate (G3P) acylation, which is the first step in the formation of phosphatidic acid, the key intermediate in membrane phospholipid synthesis. In Escherichia coli, acylation of the 1-position of G3P is carried out by PlsB; however, the majority of bacteria lack a plsB gene and in others it is not essential. We describe a two-step pathway that utilizes a new fatty acid intermediate for the initiation of phospholipid formation. First, PlsX produces a unique activated fatty acid by catalyzing the synthesis of fatty acyl-phosphate from acyl-acyl carrier protein, and then PlsY transfers the fatty acid from acyl-phosphate to the 1-position of G3P. The PlsX/Y pathway defines the most widely distributed pathway for the initiation of phospholipid formation in bacteria and represents a new target for the development of antibacterial therapeutics.

Bacterial Proteins↗

PubMed2006

Placental thrombosis and spontaneous fetal death in mice deficient in ethanolamine kinase 2.

Ethanolamine kinase catalyzes the first step in the CDP-ethanolamine pathway for the formation of the major membrane phospholipid phosphatidylethanolamine (PtdEtn). In this work, the predicted Etnk2 cDNA was established as a soluble protein with ethanolamine-specific kinase activity that was most highly expressed in liver. Mice with an inactivated Etnk2 gene were derived, and its absence reduced the rate of PtdEtn synthesis from exogenous ethanolamine in hepatocytes. PtdEtn is a major precursor to phosphatidylcholine in liver; however, Etnk2(-/-) mice did not have reduced amounts of either PtdEtn or phosphatidylcholine or an altered phospholipid molecular species distribution. The knock-out animals were able to adapt to a choline-deficient diet. The Etnk2(-/-) mice exhibited a maternal-specific intrauterine growth retardation phenotype that resulted in a 33% reduction in litter size and frequent perinatal death. Histological analysis of pregnant Etnk2(-/-) females showed that fetal development failed at the late stage of pregnancy in a significant percentage of embryos because of the appearance of extensive placental thrombosis. These results illustrate a non-redundant role for EtnK2 expression in regulating placental hemostasis.

Amino Acid Sequence↗

PubMed2006

Inhibiting bacterial fatty acid synthesis.

The type II fatty acid synthase consists of a series of individual enzymes, each encoded by a separate gene, that catalyze discrete steps in chain elongation. The formation of fatty acids is vital to bacteria, and each of the essential enzymes and their acyl group carriers represent a potential target for the development of novel antibacterial therapeutics. High resolution x-ray and/or NMR structures of representative members of every enzyme in the type II pathway are now available, and these structures are a valuable resource to guide antibacterial drug discovery. The role of each enzyme in regulating pathway activity and the diversity in the components of the pathway in the major human pathogens are important considerations in deciding the most suitable targets for future drug development.

Acetyltransferases↗

PubMed2006

Structure of RhlG, an essential beta-ketoacyl reductase in the rhamnolipid biosynthetic pathway of Pseudomonas aeruginosa.

Rhamnolipids are extracellular biosurfactants and virulence factors secreted by the opportunistic human pathogen Pseudomonas aeruginosa that are required for swarming motility. The rhlG gene is essential for rhamnolipid formation, and the RhlG enzyme is thought to divert fatty acid synthesis intermediates into the rhamnolipid biosynthetic pathway based on its similarity to FabG, the beta-ketoacyl-acyl carrier protein (ACP) reductase of type II fatty acid synthesis. Crystallographic analysis reveals that the overall structures of the RhlG.NADP+ and FabG.NADP+ complexes are indeed similar, but there are key differences related to function. RhlG does not undergo the conformational changes upon NADP(H) binding at the active site that in FabG are the structural basis of negative allostery. Also, the acyl chain-binding pocket of RhlG is narrow and rigid compared with the larger, flexible substrate-binding subdomain in FabG. Finally, RhlG lacks a positively charged/hydrophobic surface feature adjacent to the active site that is found on enzymes like FabG that recognize the ACP of fatty acid synthesis. RhlG catalyzed the NADPH-dependent reduction of beta-ketodecanoyl-ACP to beta-d-hydroxydecanoyl-ACP. However, the enzyme was 2000-fold less active than FabG in carrying out the same reaction. These structural and biochemical studies establish RhlG as a NADPH-dependent beta-ketoacyl reductase of the SDR protein superfamily and further suggest that the ACP of fatty acid synthesis does not carry the substrates for RhlG.

3-Oxoacyl-(Acyl-Carrier-Protein) Reductase↗

PubMed2006

Roles of the active site water, histidine 303, and phenylalanine 396 in the catalytic mechanism of the elongation condensing enzyme of Streptococcus pneumoniae.

beta-Ketoacyl-ACP synthases catalyze the condensation steps in fatty acid and polyketide synthesis and are targets for the development of novel antibiotics and anti-obesity and anti-cancer agents. The roles of the active site residues in Streptococcus pneumoniae FabF (beta-ketoacyl-ACP synthase II; SpFabF) were investigated to clarify the mechanism for this enzyme superfamily. The nucleophilic cysteine of the active site triad was required for acyl-enzyme formation and the overall condensation activity. The two active site histidines in the elongation condensing enzyme have different electronic states and functions. His337 is essential for condensation activity, and its protonated Nepsilon stabilizes the negative charge developed on the malonyl thioester carbonyl in the transition state. The Nepsilon of His303 accelerated catalysis by deprotonating a structured active site water for nucleophilic attack on the C3 of malonate, releasing bicarbonate. Lys332 controls the electronic state of His303 and also plays a critical role in the positioning of His337. Phe396 functions as a gatekeeper that controls the order of substrate addition. These data assign specific roles for each active site residue and lead to a revised general mechanism for this important class of enzymes.

Amino Acid Sequence↗

PubMed2006

Structure-activity relationships at the 5-position of thiolactomycin: an intact (5R)-isoprene unit is required for activity against the condensing enzymes from Mycobacterium tuberculosis and Escherichia coli.

Thiolactomycin inhibits bacterial cell growth through inhibition of the beta-ketoacyl-ACP synthase activity of type II fatty acid synthases. The effect of modifications of the 5-position isoprenoid side chain on both IC(50) and MIC were determined. Synthesis and screening of a structurally diverse set of 5-position analogues revealed very little tolerance for substitution in purified enzyme assays, but a few analogues retained MIC, presumably through another target. Even subtle modifications such as reducing one or both double bonds of the diene were not tolerated. The only permissible structural modifications were removal of the isoprene methyl group or addition of a methyl group to the terminus. Cocrystallization of these two inhibitors with the condensing enzyme from Escherichia coli revealed that they retained the TLM binding mode at the active site with reduced affinity. These results suggest a strict requirement for a conjugated, planar side chain inserting within the condensing enzyme active site.

3-Oxoacyl-(Acyl-Carrier-Protein) Synthase↗

PubMed2006

Serum opacity factor, a streptococcal virulence factor that binds to apolipoproteins A-I and A-II and disrupts high density lipoprotein structure.

Serum opacity factor (SOF) is a virulence determinant of group A streptococci that opacifies mammalian sera. We analyzed the specificity and mechanism of the opacity reaction using a recombinant form of the amino-terminal opacification domain of SOF, rSOF. Our data indicate that rSOF is neither a protease nor a lipase, but rather it is the binding of rSOF to high density lipoprotein (HDL) that triggers the opacity reaction. rSOF did not opacify plasma from apoA-I(-/-) mice or purified low or very low density lipoproteins but readily opacified HDL. rSOF binding to HDL was characterized by two high affinity binding sites; it bound to apoA-I (K(d) = 6 nm) and apoA-II (K(d) = 30 nm), and both apoA-I and apoA-II blocked the binding of rSOF to HDL. Electron microscopic examination and biochemical analyses of HDL treated with rSOF revealed the formation of lipid droplets devoid of apolipoproteins. Thus, SOF interacts with HDL in human blood by binding to apoA-I and apoA-II and causing the release of HDL lipid cargo, which coalesces to form lipid droplets, resulting in opacification. The disruption of HDL may attenuate its anti-inflammatory functions and contribute to the pathogenesis of group A streptococcal infections.

Animals↗

PubMed2006

Prokaryotic type II and type III pantothenate kinases: The same monomer fold creates dimers with distinct catalytic properties.

Three distinct isoforms of pantothenate kinase (CoaA) in bacteria catalyze the first step in coenzyme A biosynthesis. The structures of the type II (Staphylococcus aureus, SaCoaA) and type III (Pseudomonas aeruginosa, PaCoaA) enzymes reveal that they assemble nearly identical subunits with actin-like folds into dimers that exhibit distinct biochemical properties. PaCoaA has a fully enclosed pantothenate binding pocket and requires a monovalent cation to weakly bind ATP in an open cavity that does not interact with the adenine nucleotide. Pantothenate binds to an open pocket in SaCoaA that strongly binds ATP by using a classical P loop architecture coupled with specific interactions with the adenine moiety. The PaCoaA*Pan binary complex explains the resistance of bacteria possessing this isoform to the pantothenamide antibiotics, and the similarity between SaCoaA and human pantothenate kinase 2 explains the molecular basis for the development of the neurodegenerative phenotype in three mutations in the human protein.

Adenosine Triphosphate↗

PubMed2006

Transcriptional regulation of fatty acid biosynthesis in Streptococcus pneumoniae.

The transcriptional regulation of membrane fatty acid composition in the human pathogen Streptococcus pneumoniae is distinct from the systems utilized in the model organisms Escherichia coli and Bacillus subtilis. The genes encoding the components of type II fatty acid biosynthesis cluster at a single location within the S. pneumoniae genome, and the second gene in this cluster (SPR0376) encodes a transcription factor (FabT) that belongs to the MarR superfamily. Derivatives of S. pneumoniae strain D39 were constructed that lacked functional FabT. This strain had significantly elevated levels of saturated fatty acids and longer chain lengths than the control strain, was unable to grow at pH 5.5 and had increased sensitivity to detergent. Eliminating FabT function increased the expression levels of all of fab genes with the notable exception of fabM. FabT was purified and bound to the DNA palindrome located within the promoter regions of the fabT and fabK genes within the cluster. The analysis of cells with increased expression of individual genes leads to a model where the physical properties of the S. pneumoniae membrane is controlled primarily by the activity of FabK, the enoyl reductase, which diverts intermediates to saturated fatty acid formation, in contrast to E. coli where FabB, an elongation condensing enzyme, pulls the pathway in the direction of unsaturated acid synthesis.

Bacterial Proteins↗

PubMed2006

Two aerobic pathways for the formation of unsaturated fatty acids in Pseudomonas aeruginosa.

The double bond in anaerobic unsaturated fatty acid (UFA) biosynthesis is introduced by the FabA dehydratase/isomerase of the bacterial type II fatty acid biosynthetic pathway. A DeltafabA mutant of Pseudomonas aeruginosa grew aerobically, but required a UFA supplement for anaerobic growth. Wild-type cells produced 18:1Delta11 as the principal UFA, whereas the DeltafabA strain produced only 16:1Delta9. The double bond in the 16:1Delta9 was introduced after phospholipid formation and was localized in the sn-2 position. Two predicted membrane proteins, DesA and DesB, possessed the conserved histidine clusters characteristic of fatty acid desaturases. The DeltafabADeltadesA double mutant required exogenous fatty acids for growth but the DeltafabAdesB double mutant did not. Exogenous stearate was converted to 18:1Delta9 and supported the growth of DeltafabADeltadesA double mutant. A DeltafabADeltadesAdesB triple mutant was unable to desaturate exogenous stearate and was an UFA auxotroph. We detected a 2.5-fold increase in desA expression in DeltafabA mutants, whereas desB expression was derepressed by the deletion of the gene encoding a transcriptional repressor DesT. These data add two aerobic desaturases to the enzymes used for fatty acid metabolism in proteobacteria: DesA, a 2-position phospholipid Delta9-desaturase that supplements the anaerobic FabA pathway, and DesB, an inducible acyl-CoA Delta9-desaturase whose expression is repressed by DesT.

Aerobiosis↗

PubMed2005

Biochemical properties of human pantothenate kinase 2 isoforms and mutations linked to pantothenate kinase-associated neurodegeneration.

The PANK2 gene encodes the human pantothenate kinase 2 protein isoforms, and PANK2 mutations are linked to pantothenate kinase-associated neurodegeneration. Two PanK2 protein forms are proteolytically processed to form a mitochondrially localized, mature PanK2. Another isoform arose from a proposed initiation at a leucine codon and was not processed further. The fifth isoform was postulated to arise from an alternative splicing event and was found to encode an inactive protein. Fourteen mutant PanK2 proteins with single amino acid substitutions, associated with either early or late onset disease, were evaluated for activity. The PanK2(G521R), the most frequent mutation in pantothenate kinase-associated neurodegeneration, was devoid of activity and did not fold properly. However, nine of the mutant proteins associated with disease possessed catalytic activities that were indistinguishable from wild type, including the frequently encountered PanK2(T528M) missense mutation. PanK2 was extremely sensitive to feedback inhibition by CoA thioesters (IC50 values between 250 and 500 nM), and the regulation of the active PanK2 mutants was comparable with that of the wild-type protein. Coexpression of the PanK2(G521R) and wild-type PanK2 did not interfere with wild-type enzyme activity, arguing against a dominant negative effect of the PanK2(G521R) mutation in heterozygous patients. These data described the unique biochemical features of the PanK2 isoforms and suggested that catalytic defects may not be the sole cause for the neurodegenerative phenotype.

Alternative Splicing↗

PubMed2005

Structure-activity relationships and enzyme inhibition of pantothenamide-type pantothenate kinase inhibitors.

A set of novel pantothenamide-type analogues of the known Staphylococcus aureus pantothenate kinase (SaPanK) inhibitors, N-pentyl, and N-heptylpantothenamide, was synthesized in three series. The first series of analogues (1-3) were designed as molecular probes of the PanK binding site to elucidate important structure-activity relationships (SAR). The second series of analogues (4-16) were designed using structural information obtained from the Escherichia coli PanK (EcPanK) structure by targeting the pantothenate binding site and the adjacent phenylalanine-lined lipophilic pocket. Insight into the antimicrobial effect of N-pentylpantothenamide (N5-Pan) through its conversion to the antimetabolite ethyldethia-CoA and further incorporation into an inactive acyl carrier protein analogue drove the development of the third series of analogues (17-25) to enhance this effect using substrate-like substitutions. Each of the analogues was screened for enzyme inhibition activity against a panel of pantothenate kinases consisting of EcPanK, Aspergillus nidulans (AnPanK), SaPanK, and the murine isoform (MmPanK1alpha). Series 1 demonstrated only modest inhibitory activity, but did reveal some important SAR findings including stereospecific binding. Series 2 demonstrated a much higher inhibition rate for the entire series and significant inhibition was seen with analogues containing alkyl substituents. Series 3 demonstrated the most preferential inhibition profile, with the highest inhibitory activity against the SaPanK and MmPanK1alpha. The MmPanK1alpha protein was inhibited by a broad spectrum of the compounds, whereas the E. coli enzyme showed greater selectivity. The overall activity data from these analogues suggest a complex and non-enzyme specific SAR for pantothenamide substrate/inhibitors of the different PanK enzymes.

Binding Sites↗

PubMed2005

Feedback regulation of murine pantothenate kinase 3 by coenzyme A and coenzyme A thioesters.

Pantothenate kinase catalyzes a key regulatory step in coenzyme A biosynthesis, and there are four mammalian genes that encode isoforms of this enzyme. Pantothenate kinase isoform PanK3 is highly related to the previously characterized PanK1beta isoform (79% identical, 91% similar), and these two almost identical proteins are expressed most highly in the same tissues. PanK1beta and PanK3 had very similar molecular sizes, oligomeric form, cytoplasmic cellular location, and kinetic constants for ATP and pantothenate. However, these two PanK isoforms possessed distinct regulatory properties. PanK3 was significantly more sensitive to feedback regulation by acetyl-CoA (IC50 = 1 microm) than PanK1beta (IC50 = 10 microm), and PanK3 was stringently regulated by long-chain acyl-CoA (IC50 = 2 microm), whereas PanK1beta was not. Domain swapping experiments localized the difference in the two proteins to a 48-amino-acid domain, where they are the most divergent. Consistent with these more stringent regulatory properties, metabolic labeling experiments showed that coenzyme A (CoA) levels in cells overexpressing PanK3 were lower than in cells overexpressing an equivalent amount of PanK1beta. Thus, the distinct regulatory properties exhibited by the family of the pantothenate kinases allowed the rate of CoA biosynthesis to be controlled by regulatory signals from CoA thioesters involved in different branches of intermediary metabolism.

Adenosine Triphosphate↗

PubMed2005

Domain swapping between Enterococcus faecalis FabN and FabZ proteins localizes the structural determinants for isomerase activity.

Anaerobic unsaturated fatty acid synthesis in bacteria occurs through the introduction of a double bond into the growing acyl chain. In the Escherichia coli model system, FabA catalyzes both the dehydration of beta-hydroxydecanoyl-ACP and the isomerization of trans-2-decenoyl-ACP to cis-3-decenoyl-ACP as the essential step. A second dehydratase, FabZ, functions in acyl chain elongation but cannot carry out the isomerization reaction. Enterococcus faecalis has two highly related FabZ homologs. One of these, termed EfFabN, carries out the isomerization reaction in vivo, whereas the other, EfFabZ, does not (Wang, H., and Cronan, J. E. (2004) J. Biol. Chem. 279, 34489-34495). We carried out a series of domain swapping and mutagenesis experiments coupled with in vitro biochemical analyses to define the structural feature(s) that specify the catalytic properties of these two enzymes. Substitution of the beta3 and beta4 strands of EfFabZ with the corresponding strands from EfFabN was necessary and sufficient to convert EfFabZ into an isomerase. These data are consistent with the hypothesis that the isomerase potential of beta-hydroxyacyl-ACP dehydratases is determined by the properties of the beta-sheets that dictate the orientation of the central alpha-helix and thus the shape of the substrate binding tunnel rather than the catalytic machinery at the active site.

Amino Acid Sequence↗

PubMed2005

Coenzyme A: back in action.

Coenzyme A (CoA) is a ubiquitous essential cofactor that plays a central role in the metabolism of carboxylic acids, including short- and long-chain fatty acids. In the last few years, all of the genes encoding the CoA biosynthetic enzymes have been identified and the structures of several proteins in the pathway have been determined. CoA is assembled in five steps from pantothenic acid and pathway intermediates are common to both prokaryotes and eukaryotes. In spite of the identical biochemistry, remarkable sequence differences among some of the prokaryotic and eukaryotic enzymes have been revealed by comparative genomics. Renewed interest in CoA has arisen from the realization that the biosynthetic pathway is a target for antibacterial drug discovery and from the unexpected association of a human neurodegenerative disorder with mutations in pantothenate kinase. The purpose of this review is to integrate previous knowledge with the most recent findings in the genetics, enzymology and regulation of CoA biosynthesis in bacteria, plants and mammals.

Animals↗

PubMed2005

Lysophospholipid flipping across the Escherichia coli inner membrane catalyzed by a transporter (LplT) belonging to the major facilitator superfamily.

The transfer of phospholipids across membrane bilayers is protein-mediated, and most of the established transporters catalyze the energy-dependent efflux of phospholipids from cells. This work identifies and characterizes a lysophospholipid transporter gene (lplT, formally ygeD) in Escherichia coli that is an integral component in the 2-acylglycerophosphoethanolamine (2-acyl-GPE) metabolic cycle for membrane protein acylation. The lplT gene is adjacent to and in the same operon as the aas gene, which encodes the bifunctional enzyme 2-acyl-GPE acyltransferase/acyl-acyl carrier protein synthetase. In some bacteria, acyltransferase/acyl-ACP synthetase (Aas) and LplT homologues are fused in a single polypeptide chain. 2-Acyl-GPE transport to the inside of the cell was assessed by measuring the Aas-dependent formation of phosphatidylethanolamine. The Aas-dependent incorporation of [3H]palmitate into phosphatidylethanolamine was significantly diminished in deltalplT mutants, and the LplT-Aas transport/acylation activity was independent of the proton motive force. The deltalplT mutants accumulated acyl-GPE in vivo and had a diminished capacity to transport exogenous 2-acylglycerophosphocholine into the cell. Spheroplasts prepared from wild-type E. coli transported and acylated fluorescent 2-acyl-GPE with an apparent K(d) of 7.5 microM, whereas this high-affinity process was absent in deltalplT mutants. Thus, LplT catalyzes the transbilayer movement of lysophospholipids and is the first example of a phospholipid flippase that belongs to the major facilitator superfamily.

Carbon-Sulfur Ligases↗

PubMed2004

A pantothenate kinase from Staphylococcus aureus refractory to feedback regulation by coenzyme A.

The key regulatory step in CoA biosynthesis in bacteria and mammals is pantothenate kinase (CoaA), which governs the intracellular concentration of CoA through feedback regulation by CoA and its thioesters. CoaA from Staphylococcus aureus (SaCoaA) has a distinct primary sequence that is more similar to the mammalian pantothenate kinases than the prototypical bacterial CoaA of Escherichia coli. In contrast to all known pantothenate kinases, SaCoaA activity is not feedback-regulated by CoA or CoA thioesters. Metabolic labeling of S. aureus confirms that CoA levels are not controlled by CoaA or at steps downstream from CoaA. The pantothenic acid antimetabolite N-heptylpantothenamide (N7-Pan) possesses potent antimicrobial activity against S. aureus and has multiple cellular targets. N7-Pan is a substrate for SaCoaA and is converted to the inactive butyldethia-CoA analog by the downstream pathway enzymes. The analog is also incorporated into acyl carrier protein and D-alanyl carrier protein, the prosthetic groups of which are derived from CoA. The inactivation of acyl carrier protein and the cessation of fatty acid synthesis are the most critical causes of growth inhibition by N7-Pan because the toxicity of the drug is ameliorated by supplementing the growth medium with fatty acids. The absence of feedback regulation at the pantothenate kinase step allows the accumulation of high concentrations of intracellular CoA, consistent with the physiology of S. aureus, which lacks glutathione and relies on the CoA/CoA disulfide reductase redox system for protection from oxidative damage.

Amino Acid Sequence↗